HIV-1 Vpr antagonizes innate immune activation by targeting ...

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HIV-1 Vpr antagonizes innate immune activation by targeting karyopherin-mediated NF-1

κB/IRF3 nuclear transport 2

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Hataf Khan1,2,+, Rebecca P. Sumner1,+, Jane Rasaiyaah1,3, Choon Ping Tan1,4, Maria Teresa 4

Rodriguez-Plata1,5, Chris van Tulleken1, Douglas Fink1, Lorena Zuliani-Alvarez1, Lucy Thorne1, 5

David Stirling1,6 Richard S. B. Milne1 & Greg J. Towers1 6

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1Division of Infection and Immunity, University College London, 90 Gower Street, London, UK 8

2Current address: Department of Infectious Diseases, King’s College London, London, UK 9

3Current address: Molecular and Cellular Immunology Unit, UCL Great Ormond Street Institute of 10

Child Health, London, UK. 11

4Current address: Translation & Innovation Hub, 80 Wood Lane, London, UK 12

5Current address: Black Belt TX Ltd, Stevenage Bioscience Catalyst, Gunnels Wood Rd, 13

Stevenage, UK 14

6Current address: Broad Institute of MIT and Harvard University, Cambridge, MA, USA. 15

+equal contribution 16

*Correspondence: [email protected] 17

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Abstract 19

HIV-1 must replicate in cells that are equipped to defend themselves from infection through 20

intracellular innate immune systems. HIV-1 evades innate immune sensing through encapsidated 21

DNA synthesis and encodes accessory genes that antagonize specific antiviral effectors. Here we 22

show that both particle associated, and expressed HIV-1 Vpr, antagonize the stimulatory effect of 23

a variety of pathogen associated molecular patterns by inhibiting IRF3 and NF-κB nuclear 24

transport. Phosphorylation of IRF3 at S396, but not S386, was also inhibited. We propose that, 25

rather than promoting HIV-1 nuclear import, Vpr interacts with karyopherins to disturb their import 26

of IRF3 and NF-κB to promote replication in macrophages. Concordantly, we demonstrate Vpr 27

dependent rescue of HIV-1 replication in human macrophages from inhibition by cGAMP, the 28

product of activated cGAS. We propose a model that unifies Vpr manipulation of nuclear import 29

and inhibition of innate immune activation to promote HIV-1 replication and transmission. 30

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Key words: HIV-1, Vpr, DNA sensing, cGAS, Karyopherin, IRF3, NF- κB, nuclear transport 32

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mailto:[email protected]

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Introduction 39

Like all viruses, lentiviruses must navigate the hostile environment of the host cell in order to infect, 40

produce new viral particles, and transmit to new cells. A principal feature of cellular defences is 41

detection or sensing of incoming viruses and subsequent production of inflammatory cytokines, 42

particularly type 1 interferons (IFNs). All viral infections have the potential to trigger IFN in vivo 43

through viral pathogen associated molecular patterns (PAMPs) activating pattern recognition 44

receptors (PRR). The degree to which each virus does this, and their capacity to antagonize IFN 45

activity and its complex effects, are key in determining transmission mechanism, host range and 46

disease pathogenesis. Like other viruses, lentiviruses also antagonize specific host proteins or 47

pathways that would otherwise suppress infection. Lentiviruses typically do this through accessory 48

gene function. For example, HIV-1 antagonizes IFN induced restriction factors through accessory 49

genes encoding Vif (APOBEC3G/H), Vpu (tetherin) and Nef (tetherin/SERINC3/5) reviewed in 50

(Foster et al., 2017; Sumner et al., 2017). 51

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The HIV-1 accessory protein Vpr interacts with and manipulates many proteins including its 53

cofactor DCAF1 (Zhang et al., 2001), karyopherin alpha 1 (KPNA1, importin α) (Miyatake et al., 54

2016) the host enzyme UNG2 (Wu et al., 2016) as well as HTLF (Lahouassa et al., 2016; Yan et 55

al., 2019), SLX4 (Laguette et al., 2014) and CCDC137 (Zhang & Bieniasz, 2019). Indeed, Vpr has 56

been shown to significantly change infected cell protein profiles, affecting the level of hundreds of 57

proteins in proteomic studies, likely indirectly in most cases, consistent with manipulation of central 58

mechanisms in cell biology (Greenwood et al., 2019). Vpr has also been shown to both enhance 59

(Liu et al., 2014; Liu et al., 2013; Vermeire et al., 2016) or decrease, NF-κB activation (Harman et 60

al., 2015; Trotard et al., 2016) in different contexts and act as a cofactor for HIV-1 nuclear entry, 61

particularly in macrophages (Vodicka et al., 1998). However, despite this work the mechanistic 62

details of Vpr promotion of HIV replication are poorly understood and many studies seem 63

contradictory. This is partly because the mechanisms of Vpr-dependent enhancement of HIV-1 64

replication are context dependent, and cell type specific, although most studies agree that Vpr is 65

more important for replication in macrophages than in T cells or PBMC (Connor et al., 1995; 66

Dedera et al., 1989; Fouchier et al., 1998; Hattori et al., 1990; Mashiba et al., 2014). Manipulation 67

of host innate immune mechanisms by Vpr to facilitate replication in macrophages has been 68

suggested by various studies although there has been no clear mechanistic model or 69

understanding how particular Vpr target proteins link to innate immune manipulation (Harman et 70

al., 2015; Liu et al., 2014; Okumura et al., 2008; Trotard et al., 2016; Vermeire et al., 2016). 71

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Many viruses have been shown to manipulate innate immune activation by targeting transcription 73

factor nuclear entry downstream of PRR. For example, Japanese encephalitis virus NS5 targets 74

KPNA2, 3 and 4 to prevent IRF3 and NF-ĸB nuclear translocation (Ye et al., 2017). Hantaan virus 75

nucleocapsid protein inhibits NF-ĸB p65 translocation by targeting KPNA1, -2, and -4 (Taylor et 76

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al., 2009). Most recently, vaccinia virus protein A55 was shown to interact with KPNA2 to disturb 77

its interaction with NF-ĸB (Pallett et al., 2019). Hepatitis C virus NS3/4A protein restricts IRF3 and 78

NF-κB translocation by cleaving KPNB1 (importin-β) (Gagne et al., 2017). 79

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HIV-1 Vpr has also been linked to Karyopherins and manipulation of nuclear import. Vpr has been 81

shown to interact with a variety of mouse (Miyatake et al., 2016), yeast (Vodicka et al., 1998) and 82

human karyopherin proteins including human KPNA1, 2 and 5 (Nitahara-Kasahara et al., 2007). 83

Indeed, the structure of a C-terminal Vpr peptide (residues 85-96) has been solved in complex with 84

mouse importin α2 (Miyatake et al., 2016). Here demonstrate that Vpr inhibits innate immune 85

activation downstream of a variety of viral and non-viral PAMPs by inhibiting nuclear transport of 86

IRF3 and NF-ĸB by KPNA1. We confirm Vpr interaction with KPNA1 by co-immunoprecipitation 87

and link Karyopherin binding and inhibition of innate immunity by showing that Vpr prevents 88

interaction between KPNA1 and IRF3/NF-ĸB in vitro. Critically, we show that Vpr (F34I/P35N) fails 89

to inhibit nuclear transport of IRF3 and NF-ĸB, fails to antagonise innate immune sensing, and fails 90

to interact with KPNA1. We demonstrate that Vpr mutants that do not recruit to the nuclear 91

envelope, cannot to antagonize innate sensing, but retain induction of cell cycle arrest, genetically 92

separating key Vpr functions. Importantly, by targeting activated transcription factors, Vpr prevents 93

innate immune activation by a wide range of non-viral agonists suggesting Vpr has roles beyond 94

inhibiting innate immune activation of PAMPs derived from the virus itself. Our new findings support 95

a unifying model of Vpr function, consistent with much of the Vpr literature, in which Vpr associated 96

with incoming viral particles suppresses nuclear entry of activated inflammatory transcription 97

factors to facilitate HIV-1 replication in innate immune activated macrophages. 98

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Results 100

HIV-1 replication in cGAMP-stimulated MDMs requires Vpr 101

A considerable body of evidence suggests an important role for Vpr in supporting HIV-1 replication 102

in macrophages but the relevant Vpr mechanisms for this function have been enigmatic. We set 103

out to investigate the role of Vpr in manipulating host innate immune mechanisms during HIV-1 104

infection of primary human cells. We prepared human monocyte-derived macrophages (MDM) by 105

purifying monocytes from peripheral blood by adherence and treating with M-CSF (Rasaiyaah et 106

al., 2013). Macrophages prepared in this way are particularly permissive to HIV-1 replication 107

facilitating study of HIV-1 biology in a primary myeloid cell type. We found that wild type HIV-1 and 108

HIV-1∆Vpr replicated equally well in (MDM)(Figure 1A) (Rasaiyaah et al., 2013) Consistent with 109

previous studies, Wild type HIV-1, and HIV-1 deleted for Vpr replicated equally well in activated 110

primary human CD4+ T cells (Figure 1-figure supplement 1A) (Dedera et al., 1989; Fouchier et al., 111

1998). 112

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Vpr has been shown to antagonize innate immune signaling in HeLa cells reconstituted for DNA 114

sensing by STING expression (Trotard et al., 2016), so we hypothesized that Vpr might be 115

particularly important when DNA sensing is activated. To test this, we mimicked activation of the 116

DNA sensor cGAS by treating MDM with cGAMP, the product of activated cGAS. In the presence 117

of cGAMP, HIV-1 replication in MDM was, indeed, Vpr-dependent. 1µg/ml cGAMP specifically 118

suppressed HIV-1∆Vpr more potently than wild type virus and 4µg/ml cGAMP overcame Vpr 119

activity and suppressed replication of both wild type and mutant viruses (Figure 1A). Intriguingly, 120

Vpr did not rescue HIV-1 replication from cGAMP-mediated inhibition in primary human CD4+ T 121

cells, and cGAMP had only minimal effect on HIV-1 replication in Jurkat T cells (Figure 1-figure 122

supplement 1A). These data demonstrate that HIV-1 replication in cGAMP-stimulated MDM is Vpr 123

dependent. They are consistent with previous observations suggesting Vpr is more important in 124

macrophages than T cells and that the consequences of cGAMP treatment differ between these 125

cell types (Gulen et al., 2017; Xu et al., 2016). 126

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HIV-1 particle delivered Vpr inhibits gene expression stimulated by DNA sensing 128

We next investigated the effect of particle-associated Vpr on innate immune activation. The 129

myeloid cell line THP-1 expresses cGAS and STING and has a functional DNA sensing pathway 130

(Mankan et al., 2014). We used THP-1 cells expressing the Gaussia luciferase gene under the 131

control of the endogenous IFIT1 promoter (herein referred to as THP-1 IFIT1-luc) (Mankan et al., 132

2014) to measure the effect of Vpr on cGAMP-induced IFIT1-luc expression. IFIT1 (ISG56) is a 133

well-characterized ISG that is highly sensitive to cGAMP and type 1 IFN. Treatment of THP-1 IFIT-134

luc cells with cGAMP induced IFIT1-luc expression by two orders of magnitude. This activation 135

was significantly suppressed if cells were infected with VSV-G pseudotyped, genome-free, HIV-136

particles bearing Vpr, (referred to here as virus-like particles or VLP), but not by VLP lacking Vpr, 137

immediately prior to cGAMP addition (Figure 1B). IFIT1-Luc was measured 6, 8 and 24 hours after 138

cGAMP addition/infection. 139

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In this experiment, doses of VLP required to suppress IFIT1-luc expression were high, equivalent 141

to a multiplicity of infection of 20 as measured by correlating VLP reverse transcriptase levels (SG-142

PERT) (Jolien Vermeire et al., 2012), with HIV-1 GFP titers on THP-1. We assume that such a 143

high dose of Vpr-bearing VLP is required because cGAMP treatment activates numerous STING 144

complexes in most of the cGAMP-treated cells. If this effect of Vpr is relevant to infection, we 145

expect that cGAS/STING activated by the incoming HIV genome should be sensitive to the amount 146

of Vpr contained in an individual particle. To test this, we activated DNA sensing using high dose 147

infection by VSV-G pseudotyped HIV-1 vectors bearing GFP-encoding genome. We used an HIV-148

1 packaging plasmid, derived from HIV-1 clone R9, encoding Gag-Pol, Tat and Rev (p8.91) or 149

Gag-Pol, Tat and Rev and Vpr, Vpu, Vif and Nef (p8.2) (Zufferey et al., 1997). Strikingly, although 150

Vpr positive and negative HIV-1 GFP stocks infected THP-1 cells to similar levels (Figure 1D), 151

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induction of inflammatory cytokine, and ISG, CXCL10 was reduced if the HIV-1 GFP carried Vpr 152

(Figure 1C). This indicates that Vpr can inhibit the consequences of sensing driven by the Vpr 153

bearing virus particles themselves. 154

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Genome-free, non-infectious, HIV-1 particles did not induce CXCL10 expression (Figure 1E, F), 156

evidencing the importance of viral DNA in this response. Furthermore, CXCL10 expression was 157

not induced after infection of THP-1 cGAS knock out cells, consistent with CXCL10 induction being 158

cGAS-dependent (Figure 1G). Knock out of the RNA sensing adaptor protein MAVS had no effect 159

on induction of CXCL10 (Figure 1G). cGAS and MAVS knock out were confirmed by immunoblot 160

(Figure 1-figure supplement 1C). 161

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As expected, a lower dose of virus was required to see the effect of Vpr when the particles 163

themselves activated sensing, and in this latter experiment, Vpr effects were clear at MOIs of 3 164

(Figure 1C, E). Moreover, single round titer of HIV-1 GFP was not affected by cGAS or MAVS 165

knock out, confirming that sensing activation does not impact single round infectivity of HIV-1 GFP 166

VSV-G pseudotypes in this assay consistent with HIV-1 vector not being particularly sensitive to 167

IFN (Figure 1H, Figure 1-figure supplement 1B). 168

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HIV-1 Vpr expression inhibits innate immune activation 170

We next tested whether Vpr expressed in isolation can suppress innate immune activation by 171

cGAMP. Vpr from the primary founder HIV-1 clone SUMA (Fischer et al., 2010) was expressed in 172

THP-1 IFIT1-luc cells using an HIV-1 vector we called pCSVIG (Figure 2-figure supplement 1A, 173

S2B). Vpr was expressed using MOIs of approximately 0.2-1. Forty hours after transduction, cells 174

were treated with cGAMP (5µg/ml), and IFIT1-luc was measured 8 hours later. Prior expression of 175

Vpr reduced IFIT1-luc responses in a dose-dependent manner whilst the highest dose of empty 176

vector had no effect, measured as a negative control (Figure 2A; infection data in Figure 2-figure 177

supplement 1C). Vpr expression (MOI=1, Figure 2-figure supplement 1D) also suppressed 178

cGAMP-mediated induction of endogenous ISG mRNA expression, measured by qRT-PCR for 179

MxA, CXCL10, IFIT2 and viperin (Figure 2B) and inhibited cGAMP induced CXCL10 secretion 180

(Figure 2C; infection data to gauge MOI in Figure 2-figure supplement 1E). 181

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IFIT1-luc expression stimulated by transfection of herring testis (HT) DNA was also inhibited by 183

Vpr expression, consistent with the notion that Vpr antagonizes DNA sensing (Figure 2D, Figure 184

2-figure supplement 1F). Strikingly, Vpr also reduced Sendai virus induced activation of IFIT1-luc, 185

which is mediated by MDA5 and RIGI RNA sensing (Andrejeva et al., 2004; Rehwinkel et al., 2010) 186

(Figure 2E, Figure 2-figure supplement 1G) and IFIT1-luc activation after stimulation with the TLR4 187

ligand LPS (Figure 2F, Figure 2-figure supplement 1H). Thus, Vpr expression appeared to mediate 188

a generalized suppression of innate immune activation. 189

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Vpr inhibition of innate immune activation is dependent on DCAF1 but independent of cell 191

cycle arrest 192

In order to separate innate immune antagonism from other Vpr functions, we used three Vpr 193

mutants with distinct functional deficits. Vpr R80A, is defective in inducing cell cycle arrest 194

(Laguette et al., 2014); Vpr Q65R fails to recruit DCAF1 and so cannot degrade target proteins 195

(Laguette et al., 2014); and Vpr F34I/P35N fails to bind cyclophilin A and does not localize to the 196

nuclear membrane (Vodicka et al., 1998; Zander et al., 2003). 197

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All three mutant Vprs were efficiently incorporated into HIV-1 GFP particles (Figure 3A). When 199

delivered by viral particles, Vpr R80A effectively suppressed IFIT1-luc induction by cGAMP in THP-200

1 cells, however Vpr Q65R and Vpr F34I/P35N had little if any suppressive effect (Figure 3B). In 201

these experiments, cGAMP was added to the target cells directly after the virus. Suppression of 202

IFIT1-luc induction by Vpr R80A suggested that cell cycle arrest was not required for innate 203

immune antagonism. To further test this, we measured the effect of all three Vpr mutants on cell 204

cycle progression. As reported, WT Vpr expression in THP-1 cells induced a significant increase 205

of cells in G2/M phase of cell cycle and Vpr R80A had no effect (Figure 3C,Figure 3-figure 206

supplement 1G) (Laguette et al., 2014). Vpr F34I/P35N, which cannot effectively suppress cGAMP 207

mediated IFIT1-luc/ISG expression (Figure 3B, 3G), also induced G1/M cell cycle arrest, albeit 208

slightly less efficiently than wild type Vpr protein, as previously described (Vodicka et al., 1998) 209

(Figure 3C). The DCAF1 Vpr binding mutant Q65R did not inhibit cell cycle, as reported (Figure 210

3C) (Laguette et al., 2014). These data genetically separate the effects of Vpr expression on cell 211

cycle, and on inhibition of innate immune activation, suggesting that these functions depend on 212

manipulation of different target proteins. It is striking that amino acids at positions 34/35 and 80 213

are close in Vpr structures and distant from the UNG2 binding site, suggesting an additional target 214

binding interface, as seen in the highly related Vpx protein (Figure 3-figure supplement 1B, C) 215

(Morellet et al., 2003; Schwefel et al., 2014; Wu et al., 2016). 216

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We next asked whether DCAF-1 was required for innate immune antagonism, as suggested by 218

the Vpr Q65R mutant, which fails to recruit DCAF1, and cannot suppress cGAMP-induced IFIT1-219

luc expression (Figure 3B). Depletion of DCAF1 in THP-1 cells by shRNA prevented Vpr from 220

inhibiting cGAMP induction of IFIT1-luc (Figure 3D). Neither DCAF1 depletion, nor cGAMP 221

treatment reduced infectivity of HIV-1 GFP vector (Figure 3-figure supplement 1A). Vpr was active 222

in cells expressing a non-targeting shRNA (shControl) and suppressed IFIT1-luc induction (Figure 223

3D). Expression of empty (no Vpr) vector had no effect on IFIT1-luc induction (Figure 3D). Effective 224

depletion of DCAF1 was evidenced by immunoblot (Figure 3E). Thus, Vpr inhibition of innate 225

immune activation requires DCAF1. 226

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Expressed Vpr had similar mutation sensitivity as Vpr delivered by HIV-1 particles (compare 228

Figures 3F, G and 3B). Expression of wild type Vpr, or Vpr R80A, prevented cGAMP activation of 229

the IFIT1-luc reporter (Figure 3F), and of endogenous MxA in THP-1 cells (Figure 3G, Figure 3-230

figure supplement 1D). HT DNA transfection, but not lipofectamine alone, activated IFIT1-luc 231

reporter expression, as expected, and this was also sensitive to wild type and VprR80A expression, 232

but not expression of Vpr F34I/P35N (Figure 3-figure supplement 1E, F). Vpr Q65R had only a 233

small inhibitory effect consistent with data in Figure 3B. 234

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Wild Type Vpr, but not sensing antagonism inactive Vpr mutants, colocalize with nuclear 236

pores 237

Having identified Vpr mutants defective for antagonism of innate immune sensing, we sought 238

further clues about Vpr mechanism by examining wild type and mutant Vpr location within cells. 239

Vpr expressed in isolation is found in the nucleus and associated with nuclear pores (Fouchier et 240

al., 1998; Le Rouzic et al., 2002). Concordantly, we found FLAG-Vpr in the nucleus, and 241

colocalized with antibody staining the nuclear pore complex (NPC), when expressed by transient 242

transfection in HeLa cells (Figure 4A, B). As previously reported for the single mutant F34I (Jacquot 243

et al., 2007; Vodicka et al., 1998), we found that the double Vpr mutant F34I/P35N, as well as Vpr 244

Q65R, were mislocalized, as compared to wild type and R80A Vpr. Thus, these mutants which fail 245

to inactivate innate immune sensing, fail to localize to the nuclear membrane. Defective Vpr 246

mutants F34I/P35N and Q65R appeared qualitatively different inside the nucleus, and nuclear rim 247

staining was less well defined, suggesting that they have lost interactions with a protein(s) that 248

normally influences their position within the cell. Fluorescence intensity measurements along 249

transverse sections of nuclei in single confocal images showed two distinct peaks of nuclear pore 250

staining representing each edge of the nucleus. These peaks overlapped with WT and Vpr R80A 251

fluorescence but not with Vpr F34I/P35N or Vpr Q65R fluorescence, which was more diffuse and 252

less well defined at the nuclear rim (Figure 4C). These data link Vpr nuclear membrane association 253

with antagonism of innate immune sensing for the first time. 254

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Vpr has been described to interact with cyclophilin A (CypA) and mutating Vpr residue P35 was 256

reported to prevent this interaction (Zander et al., 2003). The nuclear pore complex has cyclophilin-257

like domains, which are structurally very similar to CypA, at the end of the Nup358 fibers that 258

protrude into the cytoplasm (Schaller et al., 2011). To test whether Nup358 was required for Vpr 259

association with the nuclear rim, we expressed FLAG-Vpr in Nup358-depleted HeLa cells (Schaller 260

et al., 2011) and stained the Vpr FLAG tag (green) and NPC (red) (Figure 4-figure supplement 1A, 261

B). Despite effective Nup358 depletion (Figure 4-figure supplement 1C), Vpr remained associated 262

with the nuclear rim suggesting that Nup358 is not required for Vpr nuclear rim association (Figure 263

4-figure supplement 1A, B, D). 264

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Vpr inhibits IRF3 nuclear translocation 266

cGAMP is produced by activated cGAS and is recruited by STING, which then forms an active 267

kinase complex in which TBK1 phosphorylates STING, TBK1 itself, and the transcription factor 268

IRF3 (Liu et al., 2015; Zhang et al., 2019). IRF3 phosphorylation promotes nuclear translocation 269

and subsequent activation of gene expression including type 1 IFNs (Chen et al., 2008). As 270

expected, transfection of THP-1 IFIT1-luc cells with HT DNA induced phosphorylation of STING, 271

TBK1 and IRF3-S386 (Figure 5A). Measurement of IFIT1-luc expression, in the same samples, 272

three hours after stimulation, indicated induction of IFIT1-luc by HT DNA but not after prior Vpr 273

expression using a lentiviral vector (Figure 5B). Strikingly, Vpr expression for 48 hours did not 274

impact STING, TBK1 or IRF3 protein levels, or their phosphorylation status 3 hours after DNA 275

transfection, measuring IRF3 phosphorylation at S386 (Figure 5A). Empty vector expression had 276

no detectable effect on protein levels or phosphorylation (Figure 5A). Actin was detected as a 277

loading control and Vpr/empty vector were used at a vector MOI of about 1 (Figure 5-figure 278

supplement 1A). A second example of this experiment is presented in Figure 5-figure supplement 279

1B-E. IRF3 is phosphorylated at multiple sites during activation including at IRF3 S396. We 280

therefore examined IRF3 S396 phosphorylation using a phospho-IRF3-S396 specific antibody and 281

flow cytometry because this antibody didn’t work well by immunoblot. We found that in this case, 282

Vpr delivery by VLP did reduce phosphorylation of IRF3-S396 after stimulation by either cGAMP 283

or HT DNA in THP-1 cells (Figure 5C). 284

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Given that Vpr is associated with the nuclear rim, and Vpr mutations that break antagonism of 286

innate sensing mislocalize Vpr, we hypothesized that rather than impacting levels of signaling 287

proteins, Vpr may act at nuclear pores to influence nuclear transport of inflammatory transcription 288

factors. This would be consistent with the broad innate immune antagonism that we have observed 289

(Figure 2), and with previous reports of Vpr influencing nuclear transport, for example, of viral 290

nucleic acids (Heinzinger et al., 1994; Miyatake et al., 2016; Popov et al., 1998), and inhibiting 291

sensing of HIV-1 (Trotard et al., 2016). We therefore investigated the effect of Vpr on cGAMP-292

induced IRF3 nuclear translocation. THP-1 were differentiated with 50ng/ml phorbol-12 myristate 293

acetate (PMA) to attach them to glass for microscopy. In these experiments, VLP with or without 294

Vpr are used to infect cells immediately after they are treated with innate immune stimulants. IRF3 295

translocation is measured three hours later by immunofluorescent labeling. VSV-G pseudotyped 296

HIV-1 GFP bearing Vpr reduced cGAMP-stimulated IRF3 nuclear translocation in a dose-297

dependent way whilst HIV-1 lacking Vpr had no effect (Figure 5D, E, Figure 5-figure supplement 298

2A). These data are consistent with a previous report in which Vpr suppressed nuclear transport 299

of IRF3-GFP on HIV-1 infection of HeLa cells in which DNA sensing had been reconstituted by 300

expression of STING (Trotard et al., 2016). Importantly, in our experiments in THP-1, suppression 301

of IRF3 nuclear translocation by Vpr was sensitive to Vpr mutation, with the same specificity as 302

before (Compare Figure 3, 4, 5F, Figure 5-figure supplement 1G-J). HIV-1 GFP bearing Vpr 303

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F34I/P35N, or Vpr Q65R, failed to efficiently suppress IRF3 nuclear localization after cGAMP 304

stimulation (Figure 5F, S5G) or after transfection of differentiated THP-1 with HT DNA (Figure 5G, 305

S5H). Conversely, HIV-1 GFP bearing wild type Vpr, or Vpr R80A, effectively suppressed IRF3 306

nuclear localization after stimulation with cGAMP or HT DNA (Figure 5F, G S5G, H). Similar 307

inhibition specificity by Vpr was also seen after activation of IRF3 nuclear translocation by 308

transfection with the RNA mimic poly I:C (Figure 5-figure supplement 1I, J) or treatment with LPS 309

(Figure 5-figure supplement 1F). Thus, suppression of IRF3 nuclear translocation correlates with 310

the capacity of Vpr mutants to antagonize innate immune activation. 311

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Vpr inhibits NF-κB p65 nuclear translocation and NF-κB sensitive plasmid expression 313

DNA sensing by cGAS is known to activate NF-κB as well as IRF3 (Fang et al., 2017). To test 314

whether Vpr influenced NF-κB activation we repeated the experiment in Figure 1C-F but using 315

THP-1 cells bearing an NF-κB -luciferase reporter (THP-1 NF-κB-luc) (Figure 6A-C). VSV-G 316

pseudotyped HIV-1 GFP vector bearing Vpr minimally activated NF-κB-luc expression, whereas 317

Vpr negative HIV-1 GFP activated NF-κB-luc expression effectively (Figure 6A). Activation was 318

dependent on viral genome because similar doses of HIV-1 VLP, made without genome, did not 319

induce NF-κB-luc expression (Figure 6A). Viral doses were equalized by measurement of RT 320

activity (SGPERT) (Vermeire et al., 2016). Vpr bearing, and Vpr negative, HIV-1 GFP were equally 321

infectious and genome-free VLP were not infectious, as expected (Figure 6B). VSV-G 322

pseudotyped HIV-1 GFP bearing Vpr, but not virus lacking Vpr, suppressed cGAMP-mediated 323

activation of the NF-κB-sensitive gene IL6 (Figure 6C). We could not detect NF-κB nuclear 324

localization in THP-1 after cGAMP treatment, perhaps due to timing, so we tested mutant Vpr 325

specificity using Poly I:C to stimulate NF-κB p65 nuclear localization. Again, we transfected 326

differentiated THP-1 cells, this time with Poly I:C and then immediately infected them with HIV-1 327

GFP bearing or lacking Vpr and fixed and stained for NF-κB p65 localisation three hours later. We 328

found Vpr inhibited NF-κB p65 nuclear localisation with similar sensitivity to mutation as for IRF3: 329

VLP bearing wild type Vpr or Vpr R80A inhibited NF-κB p65 nuclear localisation but VLP bearing 330

Vpr F34I/P35N or Vpr Q65R did not (Figure 6D, Figure 6-figure supplement 1B). Vpr also 331

suppressed NF-κB p65 nuclear localization after treatment of THP-1 with LPS (Figure 6-figure 332

supplement 1C). 333

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Previous work has shown that Vpr inhibits the activity of the human CMV major immediate early 335

promoter (MIEP) (Liu et al., 2015). We hypothesized that this effect may be due to the dependence 336

of this promoter on NF-κB (DeMeritt et al., 2004). As expected Flag-Vpr expression suppressed 337

GFP expression from a co-transfected CMV MIEP – GFP construct (Figure 6E) as well as several 338

other NF-κB sensitive constructs expressing luciferase (Figure 6-figure supplement 1A). 339

Importantly, Vpr mutants F34I/P35N, and Vpr Q65R suppressed GFP expression much less 340

effectively than WT Vpr, or Vpr R80A, consistent with this effect being due to inhibition of NF-κB 341

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nuclear entry (Figure 6E, S6D, E). To probe this further, we used two constructs lacking NF-κB 342

binding sites in which GFP is driven from the Ubiquitin C (Ub) promoter (Matsuda & Cepko, 2004) 343

or from the elongation factor 1 alpha (EF1α) promoter (Matsuda & Cepko, 2004). Expression of 344

GFP from these constructs was minimally affected by Vpr co-transfection, but GFP expression 345

from the CMV MIEP was reduced as before (Figure 6F). Importantly, CMV MIEP-GFP expression 346

was induced by activation of NF-κB with exogenous tumour necrosis factor alpha (TNFα) whereas 347

Ub-GFP and EF1α-GFP were not, providing further evidence that Vpr inhibition correlated with 348

promoter sensitivity to NF-κB (Figure 6G, Figure 6-figure supplement 1F-G). Thus, inhibition of NF-349

κB nuclear transport by Vpr likely explains the observation that Vpr suppresses expression from 350

the CMV MIEP, but not promoters that are independent of NF-κB activity for expression. This is 351

important because previous studies have used Vpr co-transfection with CMV MIEP driven 352

promoters to address Vpr function (Su et al., 2019). 353

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HIV-1 Vpr interacts with karyopherins and inhibits NF-κB (p65) and IRF3 recruitment 355

WT Vpr suppresses nuclear entry of IRF3 and NF-κB, but Vpr DCAF1 binding mutant Q65R does 356

not (Figure 5, 6). This suggested that Vpr might degrade particular nuclear transport proteins to 357

exert its effect. We therefore tested whether Vpr expression caused degradation of karyopherins 358

KPNA1, KPNA2, KPNA3, KPNA4, KPNA5, KPNA6 or KPNB1. We infected cells with Vpr encoding 359

HIV-1 vector, extracted total protein 48 hours after infection, and detected each protein using 360

immunoblot (Figure 7A). However, we did not detect reduced levels of any of these karyopherins. 361

It is possible that Vpr recruits karyopherins but does not degrade them. To test this, we sought 362

interaction between Vpr and karyopherins KPNA1, KPNA2 and KPNA3 by co-immunoprecipitation. 363

We found that immunoprecipitation of wild type HA-Vpr co-precipitated Flag-KPNA1, as has been 364

reported previously (Miyatake et al., 2016; Nitahara-Kasahara et al., 2007; Vodicka et al., 1998) 365

and to a lesser degree Flag-KPNA2 and Flag-KPNA3, but not Flag-tagged GFP (Figure 7B). In a 366

second experiment we tested whether KPNA1-3 interacted with the inactive Vpr mutant 367

F34I/P35N. WT Vpr interacted with KPNA1 as before, with less efficient interaction with KPNA2 368

and KPNA3 (Figure 7C). Importantly, KPNA1 interacted with the Vpr F34I/P35N only very weakly, 369

and much less than WT Vpr, consistent with the mutant’s reduced activity in antagonizing innate 370

immune sensing (Figure 7C). Given that Vpr expression did not cause KPNA1 degradation, we 371

sought evidence for Vpr disturbing interactions between KPNA1 and IRF3 or NF-κB p65. HA-IRF3 372

immunoprecipitated with Flag-KPNA1 as expected and this interaction was reduced by expression 373

of WT Vpr, but not inactive mutant Vpr F34I/P35N (Figure 7D). A competing immunoprecipitation 374

experiment with KPNA1 and NF-κB p65 gave similar results. Immunoprecipitation of Flag-KPNA1 375

co-precipitated NF-κB p65 and this was reduced by co-expression of WT Vpr, but not Vpr 376

F34I/P35N (Figure 7E). Thus, for the first time, we explain the interaction of Vpr with karyopherins, 377

by demonstrating that it prevents them from efficiently recruiting and transporting transcription 378

factors IRF3 and NF-κB into the nucleus after innate immune activation. This finding provides a 379

11

mechanistic basis for the broad innate immune antagonism activity of Vpr and links manipulation 380

of nuclear transport with antagonism of innate immunity rather than with infection itself. 381

382

Discussion 383

Despite many studies investigating Vpr function, a clear mechanism for how HIV-1 Vpr promotes 384

replication has not been forthcoming, partly because Vpr replication phenotypes have not been 385

clearly mechanistically linked to manipulation of specific target proteins. Early work connected 386

nuclear membrane association of Vpr with replication in macrophages, but not T cells (Connor et 387

al., 1995; Dedera et al., 1989; Fouchier et al., 1998; Hattori et al., 1990; Mashiba et al., 2014; 388

Vodicka et al., 1998). Early work also separated the effect of Vpr on cell cycle from its association 389

with the nuclear envelope using Vpr mutants, particularly Vpr F34I, which, as confirmed herein, 390

suppressed cell cycle, but did not recruit to the nuclear membrane (Jacquot et al., 2007; Vodicka 391

et al., 1998). Vpr mutants that did not localise to the nuclear membrane, did not promote 392

macrophage replication, leading the authors to reasonably conclude that Vpr contributed to nuclear 393

transport of the virus itself. This observation was consistent with the notion that Vpr-mediated 394

support of nuclear entry is expected to be more important in non-dividing cells (macrophages), 395

than rapidly dividing cells (activated T cells). Vpr is also not typically required for infection of cell 396

lines, even if they are not dividing (Yamashita & Emerman, 2005). 397

398

In complementary studies, Vpr has been associated with antagonism of innate immune sensing in 399

macrophages (Harman et al., 2015), T cells (Vermeire et al., 2016), as well as in HeLa cells 400

reconstituted for DNA sensing by STING expression (Trotard et al., 2016). Here we propose a 401

model that unifies Vpr’s role in manipulating nuclear entry with its antagonism of innate immune 402

signalling. We propose that Vpr interaction with karyopherin KPNA1 (Figure 7) (Miyatake et al., 403

2016; Nitahara-Kasahara et al., 2007; Vodicka et al., 1998) inhibits nuclear transport of activated 404

IRF3 and NF-ĸB (Figure 5-7) and subsequent gene expression changes downstream of innate 405

immune sensing (Figures 1-3). Thus, HIV-1 Vpr antagonizes the consequences of innate immune 406

activation by HIV-derived, and non-HIV derived PAMPs alike. This explains its importance for 407

maximal replication in macrophages, because activated T cells, and most cell lines, respond to 408

innate immune agonists poorly, and particularly to DNA based PAMPs (Figure 1) (Cingöz & Goff, 409

2019; de Queiroz et al., 2019; Heiber & Barber, 2012; Xia et al., 2016; Xia et al., 2016). 410

411

We propose that previous demonstrations of Vpr dependent HIV-1 replication in macrophages, 412

that depended on association of Vpr with the NPC, or with nuclear transport factors, are explained 413

by Vpr inhibition of innate immune sensing and subsequent antiviral responses (Jacquot et al., 414

2007; Vodicka et al., 1998). Indeed, we now know that induction of an innate response by HIV-1 415

lacking Vpr is expected to suppress viral nuclear entry because IFN induction of MxB in 416

macrophages causes inhibition of HIV-1 nuclear entry (Goujon et al., 2013; Kane et al., 2013). 417

12

Thus we propose that Vpr does not directly promote HIV-1 nuclear entry. Rather it prevents 418

inhibition of nuclear entry downstream of innate immune activation. We hypothesise that Vpr 419

provides an in vivo replication advantage because activation of IRF3 and NF-ĸB induces 420

expression of inflammatory cytokines, including type 1 IFNs, and subsequently restriction factors 421

for which HIV-1 does not encode antagonists. For example, in addition to MxB, IFN induces 422

IFITM1-3 (Foster et al., 2016) and TRIM5α (Jimenez-Guardeño et al., 2019) all of which can inhibit 423

HIV-1. Concordantly, accidental infection of a lab worker with a Vpr-defective HIV-1 isolate resulted 424

in delayed seroconversion, suppressed viremia and normal T-cell counts without need for anti-viral 425

treatment (Ali et al., 2018). 426

427

In most of the experiments herein, and in previous studies of Vpr function in cell lines (Yamashita 428

& Emerman, 2005), Vpr did not impact infection of single round VSV-G pseudotyped HIV-1 vectors 429

encoding GFP. We propose that this is because if antiviral inflammatory responses, e.g. IFN, are 430

triggered at around the time of infection, either by exogenous signals, or by HIV-1 itself, then the 431

activated antiviral effectors are too slow to inhibit that infection, i.e. the expression of GFP from an 432

integrated provirus. Thus, a requirement for Vpr is only revealed by spreading infection assays in 433

innate competent cells such as macrophages, which can suppress replication of subsequent 434

rounds of infection. 435

436

We and others, have argued that the wild type infectious HIV-1 genome is not efficiently sensed 437

by nucleic acid sensors, or degraded by cellular nucleases, because the capsid protects and 438

sequesters genome, while regulating the process of reverse transcription, during transport across 439

a hostile cytoplasmic environment, prior to uncoating at the NPC, or in the nucleus of infected cells 440

(Bejarano et al., 2019; Burdick et al., 2017; Francis et al., 2016; Jacques et al., 2016; Rasaiyaah 441

et al., 2013; Schaller et al., 2011; Sumner et al., 2019; Towers & Noursadeghi, 2014; N. Yan et al., 442

2010; Zila et al., 2019). Indeed, we find that Vpr can promote HIV-1 replication, even if the innate 443

immune stimulation does not originate from an HIV-1 derived PAMP, here exemplified by 444

replication assays in cGAMP treated primary human macrophages (Figure 1). We also found that 445

Vpr antagonised the effects of exposure to LPS, RNA and DNA ligands, as well as other viral 446

infections, exemplified here by Sendai virus infection, which potently activates RNA sensing and 447

IFN production in human macrophages (Matikainen et al., 2000)(Figure 2). In this way, Vpr can 448

suppress activation signals connected indirectly to infection. A series of recent studies have 449

demonstrated that infected cells produce a diverse range of endogenous RNA and DNA derived 450

PAMPs. Examples include retroelement induction by influenza infection (Schmidt et al., 2019), 451

RNA pseudogene expression after herpes simplex virus infection (Chiang et al., 2018) and RIGI 452

ligands after Kaposi’s sarcoma herpes virus infection (Zhao et al., 2018). These studies suggest 453

that viruses must be able to manage innate activation from non-viral PAMPs even when their own 454

PAMPs are sequestered. HIV-1 infection has also been described to induce retroelement 455

13

expression (Jones et al., 2013) consistent with a requirement for Vpr to suppress innate immune 456

activation downstream of endogenous PAMPs. Furthermore, HIV seroconversion has been 457

associated with a cytokine storm (Stacey et al., 2009) the anti-viral effect of which may be mitigated 458

by particle associated Vpr. Thus HIV-1 may utilise Vpr to replicate in an innate immune activated 459

environment, even when its own PAMPs are effectively sequestered. A link between escape from 460

innate sensing and successful transmission is suggested by several lines of evidence. These 461

include a generally low HIV transmission frequency (Shaw & Hunter, 2012), the observation that 462

HIV transmitted founder clones are particularly resistant to IFN (Iyer et al., 2017), and encode 463

distinct Vpr amino acid signatures, as compared to chronic viruses (Rossenkhan et al., 2016), as 464

well as the HIV transmission-associated cytokine storm itself (Stacey et al., 2009). Concordantly, 465

Vpu, Nef and Vif, and Vpr, antagonize innate immunity to enhance viral replication, reviewed in 466

Sumner et al., 2019. 467

468

Vpr has been suggested to cause IRF3 degradation (Okumura et al., 2008) but we did not detect 469

IRF3 degradation in THP-1 cells under conditions when gene expression and IRF3 nuclear 470

transport were strongly suppressed (Figure 5). Furthermore, in addition to suppressing IRF3 471

nuclear transport, we found that Vpr reduced IRF3 phosphorylation at S396 but not at S386 (Figure 472

5). Previous studies have suggested that phosphorylation of IRF3 at S386 is necessary and 473

sufficient for IRF3 activation (Lin et al., 1999; Mori et al., 2004; Schirrmacher, 2015; Servant et al., 474

2003; Suhara et al., 2000; Yoneyama et al., 1998). Thus our data are consistent with a more 475

complex picture of IRF3 activation by phosphorylation. It is possible that phosphorylation at S396 476

occurs in a karyopherin or NPC-dependent way that is occluded by Vpr recruitment to karyopherin. 477

Phosphorylation of IRF3 at S396 has been associated with enhanced association and 478

multimerization with transcriptional coactivator CREB binding protein (CBP/p300) suggesting a 479

later role than phosphorylation at S386 (Chen et al., 2008). It is possible that the lack of S396 IRF3 480

phosphorylation is a consequence of IRF3 dephosphorylation at S396 as nuclear entry is 481

prevented. 482

483

Inhibition of IRF3 phosphorylation is also consistent with reported inhibition of TBK1 by Vpr, 484

although this study detected inhibition of TBK phosphorylation, whereas we did not (Harman et al., 485

2015). In that study, Vpr promoted infection in macrophages and dendritic cells, despite HIV 486

induced formation of innate immune signalling complexes containing TBK1, IRF3 and TRAF3, 487

visualised by immunofluorescence staining. Thus TBK1 inhibition by Vpr may occur in addition to 488

Vpr activity on nuclear transport, because TBK1 is seen in the cytoplasm, not at the nuclear 489

envelope, in these HIV infected cells (Harman et al., 2015). IRF3 degradation was not detected in 490

this study and nor was HIV-1 induced IRF3 phosphorylation, although the impact of infection on 491

IRF3 by wild type HIV-1 and HIV-1 deleted for Vpr were not compared. 492

493

14

The regulation of the nuclear import of NF-ĸB and IRF3 by multiple karyopherins is expected to be 494

complex (Fagerlund et al., 2005, 2008; Kumar et al., 2002; Liang et al., 2013). Targeting 495

karyopherins is a typical viral strategy for manipulation of cellular responses but the different ways 496

viruses perform this function hints at the complexity required to inhibit innate responses whilst 497

avoiding shutting down viral transcription. We propose that the different mechanisms of NF-498

κB/IRF3 manipulation by different viruses reflect their reliance on transcriptional activation while 499

simultaneously depending on inhibition of the same transcription factors activated by defensive 500

processes. We hypothesise that each virus has specifically adapted to facilitate replication while 501

dampening activation of inhibitory effectors. Failure to degrade karyopherin proteins suggests that 502

some KPNA1 nuclear import function may be left intact by HIV to facilitate a more subtle 503

manipulation of host cell biology (Figure 7). A similar model of inhibition of KPNA target binding to 504

manipulate nuclear import has been suggested by a crystal structure of Ebola Virus VP24 protein 505

in complex with KPNA5. This study proposed that VP24 targets a KPNA5 NLS binding site to 506

specifically inhibit nuclear import of phosphorylated STAT1 (Xu et al., 2014). 507

508

Cell type clearly also plays a role in Vpr function. For example, in myeloid cells (Kogan et al., 2013; 509

Miller et al., 2017), and T cells (Ayyavoo et al., 1997), Vpr has been reported to inhibit NF-ĸB. 510

Other studies in T cells suggest NF-ĸB activation by Vpr to drive viral transcription (Liu et al., 2014; 511

Vermeire et al., 2016). In a more recent study, Hotter and colleagues showed that expression of 512

diverse primate immunodeficiency virus Vprs in 293T cells could activate or inhibit NF-ĸB activity 513

depending on the assay (Hotter et al., 2017). For example, Vpr expression in 293T cells activated 514

baseline, and TNF stimulated, expression of a transfected NF-ĸB sensitive reporter, but inhibited 515

activation of reporter by transfected IKK. The authors proposed that Vpr mediated inhibition of 516

NF-ĸB was relevant because Vpr inhibited an IFN reporter activated by Sendai Virus infection, 517

consistent with results presented herein. We propose that cell type, and the stage of the viral life 518

cycle, influence the effect of Vpr on transcription factor activation. One possibility is that incoming 519

particle associated Vpr is active against NF-ĸB, to mitigate innate sensing, but Vpr expressed from 520

the provirus in an infected cell is bound by Gag, which sequesters Vpr, reducing further inhibition 521

of the activated NF-ĸB that is required for on-going viral transcription (Belzile et al., 2010). 522

523

Our data also explain previous reports of the suppression of expression from co-transfected CMV 524

MIEP-driven plasmids by Vpr (Liu et al., 2015). Vpr inhibition of NF-ĸB transport into the nucleus 525

to activate the MIEP likely explains these data, but another possibility is that transcription factor 526

bound to cytoplasmic plasmid DNA has a role in importing plasmid into the nucleus, and it is 527

plasmid transport that is inhibited (Mesika et al., 2001). Vpr insensitivity of NF-ĸB-independent 528

ubiquitin and EF1α promoters (Figure 6) is consistent with this model, summarized in Figure 7 529

figure supplement 1A. This is important because inhibition of transfected plasmid driven protein 530

expression may explain the effect of cotransfected SIV Vpr on STING and cGAS signaling reported 531

15

recently (Su et al., 2019). Note that STING expression was not affected by Vpr co-expression but 532

STING was expressed from the Vpr and NF-ĸB-insensitive EF1α promoter (Figure 6), whereas 533

cGAS, which was not measured by western blot, was expressed from a Vpr and NF-ĸB-sensitive 534

(Figure 6) CMV driven plasmid VR1012 (Hartikka et al., 1996). Some experiments in (Hotter et al., 535

2017) may also have been influenced by this phenomenon. 536

537

Importantly, our data are consistent with reports that manipulation of cell cycle by Vpr is 538

independent of interaction with karyopherin proteins. The Vpr R80A mutant, which does not arrest 539

cell cycle, or manipulate SLX4 complex (Gaynor & Chen, 2001; Laguette et al., 2014) was 540

functional in inhibition of innate sensing (Figures 3, 5, 6). Thus we assume that SLX4 interaction 541

does not play a role in the innate immune antagonism shown herein. Mapping the residues of Vpr 542

that are important for innate immune inhibition onto structures resolved by NMR and X-ray 543

crystallography reveals a potentially distinct interface from that targeting UNG2 because residues 544

Vpr 34/35 are distant from the UNG2 binding site (Figure 3-figure supplement 1B, C). Further, 545

UNG2 has not been associated with innate immune sensing. Given that Vpr has been shown to 546

bind FxFG motif in p6 of Gag during virion incorporation (Zhu et al., 2004), and FG motifs at the 547

NPC (Fouchier et al., 1998) it is possible that interaction of Vpr with nuclear pore proteins via the 548

FG motifs contribute to Vpr mediated inhibition of IRF3 and NF-ĸB nuclear import. 549

550

In vitro, primary myeloid cells behave according to the stimuli they have received. Thus, 551

inconsistent results between studies, for example the requirement here for cGAMP, but not in other 552

studies, to cause Vpr dependent replication in macrophages (Figure 1), could be explained by 553

differences in myeloid cell stimulation due to differences in cell purification and differentiation 554

methods or reagents used. Methods of virus preparation, here viruses were purified by 555

centrifugation through sucrose, may also be a source of target cell activation and experimental 556

variation. We hypothesise that cGAMP induced Vpr dependence in MDM (Figure 1) because cells 557

were not activated prior to cGAMP addition, whereas in other studies basal activation produced 558

Vpr dependent replication. Replication in activated primary CD4+ T cells was, in our hands, 559

independent of Vpr in the presence and absence of cGAMP, which was inhibitory, suggesting that 560

Vpr cannot overcome signalling downstream of cGAMP in these cells. This implies that activated 561

T-cells respond differently to cGAMP than macrophages, consistent with observations that in T 562

cell/macrophage mixed cultures, the negative effects of cGAMP on HIV-1 replication were 563

principally mediated via macrophages (Xu et al., 2016). Vpr sensitive, cGAS dependent, IFN 564

production from T cells has been reported suggesting that in the right circumstances, T cells can 565

sense HIV-1 DNA, via cGAS, in T cells (Vermeire et al., 2016). Importantly, this study used 566

integration inhibition to demonstrate provirus-dependent detection of HIV-1 suggesting that 567

incoming HIV-1 DNA is not the cGAS target in this study. The nature of the PAMP in these 568

16

experiments remains unclear. Certainly, further work is required to understand the different 569

requirements for Vpr function in T cells and macrophages. 570

571

Sensing of HIV-1 is clearly viral dose, and therefore PAMP dose, dependent. For example, Cingoz et 572

al reported failure of VSV-G pseudotyped HIV-1 (∆Env, ∆Nef, ∆Vpr) to activate sensing in a variety 573

of cell lines (Cingöz & Goff, 2019). However, other studies have demonstrated sensing of wild type 574

HIV-1 DNA by cGAS (Gao et al., 2013; Lahaye et al., 2013), and here we observed cGAS-575

dependent, Vpr-sensitive, induction of CXCL10 or NF-ĸB reporter by high dose (MOI 1-3) VSV-G 576

pseudotyped single round HIV-1 GFP vector in THP-1 cells (Figures 1, 6). The effect of dose is 577

illustrated in Figure 1 in which MOI (0.1-0.3) had little effect on CXCL10 expression in THP-1 cells. 578

However, higher doses activated CXCL10 expression, unless the virions carried Vpr, in which case 579

CXCL10 induction was suppressed. Cingoz used luciferase to measure infection and therefore 580

MOIs are obscure making dose comparison difficult. Note that herein, MOI calculated by GFP 581

expression are included in supplementary data for most experiments. Given that infection typically 582

depends on exposing cells to more than one viral particle, requiring tens of particles in even the 583

most conservative estimates, it is likely that Vpr delivered by particles, that do not eventually form 584

a provirus, contributes to suppression of sensing. Certainly, a lower MOI is required for Vpr activity 585

when the stimulation comes from the Vpr bearing viral particles themselves (MOI 3 required, Figure 586

1C), compared to from external stimulus (MOI 20 required, Figure 1B). It is hard to know what MOI 587

are really relevant to replication in vivo but it is important to note that in our experiments, high MOI 588

above 1 are required for innate immune activation and Vpr dependent antagonism. This suggests 589

that low MOI infection depends on sensor evasion by viral PAMP sequestration within intact 590

capsids (Jacques et al., 2016) but higher MOI infections can rely on particle associated Vpr to 591

suppress the activation of any exposed viral PAMPs and any endogenous PAMPs that are 592

induced. 593

594

In summary our findings connect Vpr manipulation of nuclear transport with inhibition of innate 595

immune sensing, rather than viral nuclear import. They highlight the crucial role of particle 596

associated Vpr in inhibiting innate immune activation during the early stages of the viral life cycle 597

and unify a series of studies explaining previously apparently unconnected observations. Given 598

the complexity of NF-kB activation, and the different ways each virus manipulates defensive 599

transcriptional responses, we propose that the further study of viral inhibition of PAMP-driven 600

inflammatory responses will lead to a better understanding of the biology of the transcription factors 601

involved and highlight novel, tractable targets for therapeutic anti-inflammatory development. 602

603

Acknowledgements 604

We thank Veit Hornung for providing THP-1-IFIT-1 cells wild type and knock outs, Neil Perkins for 605

providing NF-ĸB constructs and advice, Geoffrey Smith for providing constructs encoding KPNA1-606

17

3 and Clare Jolly and Richard Sloan for providing NL4.3∆Vpr. This work was funded through an 607

MRC PhD studentship (HK) an MRC Clinical Training Fellowship (CVT), a Wellcome Trust clinical 608

training fellowship (DF), a Wellcome Trust Senior Biomedical Research Fellowship (GJT), the 609

European Research Council under the European Union’s Seventh Framework Programme 610

(FP7/2007-2013)/ERC (grant HIVInnate 339223) (GJT), a Wellcome Trust Collaborative award 611

(GJT) and was supported by the National Institute for Health Research University College London 612

Hospitals Biomedical Research Centre. 613

614

615

Methods 616

Key Resources Table

Reagent type (species) or resource

Designation Source or reference

Identifiers Additional information

antibody anti-FXFG repeats (mouse monoclonal

Abcam Cat# ab24609 IF (1:1000)

antibody Anti-FLAG tag (mouse monoclonal)

Sigma Cat# F3165 IF (1:1000)

antibody Anti-IRF3 (rabbit polyclonal)

Santa Cruz biotechnology

Cat# sc-9082 IF (1:400)

antibody Anti-rabbit alexa fluor 488 IgG (goat polyclonal)

Invitrogen Cat# A-11008 IF (1:500)

antibody Anti-mouse Alexa Fluor 546 IgG (goat polyclonal)

Invitrogen Cat# A-11030 IF (1:500)

antibody anti-VSV-G (rabbit polyclonal)

Sigma Cat# V4888 WB (1:20000)

antibody anti-HIV-1 p24 (mouse monoclonal)

NIH AIDS reagent program

Cat# 3537 WB (1:1000)

antibody anti-STING (Rabbit monoclonal)

Cell Signaling Cat# 13647 WB (1:1000)

18

antibody Anti-phospho STING (Rabbit monoclonal)


antibody Anti-TBK1 (Rabbit monoclonal)

Cell Signaling Cat# 3504S WB (1:1000)

antibody Anti-phospho TBK1 (Rabbit monoclonal)


antibody Anti-IRF3 (Rabbit monoclonal)


antibody Anti-phospho -IRF3 S386 (Rabbit monoclonal)

Abcam Cat# ab76493 WB (1:1000)

antibody Anti-phospho- IRF3 S396 (Rabbit monoclonal)

Cell signaling Cat# D6O1M Flow Cytometry (1:50)

antibody Anti-actin (mouse polyclonal)


antibody Anti-cGAS (rabbit monoclonal)

Cell Signaling Technology

Cat# 15102 WB (1:1000)

antibody Anti-MAVS (mouse polyclonal)

Cell Signaling Technology

Cat# 3993 WB (1:1000)

antibody Anti-DCAF1(rabbit polyclonal)

Bethyl Cat# A301-887A

WB (1:1000)

antibody Anti-Nup358 (rabbit polyclonal)


antibody Anti-FLAG (mouse monoclonal)

Sigma Cat# F3165 WB (1:1000)

antibody Anti GFP (rabbit polyclonal)


antibody Anti KPNA1 (rabbit polyclonal)

ABclonal Cat# A1742 WB (1:1000)





19







antibody Anti KPNB1 (rabbit polyclonal)


antibody Anti CypB (rabbit polyclonal)


antibody Anti HA (rabbit polyclonal)

Sigma Cat# H6908 WB (1:1000)

antibody Anti Vpr (rabbit polyclonal

NIH AIDS reagents programme

Cat# 11836 WB (1:1000)

antibody Anti mouse IgG IRdye 800CW (goat poly clonal)

LI-COR Biosciences

Cat# 926-32210

WB (1:10000)

antibody Anti rabbit IgG IRdye 800CW (goat poly clonal)

LI-COR Biosciences

Cat# 926-32211

WB (1:10000)

other Herring testes DNA Sigma Cat# D6898

amount used stated in text

other cGAMP Invivogen Cat code (tlrl-nacga23-1)


other Poly I:C Invivogen

Cat code (tlrl-pic)


other Lipopolysaccaride Invivogen

Cat code (tlrl-smlps)


617

Cells and reagents 618

HEK293T cells were maintained in DMEM (Gibco) supplemented with 10 % foetal calf serum (FCS, 619

Labtech) and 100 U/ml penicillin and 100 μg/ml streptomycin (Pen/Strep; Gibco). THP-1 cells were 620

maintained in RPMI (Gibco) supplemented with 10% FCS and Pen/Strep. THP-1-IFIT-1 luciferase 621

reporter cells express Gaussia luciferase under the control of the endogenous IFIT1 promoter have 622

been described (Mankan et al., 2014). THP-1 CRISPR control, cGAS-/- and MAVS -/- knock out 623

cells have been described (Mankan et al., 2014). Nup358 depleted HeLa cells have been 624

described (Schaller et al., 2011). Lipopolysaccharide, poly I:C and TNF𝛼 were obtained from 625

PeproTech. Sendai virus was obtained from Charles River Laboratories. Herring-testis DNA was 626

20

obtained from Sigma. cGAMP was obtained from Invivogen. NF-ĸB Lucia THP-1 reporter cells 627

were obtained from Invivogen. All cell lines were tested negative for mycoplasma. 628

629

Cloning and plasmids 630

The Vpr gene from HIV-1 founder clone SUMA (Fischer et al., 2010) was codon optimised and 631

synthesised by GeneArt. To generate the HIV-1 vector encoding Vpr (pCSVIG), the codon 632

optimised SUMA Vpr gene was cloned into pSIN-BX-IRES-Em between BamHl and Xhol sites 633

under the control of the SFFV LTR promoter. pSIN-BX-IRES-Em was obtained from Dr Yasuhiro 634

Takeuchi. EF1α-GFP and UB-GFP were obtained from Addgene (Matsuda & Cepko, 2004). The 635

CMV-GFP construct was pEGFPC1 (Clontech). HIV-1 bearing a Ba-L envelope gene has been 636

described (Rasaiyaah et al., 2013). Flag- KPNA1-3 plasmids were obtained from Prof. Geoffrey 637

Smith. HIV-1∆Vpr was a gift from Richard Sloan and encoded an 17 nucleotide insertion (Vpr 64-638

81) that destroys the Vpr coding sequence. 639

640

Production of virus in HEK293T cells 641

Replication competent HIV-1 and VSV-G pseudotyped HIV-1 GFP vectors were produced by 642

transfection of HEK293T cells in T150 flasks using Fugene 6 transfection reagent (Promega) 643

according to the manufacturer's instructions. Briefly, just-subconfluent T150 flasks were 644

transfected with 8.75 μg of HIV-1 YU2 or HIV-1 YU2 lacking Vpr (HIV-1 YU2 ∆Vpr) and 30 µl 645

Fugene 6 in 500 µl Optimem (Thermofisher Scientific). To make VSV-G pseudotyped HIV-1 GFP, 646

each T150 flask was transfected with 2.5 μg of vesicular stomatitis virus-G glycoprotein encoding 647

plasmid (pMDG) (Genscript), 2.5 μg of packaging plasmid, p8.91 (encoding Gag-Pol, Tat and Rev) 648

or p8.2 (encoding Gag-Pol, Tat and Rev and Vif, Vpr, Vpu and Nef) (Zufferey et al., 1997), and 649

3.75 μg of GFP encoding genome plasmid (pCSGW) using 30 µl Fugene 6 in 500µl optimum. To 650

make Vpr encoding HIV-1 GFP, 3.75 μg pCSVIG was transfected with 2.5 μg of pMDG and 2.5 μg 651

of p8.91. To make HIV-1 GFP particles bearing Vpr, 1 μg of Vpr expressing pcDNA3.1 (wild type 652

SUMA Vpr or Vpr mutants) was transfected with 2.5 μg of pMDG and 2.5 μg of p8.91 in 30ul 653

Fugene-6 and 500µl Optimem. All virus supernatants were harvested at 48 and 72 h post-654

transfection, replicate flasks were pooled, and supernatants subjected to ultracentrifugation 655

through a 20% sucrose cushion at 23000 rpm for 2 hours in a 30 ml swingout rotor (Sorvall) 656

(72000G). Viral particles were resuspended in RPMI supplemented with 10% FCS. HIV-GFP 657

produced with p8.91 or p8.2 used in Figure 1 were DNase treated for 2 hours at 37oC (DNaseI, 658

Sigma) prior to ultracentrifugation. Viruses were titrated by infecting THP-1 cells (2x105 cells/ml) 659

with dilutions of sucrose purified virus in the presence of polybrene (8 μg/ml, Sigma) and incubating 660

for 48 h. GFP-positive, infected cells were counted by flow cytometry using a BD Accuri C6 661

(BDBiosciences). HIV-1 vector encoding shRNA targeting DCAF1 has been described and was 662

prepared as above (Berger et al., 2015). 663

664

21

SG-PERT 665

Viral doses were determined by measuring reverse transcriptase activity of virus preparations by 666

qPCR using a SYBR Green-based product-enhanced PCR assay (SG-PERT) as described (Jolien 667

Vermeire et al., 2012). 668

669

Isolation of primary monocyte-derived macrophages and CD4+ T cells from peripheral 670

blood 671

Primary monocyte-derived macrophages (MDM) were prepared from fresh blood from healthy 672

volunteers. This study was approved by the UCL/UCLH Committees on the Ethics of Human 673

Research, Committee Alpha reference (06/Q0502/92). All participants provided written informed 674

consent and consent for publication. Primary CD4+ T cells were obtained from leukocyte cones 675

from healthy donors purchased from the National Blood Service UK. Peripheral blood mononuclear 676

cells (PBMCs) were isolated by density gradient centrifugation using Lymphoprep (Stemcell 677

Technologies). For MDM preparation, healthy donor PBMCs were washed three times with PBS 678

and plated to select for adherent cells. Non-adherent cells were washed away after 1.5 h and the 679

remaining cells incubated in RPMI (Gibco) supplemented with 10 % heat-inactivated pooled 680

human serum (Sigma) and 40 ng/ml macrophage colony stimulating factor (R&D systems). Cells 681

were further washed after 3 days and the medium changed to RPMI supplemented with 10% heat-682

inactivated human serum (Sigma). MDM were then infected 3-4 days later at low multiplicity of 683

infection. Spreading infection was detected by Gag staining and counting Gag positive cells as 684

described (Rasaiyaah et al., 2013). For CD4+ T cells, untouched CD4+ T cells were purified from 685

PBMCs with an indirect magnetic labeling system (MACS, Miltenyi Biotec), according to 686

manufacturer’s instructions. Cells were then cultured with 2 μg/ml of plate-bound anti-CD3 and 687

anti-CD28 monoclonal antibodies (αCD3αCD28 stimulation) (mAbs) (eBioscience) and 25 U/ml of 688

recombinant human interleukin-2 (IL-2; Roche Applied Science) at a concentration of 1.5-2 x 106 689

cells/ml in RPMI supplemented with 10% heat-inactivated Human Serum (HS) (SigmaAldrich). 690

Cells were maintained at 37°C in 5% CO2 in a humidified incubator for 72 h. CD4+ T cells were 691

then assessed for spreading infection of CXCR4-tropic HIV-1 NL4.3 WT and ΔVPR at low 692

multiplicity of infection (300 mU of HIV-1 RT Activity per 1x106 cells). Percentage of HIV-1-infected 693

primary CD4+ T cells was determined by flow cytometry measuring p24Gag antigen employing 694

the monoclonal antibody p24Gag-FITC (HIV-1 p24 (24-4), Santa Cruz Biotechnology). 695

696

Innate immune sensing assays 697

THP-1 cells were seeded in 96 well plates (5x105 cells/ml). For Vpr expression, cells were infected 698

with an empty or Vpr expressing (pCSVIG) lentiviral vectors for 40 hours. Cell viabilities were 699

similar at 40 hours as assessed by eye, for an example see Fig S5K. For stimulation of cells with 700

HT-DNA or poly I:C, 0.2 μl of lipofectamine and 25 μl of Optimem were incubated with HT-DNA or 701

poly I:C (amounts stated in figure legends) for 20 minutes and added to cells. Lipopolysaccharide 702

22

(1 μg/ml), TNF𝛼 (200 ng/ml), Sendai virus (200 HA U/ml) or cGAMP (5 μg/ml) were added directly 703

to the media. For experiments with virion delivered/associated Vpr, cells were stimulated at the 704

time of infection. Gaussia/Lucia luciferase activities were measured 8 hours post cell 705

stimulation/infection by transferring 10 μl supernatant to a white 96 well assay plate, injecting 50 706

μl per well of coelenterazine substrate (Nanolight Technologies, 2 μg/ml) and analysing 707

luminescence on a FLUOstar OPTIMA luminometer (Promega). Data were normalized to a mock-708

treated control to generate a fold induction. 709

710

ELISA 711

Cell supernatants were harvested for ELISA at 8 h post-stimulation and stored at -80 oC. CXCL-712

10 protein was measured using Duoset ELISA reagents (R&D Biosystems) according to the 713

manufacturer’s instructions. 714

715

ISG qPCR 716

RNA was extracted from THP-1 cells using a total RNA purification kit (Norgen) according to the 717

manufacturer’s protocol. Five hundred ng RNA was used to synthesise cDNA using Superscript III 718

reverse transcriptase (Invitrogen), also according to the manufacturer’s protocol. cDNA was diluted 719

1:5 in water and 2 μl was used as a template for real-time PCR using SYBR® Green PCR master 720

mix (Applied Biosystems) and a 7900HT Real-Time PCR machine (Applied Biosystems). 721

Expression of each gene was normalised to an internal control (GAPDH) and these values were 722

then normalised to mock-treated control cells to yield a fold induction. The following primers were 723

used: 724

GAPDH: Fwd 5’-GGGAAACTGTGGCGTGAT-3’, Rev 5’-GGAGGAGTGGGTGTCGCTGTT-3’ 725

CXCL-10: Fwd 5’-TGGCATTCAAGGAGTACCTC-3’, Rev 5’-TTGTAGCAATGATCTCAACACG-3’ 726

IFIT-2: Fwd 5’-CAGCTGAGAATTGCACTGCAA-3’, Rev 5’-CGTAGGCTGCTCTCCAAGGA-3’ 727

MxA: Fwd 5’-ATCCTGGGATTTTGGGGCTT-3’, Rev 5’-CCGCTTGTCGCTGGTGTCG-3’ 728

Viperin: Fwd 5’-CTGTCCGCTGGAAAGTG-3’, Rev 5’-GCTTCTTCTACACCAACATCC-3’ 729

IL-6: Fwd 5’- AAATTCGGTACATCCTCGACG-3’, Rev 5’- GGAAGGTTCAGGTTGTTTTCT-3’ 730

731

Immunofluorescence 732

For confocal microscopy, HeLa cells (5x104 cells/ml) were seeded into 24-well plates containing 733

sterile glass coverslips. For nuclear translocation assays, we used THP-1 cells (4x105 cells/ml) 734

adhered in an optical 96-well plate (PerkinElmer) with 50 ng/ml phorbol 12-myristate 13-acetate 735

(PMA, Peprotech) for 48 hours. Where cells were infected and transfected (DNA, PolyI:C) or 736

treated (cGAMP) with innate immune stimulants, the cells were treated or transfected first, and 737

then viral supernatant added to the cultures. Cells were then fixed and stained three hours after 738

this. For fixation, HeLa or adhered THP-1 cells were washed twice with ice-cold PBS and fixed in 739

4% (vol/vol) paraformaldehyde. Autofluorescence was quenched in 150 mM ammonium chloride, 740

23

the cells permeabilized in 0.1% (vol/vol) Triton X-100 in PBS and blocked for 30 min in 5% (vol/vol) 741

FCS in PBS. Cells were incubated with primary Ab for 1 hour followed by incubation with secondary 742

Ab for 1 hour. Cells were washed with PBS three times between each step. The coverslips were 743

placed on a slide prepared with a 30 μl drop of mounting medium (Vectashield, containing 4',6-744

diamidino-2-phenylindole (DAPI)) and allowed to set before storing at 4o C. Images were taken on 745

a Leica TCS SPE confocal microscope and analyzed in ImageJ. For IRF3/NF-κB(p65) 746

translocation, images were taken on Hermes WISCAN (IDEA Bio-Medical) and analyzed with 747

Metamorph software (Molecular Devices). Metamorph calculated a translocation coefficient 748

representing the proportion of staining in nuclear versus cytoplasmic compartments. A value of 1 749

represents "all staining in the nucleus", -1 is "exclusively in cytoplasm" and 0 is "equally 750

distributed”. 751

752

Immunoblotting 753

For immunoblotting of viral particles, sucrose purified (as described above) virions (1×1011 RT 754

units) were boiled for 10 min in 6X Laemmli buffer (50 mM Tris-HCl (pH 6.8), 2 % (w/v) SDS, 10% 755

(v/v) glycerol, 0.1% (w/v) bromophenol blue, 100 mM β-mercaptoethanol) before separating on 12 756

% polyacrylamide gel. Cells were lysed in lysis buffer containing 50 mM Tris pH 8, 150 mM NaCl, 757

1 mM EDTA, 10% (v/v) glycerol, 1 % (v/v) Triton X100, 0.05 % (v/v) NP40 supplemented with 758

protease inhibitors (Roche), clarified by centrifugation at 14,000 x g for 10 min and boiled in 6X 759

Laemmli buffer for 10 min. Proteins were separated by SDS-PAGE on 12% polyacrylamide gels. 760

Proteins were transferred to a Hybond ECL membrane (Amersham biosciences) using a semi-dry 761

transfer system (Biorad). 762

763

Cell cycle analysis 764

WT Vpr or Vpr mutants were expressed in THP-1 cells using pCSVIG at an MOI of 1. Cells were 765

incubated for 48 hours and then washed with PBS and fixed in 1 ml cold 70% ethanol on ice for 766

30 minutes. To ensure efficient fixing and minimise clumping, ethanol was added dropwise while 767

vortexing. Cell were pelleted in a microfuge and ethanol was removed followed by two wash steps 768

with PBS. To remove RNA from the samples, RNase A (100 𝜇g/ml) was added and the cells were 769

stained with propidium iodide (PI) (50 𝜇g/ml) to stain cellular DNA. Cells were incubated for 10 770

minutes at room temperature and DNA content analysed by flow cytometry on a BD FACSCalibur 771

(BD Biosciences). The data were analysed with FlowJo. 772

773

Generation of Vpr mutants 774

Site directed mutagenesis was performed using Pfu Turbo DNA Polymerase (Agilent) according 775

to the manufacturer’s instructions with the following primers using either pCDNA3.1 or pCSVIG 776

encoding SUMA Vpr as template. 777

VprF34I+P35N: Fwd 5’-GCCGTGCGGCACATCAACAGACCTTGGCTGCATAGC-3’, 778

24

Rev 5’GCTATGCAGCCAAGGTCTGTTGATGTGCCGCACGGC-3’ 779

VprQ65R: Fwd 5’-GCCATCATCAGAATCCTGCGGCAGCTGCTGTTCATC-3’, 780

Rev 5’-GATGAACAGCAGCTGCCGCAGGATTCTGATGATGGC-3’ 781

VprR80A: Fwd 5’-GGCTGCCGGCACAGCGCCATCGGCATCACCCCT-3’, 782

Rev 5’-AGGGGTGATGCCGATGGCGCTGTGCCGGCAGCC-3’ 783

784

Co-immunoprecipitation assays 785

For KPNA-cargo IPs HEK293T cells were grown in 10 cm dishes and co-transfected with 1 µg of 786

a plasmid expressing FLAG-tagged KPNA1, 1 µg of a plasmid expressing HA-tagged p65 or IRF3 787

and 1 µg of a plasmid expressing un-tagged SUMA VprF34I+P35N or empty vector control. To 788

account for the effects of SUMA Vpr on expression from CMV promoter-containing plasmids, for 789

IPs containing wild-type SUMA Vpr cells were co-transfected with 2 µg of a plasmid expressing 790

FLAG-tagged KPNA1, 3 µg of a plasmid expressing HA-tagged p65 or IRF3 and 1 µg of a plasmid 791

expressing un-tagged wild-type SUMA Vpr. All transfection mixes were made up to 6 µg with an 792

empty vector plasmid. After 24 h cells were lysed in lysis buffer (0.5 (v/v) % NP-40 in PBS 793

supplemented with protease inhibitors (Roche) and phosphatase inhibitors (Roche), pre-cleared 794

by centrifugation and incubated with 25 µl of mouse-anti-HA agarose beads (Millipore) or mouse-795

anti-FLAG M2 agarose affinity gel (Sigma) for 2-4 h. Immunoprecipitates were washed 3 times in 796

1 ml of lysis buffer and eluted from the beads by boiling in 20 µl of 2X sample buffer containing 797

SDS and -mercaptoethanol. Proteins were resolved by SDS-polyacrylamide gel electrophoresis 798

(NuPAGE 4-12 % Bis-Tris protein gels, Invitrogen) and detected by immunoblotting. 799

800 801

Statistical analyses 802

Data were analysed by statistical tests as indicated in the figure legends. * represent statistical 803

significance: * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001). 804

805

Figure Legends 806

Figure 1 HIV-1 replication in cGAMP stimulated MDMs requires Vpr 807

(A) Replication of WT Yu2 HIV-1 or Yu2 HIV-1ΔVpr in MDMs stimulated with 1 μg/ml, 2 μg/ml or 808

4 μg/ml cGAMP or left unstimulated, infection measured by counting Gag positive cells stained 809

with anti-p24. Mean+/-SEM n=3 1 and 2 μg/ml cGAMP; n=2 4 μg/ml cGAMP. *** = 2 way ANOVA 810

p value <0.001, * = p<0.05. (B) Fold induction of IFIT1-Luc after activation of STING by cGAMP (5 811

μg/ml) and infection with HIV-1 virus like particles (VLP) lacking genome and bearing Vpr (+Vpr) 812

or lacking Vpr (-Vpr) (1 RT U/ml) in IFIT1-Luc reporter THP-1 cells. cGAMP and virus were added 813

to cells at the same time. (C) Fold induction of CXCL10 after infection of THP-1 cells with HIV-GFP 814

-Vpr or HIV-GFP +Vpr at the indicated MOI. (D) Percentage of THP-1 cells infected by HIV-GFP -815

Vpr or HIV-GFP +Vpr in (C). (E) Fold induction of CXCL10 after infection of THP-1 cells with HIV-816

GFP -Vpr, HIV-GFP +Vpr or HIV-1 particles lacking Vpr and genome, at indicated doses measured 817

25

by reverse transcriptase SG-PERT assay. (F) Percentage of THP-1 cells infected by HIV-GFP 818

viruses in (E). (G) Fold induction of CXCL10 after infection of unmodified control, cGAS-/- or 819

MAVS-/- THP-1 knock out cells with HIV-GFP lacking Vpr (0.3 RT U/ml). (H) Percentage infection 820

of control, cGAS-/- or MAVS-/- THP-1 knockout cells infected with HIV-GFP at indicated doses of 821

RT (SG-PERT). (B-H) Data are expressed as means ± SD (n = 3) with two-way ANOVA * (p<0.05), 822

** (p<0.01), *** (p<0.001), **** (p<0.0001) compared to virus without genome (B), HIV GFP+Vpr 823

(C, E) and control (G). 824

825

Figure 1 figure supplement 1 HIV-1 replication in cGAMP stimulated MDMs requires Vpr and 826

Vpr suppresses HIV-1 innate immune sensing by cGAS 827

(A) Replication of wild type (WT) NL4-3 HIV-1, or NL4-3 HIV-1ΔVpr, in activated primary human 828

CD4+ T cells stimulated with 1, 2 or 4 μg/ml cGAMP or left unstimulated as a control. Two 829

representative examples of three are shown with virus replication measured by percentage T-cell 830

p24 positivity, measured by flow, (top panels) or supernatant RT activity (lower panels). This 831

experiment was also performed twice in Jurkat cells with virus replication measured by percentage 832

T-cell p24 positivity, measured by flow, giving similar results as shown.” Replication of WT NL4-3 833

HIV-1 or NL4-3 HIV-1ΔVpr in activated CD4+ T cells stimulated with 1 μg/ml, 2 μg/ml or 4 μg/ml 834

cGAMP or left unstimulated, measured by flow cytometry staining infected cells with anti-p24 835

antibody. (B) HIV-GFP titre in control, cGAS-/- or MAVS-/- THP-1 cells used in Figure 1 (G). (C) 836

Immunoblot detecting cGAS, MAVS, or actin as a loading control, from extracted cGAS-/- or 837

MAVS-/- knock out THP-1 cells or their CRISPR/Cas control cells. Size marker positions are shown 838

on the right (kDa). 839

840

Figure 2 HIV-1 Vpr expression inhibits interferon stimulated gene expression after 841

stimulation with various innate immune stimuli 842

(A) Fold induction of IFIT1-Luc, after activation of STING by cGAMP (5 μg/ml), in IFIT1-Luc reporter 843

THP-1 cells expressing Vpr from a lentiviral vector delivered at MOIs of 0.25, 0.5, 1, or after empty 844

vector transduction (MOI 1) or in untransduced cells. (B) Fold induction of ISGs MxA, CXCL10, 845

IFIT2 and Viperin after activation of STING by cGAMP (5 μg/ml) in cells expressing Vpr from a 846

lentiviral vector (MOI 1), or after empty vector transduction (MOI 1) or in untransduced THP-1 cells. 847

(C) Secreted CXCL10 (ELISA) after activation of STING by cGAMP (5 μg/ml) in cells expressing 848

Vpr from a lentiviral vector (MOI 0.5, 1), or after transduction with empty vector (MOI 0.5, 1) or in 849

untransduced THP-1 cells. Dotted line shows limit of detection. (D) Fold induction of IFIT1-Luc 850

after HT-DNA transfection (5 μg/ml) of cells expressing Vpr from a lentiviral vector (MOI 0.5, 1), or 851

empty vector (MOI 0.5, 1) or in untransduced IFIT1-Luc reporter THP-1 cells. (E) Fold induction of 852

IFIT1-Luc, after Sendai virus infection, of cells expressing Vpr from a lentiviral vector (MOI 0.5, 1), 853

or after transduction by empty vector (MOI 0.5, 1) or in untransduced IFIT1-Luc reporter THP-1 854

cells. (F) Fold induction of IFIT1-Luc, after LPS treatment (1 μg/ml), of cells expressing Vpr from a 855

26

lentiviral vector (MOI 0.25, 0.5, 1), after transduction by empty vector (MOI 1) or in untransduced 856

IFIT1-Luc reporter THP-1 cells. Data are expressed as mean ± SD (n = 3) analysed using two-way 857

ANOVA * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001) compared to data for empty vector. 858

n= 3 (A, D-F) or 2 (B-C) independent experiments. 859

860

Figure 2 figure supplement 1 HIV-1 Vpr expression inhibits interferon stimulated gene 861

expression after stimulation with various innate immune stimuli 862

(A) Vpr encoding lentiviral expression construct (pCSVIG) contained self-inactivating Long terminal 863

repeat (SIN LTR), Rev response element (RRE), Central polypurine tract (cPPT), Spleen focus-864

forming virus promoter (SFFV), internal ribosome entry site (IRES), green fluorescent protein 865

(GFP) and Woodchuck hepatitis virus post‐transcriptional regulatory element (WPRE). (B) 866

Immunoblot detecting VSV-G envelope, capsid (p24) and Vpr in vector supernatant and Vpr 867

additionally in target cell lysate. Size markers in kDa are indicated on the right. (C) Percentage of 868

THP-1 cells in Figure 2A transduced by the vector encoding Vpr and GFP (MOI 0.25, 0.5, 1) or 869

empty vector encoding GFP alone (MOI 1) and treated with cGAMP (5 μg/ml) or left untreated as 870

a control. (D) Percentage of THP-1 cells in Figure 2B transduced by the vector encoding Vpr and 871

GFP (MOI 1) or empty vector encoding GFP alone (MOI 1) and treated with cGAMP (5 μg/ml) or 872

left untreated as a control. (E) Percentage of THP-1 cells in Figure 2C transduced by the vector 873

encoding Vpr and GFP (MOI 0.5, 1) or empty vector expressing GFP alone (MOI 0.5, 1) and treated 874

with cGAMP (5 μg/ml) or left untreated as a control. (F) Percentage of THP-1 cells in Figure 2D 875

transduced by the vector encoding Vpr and GFP (MOI 0.5, 1) or empty vector encoding GFP alone 876

(MOI 0.5, 1) and stimulated with HT-DNA transfection (5 μg/ml) or left untreated as a control. (G) 877

Percentage of THP-1 cells in Figure 2E transduced by the vector encoding Vpr and GFP (MOI 0.5, 878

1) or empty vector expressing GFP alone (MOI 0.5, 1) and stimulated with Sendai virus infection 879

or left untreated as a control. (H) Percentage of THP-1 cells in Figure 2F transduced by the vector 880

encoding Vpr and GFP (MOI 0.25, 0.5, 1) or empty vector encoding GFP alone (MOI 1) and 881

stimulated with LPS treatment (1 μg/ml) or left untreated as a control Data are expressed as means 882

± SD (n = 3). Data are representative of three (C, F-H) or two (B, D, E) independent experiments. 883

884

Figure 3 Vpr inhibition of innate immune activation is dependent on DCAF1 but independent 885

of cell cycle arrest 886

(A) Immunoblot detecting p24 (capsid) and Vpr in pelleted VSV-G pseudotyped VLP lacking 887

genome used in (B). Size markers in kDa are indicated on the right. (B) Fold induction of IFIT1-888

Luc after activation of STING by cGAMP (5 μg/ml) and infection with VLP bearing WT or mutant 889

Vpr, or lacking Vpr (1 RT U/ml) in IFIT1-Luc reporter THP-1 cells. Cells were infected at the same 890

time as cGAMP treatment. (C) Flow cytometry plots showing cell cycle phases of THP-1 cells 891

transduced with an empty vector, WT Vpr, or mutant Vpr, encoding vector (MOI 1) or left 892

untransduced as a control and stained with propidium iodide to label DNA. Percentage cells in 893

27

each cell cycle stage are shown. (D) Fold induction of IFIT1-Luc after activation of STING by 894

cGAMP (5 μg/ml) in cells expressing Vpr from a lentiviral vector, or expressing empty vector, or in 895

untransduced IFIT1-Luc reporter THP-1 cells expressing a control, or a DCAF1 targeting shRNA. 896

Mean +/-SEM n=3 independent experiments. (E) Immunoblot detecting DCAF1, or actin as a 897

loading control, from extracted THP-1 cells expressing a non-targeting, or DCAF1-targeting, 898

shRNA. Size markers are shown in kDa on the right. (F) Fold induction of IFIT1-Luc after activation 899

of STING by cGAMP (5 μg/ml) in cells expressing WT, or mutant, Vpr from a lentiviral vector (MOI 900

1), or empty vector (MOI 1) or in untransduced IFIT1-Luc reporter THP-1 cells. (G) Fold induction 901

of MxA mRNA after activation of STING by cGAMP (5 μg/ml) in cells expressing WT, or mutant, 902

Vpr from a lentiviral vector (MOI 1), or after transduction by empty vector (MOI 1) or in 903

untransduced THP-1 cells. Data are mean ± SD (n = 3). Two-way ANOVA test: * (p<0.05), ** 904

(p<0.01), *** (p<0.001), **** (p<0.0001) compared to no Vpr or empty vector controls. Data are 905

representative of three (B-D, F) or two (A, E, G) independent experiments. 906

907

Figure 3 figure supplement 1 Vpr inhibition of innate immune activation is dependent on 908

DCAF1 but independent of cell cycle arrest 909

(A) Percentage of THP-1 cells in Figure 3C transduced by the vector encoding Vpr and GFP, or 910

empty vector encoding GFP alone, at the indicated MOI and treated with cGAMP (5 μg/ml) or left 911

untreated. (B) NMR structure of full length Vpr showing position of Vpr mutants (PDB: 1M8L). 912

White region (c-terminus) of Vpr shown in (B) is unresolved in the crystal structure (C). (C) Crystal 913

structure of Vpr (orange) with its target protein UNG2 (blue) and cofactors DCAF1(pink) and DDB1 914

(green) showing position of Vpr mutations (PDB: 5JK7). (D) Percentage of THP-1 cells in Figure 915

3F transduced by the vector encoding WT, or mutant, Vpr and GFP (MOI 1), or empty vector 916

encoding GFP alone (MOI 1), and treated with cGAMP (5 μg/ml), or left untreated as a control. (E) 917

Fold induction of IFIT1-Luc after HT-DNA (5 μg/ml) transfection in cells expressing WT, or mutant, 918

Vpr from a lentiviral vector (MOI 1), or empty vector (MOI 1), or in untransduced IFIT1-Luc reporter 919

THP-1 cells. (F) Percentage of THP-1 cells in Figure S3E transduced with HIV-1 vector encoding 920

WT, or mutant, Vpr and GFP (MOI 1), or empty vector encoding GFP alone (MOI 1), and 921

transfected with HT-DNA (5 μg/ml) or left untransfected as a control. (G) Percentage of THP-1 922

cells in G2/M phase of cell cycle after transduction with an empty vector (MOI), or vector encoding 923

WT Vpr, or mutant Vpr, (MOI 1) or left untransduced as a control. Mean+/- SEM n=2. Unless stated 924

data are expressed as means ± SD (n = 3). Data is analysed using two-way ANOVA test. * 925

(p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001) compared to empty vector. Data are 926

representative of three (A), (D) or two (E-G) independent experiments. 927

928

Figure 4 Wild Type Vpr, but not sensing antagonism inactive Vpr mutants, localise to 929

nuclear pores 930

28

(A) Immunofluorescence confocal projections of HeLa cells transfected with Flag-tagged WT, or 931

mutant, Vpr encoded by pcDNA3.1 plasmid (50 ng) and stained using antibodies detecting the 932

Flag-tag (green) or nuclear pore complex (mab414) (red). 4′,6-Diamidine-2′-phenylindole 933

dihydrochloride (DAPI) stains nuclear DNA (Blue). (B) Selected confocal images (z-section) of cells 934

in (A) showing effect of Vpr mutation on Vpr colocalization with mab414 nuclear pore staining. (C) 935

Assessment of colocalization of Vpr with mab414 nuclear pore staining. Scale bars represent 10 936

𝜇m. 937

938

Figure 4 figure supplement 1 Nup358 is not required for Vpr colocalization with mab414 939

nuclear pore staining 940

(A) Immunofluorescence images of HeLa cells expressing a control, or Nup358 targeting, shRNA 941

transfected with empty vector or Flag-tagged Vpr encoding pcDNA3.1 plasmid (50 ng) using 942

antibodies detecting the Flag-tag (green) or the nuclear pore complex (mab414) (red). 4′,6-943

Diamidine-2′-phenylindole dihydrochloride (DAPI) stains nuclear DNA (Blue). (B) Selected 944

confocal images (z-section) of cells in (A) showing effect of Nup358 depletion on colocalization of 945

Vpr with mab414 nuclear pore staining (C) Immunoblot detecting Nup358, or actin as a loading 946

control, from extracted Hela cells expressing a control, or Nup358 targeting, shRNA in cells from 947

A. Size markers are shown (kDa). (D) Assessment of colocalization of Flag-tagged Vpr and 948

mab414 stained nuclear pores in cells expressing a control, or Nup358 targeting, shRNA. Scale 949

bars represent 10 𝜇m. 950

951

Figure 5 Vpr inhibits IRF3 nuclear translocation 952

(A) Immunoblot detecting Phospho-STING (Ser366), total STING, phospho-TBK1 (Ser172), total 953

TBK1, phospho-IRF3 (Ser386), total IRF3, or actin as a loading control, from extracted THP-1 cells 954

expressing Vpr from a lentiviral vector (MOI 1), expressing empty vector, or THP-1 left 955

untransduced as a control and transfected with HT-DNA (5 μg/ml) or left untransfected as a control. 956

Size markers are shown in kDa. (B) Mean fold induction of IFIT1-Luc in cells from Figure 5A and 957

Figure S5B (C) Flow cytometry plot (forward scatter vs pIRF3-S396 fluorescence) of THP-1 cells 958

infected with Vpr bearing virus-like particles (VLP) lacking genome (1 RT U/ml), or Vpr free VLP, 959

stimulated with cGAMP (5 μg/ml) or HT-DNA transfection (5 μg/ml). Lower panel shows the flow 960

cytometry data as a bar graph, plotting pIRF3-S396 positive cells. (D) Single cell 961

immunofluorescence measurement of IRF3 nuclear translocation in PMA differentiated THP-1 962

cells treated with cGAMP, or left untreated, and infected with HIV-1 GFP bearing Vpr, lacking Vpr 963

or left untransduced. Cells were fixed and stained three hours after infection/transfection. Red line 964

shows the translocation coefficient threshold. Blue lines represent mean translocation coefficient. 965

(E) Percentage of cells in Figure 5D with IRF3 translocation coefficient greater than 0.5 (above red 966

line). (F) Single cell immunofluorescence measurement of IRF3 nuclear translocation in PMA 967

differentiated THP-1 cells stimulated with cGAMP (5 μg/ml), or left unstimulated, and infected with 968

29

HIV-1 GFP lacking Vpr or bearing WT Vpr or Vpr mutants as shown (1 RT U/ml) or left uninfected. 969

(G) Single cell immunofluorescence measurement of IRF3 nuclear translocation in PMA 970

differentiated THP-1 cells transfected with HT-DNA (5 μg/ml), or left untransfected, and infected 971

with HIV-1 GFP lacking Vpr, or bearing WT or mutant Vpr (1 RT U/ml) or left uninfected. Data in B 972

is expressed as means ± SEM (n = 2). Data is analysed using two-way ANOVA: * (p<0.05), ** 973

(p<0.01), *** (p<0.001), **** (p<0.0001) compared to data from infection with HIV-1 lacking Vpr. 974

Data are representative of three (C–G) or two (A, B) independent experiments. 975

976

Figure 5 figure supplement 1 Vpr inhibits IRF3 nuclear translocation 977

(A) Percentage of THP-1 cells in Figure 5A transduced by HIV-1 GFP vector bearing Vpr , or HIV-978

1 GFP lacking Vpr, transfected with HT-DNA (5 μg/ml) or left untransfected as a control. (B) 979

Immunoblot detecting Phosho-STING (Ser366), total STING, phospho-TBK1 (Ser172), total TBK1, 980

phospho-IRF3 (Ser386) or total IRF3 from extracted THP-1 cells expressing Vpr, empty vector or 981

left untransduced as a control, and transfected with HT-DNA (5 μg/ml), or left untransfected as a 982

control. Size markers are shown (kDa). (C) Fold induction of IFIT1-Luc in cells from gel in Figure 983

5A, expressing Vpr, or empty vector, and transfected with HT-DNA (5 μg/ml) or left untransfected 984

as a control. (D) Percentage of THP-1 cells from Figure S5B transduced by HIV-1 GFP bearing 985

Vpr, or lacking Vpr, transfected with HT-DNA (5 μg/ml) or left untransfected as a control. (E) Fold 986

induction of IFIT1-Luc in cells from second experiment (gel presented in Figure S5B) expressing 987

Vpr, or empty vector, and transfected with HT-DNA (5 μg/ml) or left untransfected as a control. (F) 988

Single cell measurement of IRF3 nuclear translocation in PMA differentiated THP-1 cells 989

stimulated with LPS, or left unstimulated, and infected with HIV-1 GFP lacking Vpr or bearing Vpr 990

(1 RT U/ml), or left uninfected (top panel). Percentage of cells with IRF3 translocation coefficient 991

greater than 0.5 plotted as a percentage (bottom panel). (G) Percentage of cells with IRF3 992

translocation coefficient greater than 0.5 plotted as a percentage from Figure 5F. (H) Percentage 993

of cells with IRF3 translocation coefficient greater than 0.5 plotted as a percentage from Figure 994

5G. (I) Single cell measurement of IRF3 nuclear translocation in PMA differentiated THP-1 cells 995

transfected with poly I:C, or left untransfected, and infected with HIV-1 GFP lacking Vpr or bearing 996

WT or mutant Vpr (1 RT U/ml), or left uninfected. (J) Percentage of cells with IRF3 translocation 997

coefficient greater than 0.5 plotted as a percentage from Figure S5I.Data is analysed using two-998

way ANOVA test: * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001) compared to empty vector. 999

Data are representative of three (F-K) or two (A-E) independent experiments. 1000

1001

Figure 5 figure supplement 2 Nuclear translocation of IRF3 after cGAMP stimulation in the 1002

presence or absence of Vpr 1003

Representative immunofluorescence images showing IRF3 (red) nuclear translocation in PMA 1004

differentiated THP-1 cells treated with cGAMP, or left untreated, and infected with HIV-1 GFP 1005

30

bearing Vpr, or lacking Vpr, or left uninfected. 4′,6-Diamidine-2′-phenylindole dihydrochloride 1006

(DAPI) stains nuclear DNA (Blue). Scale bars represent 20 𝜇m. 1007

1008

Figure 6 Vpr inhibits NF-κB p65 nuclear translocation and NF-κB sensitive plasmid 1009

expression 1010

(A) Fold induction of NF- B-Luc after infection of THP-1 cells with HIV-GFP lacking Vpr, HIV-GFP 1011

bearing Vpr, or HIV-GFP lacking Vpr and genome, at the indicated doses. (B) Percentage of THP-1012

1 cells in (A). (C) Fold induction of IL-6 after activation of STING by cGAMP (5 μg/ml) in cells 1013

expressing empty vector or Vpr encoding vector (MOI 1), or in untransduced THP-1 cells. (D) 1014

Single cell immunofluorescence measurement of NF-kB (p65) nuclear translocation in PMA 1015

differentiated THP-1 cells transfected with Poly I:C (50 ng/ml), or left untreated, and infected with 1016

HIV-1 GFP lacking Vpr, HIV-1 GFP bearing Vpr (1 RT U/ml) or left uninfected. Cells were stained 1017

three hours after transfection and infection. (E) Immunoblot detecting Flag-Vpr, GFP, or actin as a 1018

loading control, from HEK293T cells transfected with 50 ng of empty vector, Flag-tagged WT Vpr 1019

vector, or Flag-tagged mutant Vpr vector, and CMV-GFP vector (50 ng). Size markers are shown 1020

in kDa. GFP expression from two independent immunoblots was quantified by densitometry and 1021

is shown in the lower panel. (F) Immunoblot detecting Flag-Vpr, GFP, or actin as a loading control, 1022

from HEK293T cells transfected with empty vector (200 ng) or Vpr vector (50ng, 100ng, 200ng) 1023

and CMV-GFP, EF1α-GFP or Ub-GFP plasmids (50 ng). Size markers are shown in kDa. GFP 1024

expression quantified by densitometry is shown in the lower panel. (G) Immunoblot detecting GFP, 1025

or actin as a loading control, from HEK293T cells transfected with CMV-GFP, EF1α-GFP or Ub-1026

GFP plasmids (10 ng, 2 ng, 0.4 ng) and stimulated with TNFα (200 ng/ml) or left unstimulated. 1027

Size markers are shown in kDa. GFP expression, from two independent immunoblots, quantified 1028

by densitometry, is shown in the lower panel. Data in (A, B, C) is expressed as mean ± SD (n = 3). 1029

Data in (E, F, G) is expressed as mean ± SD (n=2). Two-way ANOVA: * (p<0.05), ** (p<0.01), *** 1030

(p<0.001), **** (p<0.0001) compared to empty vector or HIV GFP+Vpr. 1031

1032

Figure 6 figure supplement 1 Vpr inhibits NF-κB p65 nuclear translocation and NF-κB 1033

sensitive plasmid expression 1034

(A) Induction of luciferase reporter in HEK293T cells transfected with CSLW, CMV-Luc, TK-Luc or 1035

M5P-Luc (10ng), and empty vector, or Vpr encoding vector (50 ng, 100 ng, 200 ng). Table shows 1036

the promoters driving the luciferase reporter in each plasmid. (B) Percentage of cells in Figure 6D 1037

with translocation coefficient greater than 0.5. (C) Single cell measurement of NF- B nuclear 1038

translocation in PMA differentiated THP-1 cells stimulated with LPS, or left unstimulated, and 1039

infected with HIV-1 GFP lacking Vpr or bearing Vpr (1 RT U/ml), or left uninfected (top panel). 1040

Percentage of cells with NF- B translocation coefficient greater than 0.5 plotted as a percentage 1041

(bottom panel). Data is analysed using two-way ANOVA: * (p<0.05), ** (p<0.01), *** (p<0.001), **** 1042

(p<0.0001) compared to data from infection with HIV-1 lacking Vpr. (D) Quantification of GFP 1043

31

expression by densitometry for the immunoblot in Figure 6E. (E) Immunoblot detecting flag-Vpr, 1044

GFP or actin as a loading control from HEK293T cells transfected with empty vector, flag-tagged 1045

WT Vpr encoding vector or flag-tagged mutant Vpr encoding vector and CMV-GFP vector or left 1046

untransfected. Size markers are shown in kDa. Quantification of GFP expression by densitometry 1047

for the immunoblot is shown below. (F) Quantification of GFP expression by densitometry for the 1048

immunoblot in Figure 6G. (G) Immunoblot detecting GFP, or actin as a loading control, from 1049

HEK293T cells transfected with CMV-GFP, EF1α-GFP or Ub-GFP plasmids (10 ng, 2 ng, 0.4 ng) 1050

and stimulated with TNFα (200 ng/ml) or left unstimulated. Size markers are shown in kDa. 1051

Quantification of GFP expression by densitometry for the immunoblot is shown below. 1052

1053

Figure 7 HIV-1 Vpr interacts with karyopherins and inhibits IRF3/NF-κB(p65) recruitment to 1054

KPNA1 1055

(A) Immunoblot detecting KPNA1-6 or KPNB1 from extracted HEK293T cells infected with empty 1056

vector, or Vpr encoding vector at a dose of 0.05 RT U/ml (MOI=2). Size markers are shown in kDa. 1057

Percentage infection by HIV-1 GFP bearing Vpr encoding or empty vector is shown on the right. 1058

(B) Co-immunoprecipitation of Flag-KPNA1-3 and HA-Vpr. Input shows immunoblot detecting 1059

extracted HEK293T whole cell lysates expressing flag-KPNA1-3, flag-GFP and HA-Vpr before 1060

immunoprecipitation. Co-immunoprecipitation precipitates Vpr with HA-beads and detects Flag-1061

KPNA1-3. (C) Co-immunoprecipitation of Flag-KPNA1-3 and WT HA-Vpr or HA-Vpr F34I+P35N. 1062

Input shows immunoblots detecting HA-Vpr or Flag-KPNA1-3 in extracted HEK293T whole cell 1063

lysates (WCL) before immunoprecipitation. β-Actin is detected as a loading control. Co-1064

immunoprecipitation precipitates Vpr with HA-beads and detects Flag-KPNA1-3. (D) Co-1065

immunoprecipitation of HA-IRF3 and Flag-KPNA1 in the presence and absence of WT Vpr or Vpr 1066

F34I+P35N to detect competition between Vpr and IRF3 for KPNA1. Input shows immunoblots 1067

detecting HA-IRF3 or Flag-KPNA1 or Vpr in extracted HEK293T whole cell lysates (WCL) before 1068

immunoprecipitation. CypB is detected as a loading control. Co-immunoprecipitation precipitates 1069

KPNA1 with Flag-beads and detects HA-IRF3 in the presence and absence of WT Vpr or inactive 1070

Vpr F34I+P35N. (E) Co-immunoprecipitation of HA-NF-B p65 and Flag-KPNA1 in the presence 1071

and absence of WT Vpr or Vpr F34I+P35N to detect competition between Vpr and p65 for KPNA1. 1072

Input shows immunoblots detecting HA-p65 or Flag-KPNA1 or Vpr in extracted HEK293T whole 1073

cell lysates (WCL) before immunoprecipitation. CypB is detected as a loading control. Co-1074

immunoprecipitation precipitates KPNA1 with Flag-beads and detects HA-p65 in the presence and 1075

absence of WT Vpr or Vpr F34I+P35N. 1076

1077

Figure 7 figure supplement 1 A unifying model of Vpr function 1078

(1) Stimulation of various PRRs results in activation of transcription factors such as IRF3 and NF-1079

B. To activate ISGs or proinflammatory genes expression, NF- B and IRF3 translocate to the 1080

nucleus via the classical Karyopherin-/ dependent nuclear import pathway. (2) Nuclear import 1081

32

of a plasmid transfected into cellular cytoplasm is essential for gene expression. Transcription 1082

factors such as IRF3 and NF-B bind to their cognate response elements present in the promoter 1083

of the plasmid and allow nuclear import via the classical karyopherin-/ dependent pathway 1084

(Mesika et al., 2001) as well as transcription. (3) HIV-1 based vectors deliver genes to the nucleus 1085

in a karyopherin-/ independent manner. Vpr localises to the nuclear pores and targets 1086

karyopherin- dependent nuclear import in a DCAF1 E3 ubiquitin ligase dependent manner. This 1087

inhibits nuclear translocation of transcription factors such as IRF3 and NF-B and subsequent 1088

antiviral ISG expression. This also inhibits IRF3 and NF-B dependent plasmid expression or 1089

nuclear import but does not impact lentiviral gene delivery. 1090

1091

1092

References 1093

1094

Ali, A., Ng, H. L., Blankson, J. N., Burton, D. R., Buckheit, R. W. 3rd, Moldt, B., Fulcher, J. A., 1095

Ibarrondo, F. J., Anton, P. A., & Yang, O. O. (2018). Highly Attenuated Infection With a Vpr-1096

Deleted Molecular Clone of Human Immunodeficiency Virus-1. The Journal of Infectious 1097

Diseases, 218(9), 1447–1452. https://doi.org/10.1093/infdis/jiy346 1098

Andrejeva, J., Childs, K. S., Young, D. F., Carlos, T. S., Stock, N., Goodbourn, S., & Randall, R. E. 1099

(2004). The V proteins of paramyxoviruses bind the IFN-inducible RNA helicase, mda-5, and 1100

inhibit its activation of the IFN-β promoter. Proceedings of the National Academy of Sciences of 1101

the United States of America, 101(49), 17264–17269. https://doi.org/10.1073/pnas.0407639101 1102

Ayyavoo, V., Mahboubi, A., Mahalingam, S., Ramalingam, R., Kudchodkar, S., Williams, W. V, Green, 1103

D. R., & Weiner, D. B. (1997). HIV-1 Vpr suppresses immune activation and apoptosis through 1104

regulation of nuclear factor kappa B. Nature Medicine, 3(10), 1117–1123. 1105

https://doi.org/10.1038/nm1097-1117 1106

Bachand, F., Yao, X.-J., Hrimech, M., Rougeau, N., & Cohen, É. A. (1999). Incorporation of Vpr into 1107

human immunodeficiency virus type 1 requires a direct interaction with the p6 domain of the p55 1108

Gag precursor. Journal of Biological Chemistry, 274(13), 9083–9091. 1109

https://doi.org/10.1074/jbc.274.13.9083 1110

Bejarano, D. A., Peng, K., Laketa, V., Börner, K., Jost, K. L., Lucic, B., Glass, B., Lusic, M., Müller, B., 1111

& Kräusslich, H.-G. (2019). HIV-1 nuclear import in macrophages is regulated by CPSF6-capsid 1112

interactions at the nuclear pore complex. ELife, 8, e41800. https://doi.org/10.7554/elife.41800 1113

Belzile, J. P., Abrahamyan, L. G., Gérard, F. C. A., Rougeau, N., & Cohen, É. A. (2010). Formation of 1114

mobile chromatin-associated nuclear foci containing HIV-1 Vpr and VPRBP is critical for the 1115

induction of G2 cell cycle arrest. PLoS Pathogens, 6(9), e1001080. 1116

https://doi.org/10.1371/journal.ppat.1001080 1117

Berger, G., Lawrence, M., Hué, S., & Neil, S. J. D. (2015). G2/M cell cycle arrest correlates with 1118

primate lentiviral Vpr interaction with the SLX4 complex. Journal of Virology, 89(1), 230–240. 1119

https://doi.org/10.1128/JVI.02307-14 1120

33

Burdick, R. C., Delviks-Frankenberry, K. A., Chen, J., Janaka, S. K., Sastri, J., Hu, W. S., & Pathak, 1121

V. K. (2017). Dynamics and regulation of nuclear import and nuclear movements of HIV-1 1122

complexes. PLoS Pathogens, 13(8), e1006570. https://doi.org/10.1371/journal.ppat.1006570 1123

Chen, W., Srinath, H., Lam, S. S., Schiffer, C. A., Royer, W. E., & Lin, K. (2008). Contribution of 1124

Ser386 and Ser396 to Activation of Interferon Regulatory Factor 3. Journal of Molecular Biology, 1125

379(2), 251–260. https://doi.org/10.1016/j.jmb.2008.03.050 1126

Chiang, J. J., Sparrer, K. M. J., van Gent, M., Lässig, C., Huang, T., Osterrieder, N., Hopfner, K.-P., & 1127

Gack, M. U. (2018). Viral unmasking of cellular 5S rRNA pseudogene transcripts induces RIG-I-1128

mediated immunity. Nature Immunology, 19(1), 53–62. https://doi.org/10.1038/s41590-017-1129

0005-y 1130

Cingöz, O., & Goff, S. P. (2019). HIV-1 Is a Poor Inducer of Innate Immune Responses. MBio, 10(1), 1131

e02834-18. https://doi.org/10.1128/mBio.02834-18 1132

Connor, R. I., Chen, B. K., Choe, S., & Landau, N. R. (1995). Vpr is required for efficient replication of 1133

human immunodeficiency virus type-1 in mononuclear phagocytes. Virology, 206(2), 935–944. 1134

https://doi.org/10.1006/viro.1995.1016 1135

de Queiroz, N. M. G. P., Xia, T., Konno, H., & Barber, G. N. (2019). Ovarian Cancer Cells Commonly 1136

Exhibit Defective STING Signaling Which Affects Sensitivity to Viral Oncolysis. Molecular 1137

Cancer Research : MCR, 17(4), 974–986. https://doi.org/10.1158/1541-7786.MCR-18-0504 1138

Dedera, D., Hu, W., Vander, N., Heyden, & Ran, L. (1989). Viral Protein R of Human 1139

Immunodeficiency Virus Types 1 and 2 Is Despensable for Replication and Cytopathogenicity in 1140

Lymphoid Cells. Journal of Virology, 63(7), 3205–3208. https://doi.org/10.1007/BF02297789 1141

DeMeritt, I. B., Milford, L. E., & Yurochko, A. D. (2004). Activation of the NF-κB Pathway in Human 1142

Cytomegalovirus-Infected Cells Is Necessary for Efficient Transactivation of the Major 1143

Immediate-Early Promoter. Journal of Virology, 78(9), 498–507. 1144

https://doi.org/10.1128/jvi.78.9.4498-4507.2004 1145

Fagerlund, R., Kinnunen, L., Kohler, M., Julkunen, I., & Melen, K. (2005). NF-{kappa}B is transported 1146

into the nucleus by importin {alpha}3 and importin {alpha}4. The Journal of Biological Chemistry, 1147

280(16), 15942–15951. https://doi.org/10.1074/jbc.M500814200 1148

Fagerlund, R., Melén, K., Cao, X., & Julkunen, I. (2008). NF-κB p52, RelB and c-Rel are transported 1149

into the nucleus via a subset of importin α molecules. Cellular Signalling, 20(8), 1442–1451. 1150

https://doi.org/10.1016/j.cellsig.2008.03.012 1151

Fang, R., Wang, C., Jiang, Q., Lv, M., Gao, P., Yu, X., Mu, P., Zhang, R., Bi, S., Feng, J.-M., & Jiang, 1152

Z. (2017). NEMO–IKKβ Are Essential for IRF3 and NF-κB Activation in the cGAS–STING 1153

Pathway. The Journal of Immunology, 199(9), 3222–3233. 1154

https://doi.org/10.4049/jimmunol.1700699 1155

Fischer, W., Ganusov, V. V, Giorgi, E. E., Hraber, P. T., Keele, B. F., Leitner, T., Han, C. S., 1156

Gleasner, C. D., Green, L., Lo, C.-C., Nag, A., Wallstrom, T. C., Wang, S., McMichael, A. J., 1157

Haynes, B. F., Hahn, B. H., Perelson, A. S., Borrow, P., Shaw, G. M., … Korber, B. T. (2010). 1158

Transmission of single HIV-1 genomes and dynamics of early immune escape revealed by ultra-1159

deep sequencing. PloS One, 5(8), e12303–e12303. 1160

34

https://doi.org/10.1371/journal.pone.0012303 1161

Foster, T L, Wilson, H., Iyer, S. S., Coss, K., Doores, K., Smith, S., Kellam, P., Finzi, A., Borrow, P., 1162

Hahn, B. H., & Neil, S. J. D. (2016). Resistance of Transmitted Founder HIV-1 to IFITM-1163

Mediated Restriction. Cell Host and Microbe, 20(4), 429–442. 1164

https://doi.org/10.1016/j.chom.2016.08.006 1165

Foster, Toshana L, Pickering, S., & Neil, S. J. D. (2017). Inhibiting the Ins and Outs of HIV 1166

Replication: Cell-Intrinsic Antiretroviral Restrictions at the Plasma Membrane. Frontiers in 1167

Immunology, 8, 1853. https://doi.org/10.3389/fimmu.2017.01853 1168

Fouchier, R. A. M., Meyer, B. E., Simon, J. H. M., Fischer, U., Albright, A. V, González-Scarano, F., & 1169

Malim, M. H. (1998). Interaction of the human immunodeficiency virus type 1 Vpr protein with the 1170

nuclear pore complex. Journal of Virology, 72(7), 6004–6013. 1171

https://www.scopus.com/inward/record.uri?eid=2-s2.0-1172

0031799691&partnerID=40&md5=95c739e3621c2e60756318f4b498b0e9 1173

Francis, A. C., Marin, M., Shi, J., Aiken, C., & Melikyan, G. B. (2016). Time-Resolved Imaging of 1174

Single HIV-1 Uncoating In Vitro and in Living Cells. PLoS Pathogens, 12(6), e1005709. 1175


Gagne, B., Tremblay, N., Park, A. Y., Baril, M., & Lamarre, D. (2017). Importin beta1 targeting by 1177

hepatitis C virus NS3/4A protein restricts IRF3 and NF-kappaB signaling of IFNB1 antiviral 1178

response. Traffic (Copenhagen, Denmark), 18(6), 362–377. https://doi.org/10.1111/tra.12480 1179

Gao, D., Wu, J., Wu, Y.-T., Du, F., Aroh, C., Yan, N., Sun, L., & Chen, Z. J. (2013). Cyclic GMP-AMP 1180

synthase is an innate immune sensor of HIV and other retroviruses. Science, 341(6148), 903–1181

906. https://doi.org/10.1126/science.1240933 1182

Gaynor, E. M., & Chen, I. S. Y. (2001). Analysis of apoptosis induced by HIV-1 Vpr and examination 1183

of the possible role of the hHR23A protein. Experimental Cell Research, 267(2), 243–257. 1184

https://doi.org/10.1006/excr.2001.5247 1185

Goujon, C., Moncorgé, O., Bauby, H., Doyle, T., Ward, C. C., Schaller, T., Hué, S., Barclay, W. S., 1186

Schulz, R., & Malim, M. H. (2013). Human MX2 is an interferon-induced post-entry inhibitor of 1187

HIV-1 infection. Nature, 502(7472), 559–562. https://doi.org/10.1038/nature12542 1188

Greenwood, E. J. D., Williamson, J. C., Sienkiewicz, A., Naamati, A., Matheson, N. J., & Lehner, P. J. 1189

(2019). Promiscuous Targeting of Cellular Proteins by Vpr Drives Systems-Level Proteomic 1190

Remodeling in HIV-1 Infection. Cell Reports, 27(5), 1579-1596.e7. 1191

https://doi.org/10.1016/j.celrep.2019.04.025 1192

Gulen, M. F., Koch, U., Haag, S. M., Schuler, F., Apetoh, L., Villunger, A., Radtke, F., & Ablasser, A. 1193

(2017). Signalling strength determines proapoptotic functions of STING. Nature 1194

Communications. https://doi.org/10.1038/s41467-017-00573-w 1195

Harman, A. N., Nasr, N., Feetham, A., Galoyan, A., Alshehri, A. A., Rambukwelle, D., Botting, R. A., 1196

Hiener, B. M., Diefenbach, E., Diefenbach, R. J., Kim, M., Mansell, A., & Cunningham, A. L. 1197

(2015). HIV Blocks Interferon Induction in Human Dendritic Cells and Macrophages by 1198

Dysregulation of TBK1. Journal of Virology, 89(13), 6575–6584. 1199

https://doi.org/10.1128/jvi.00889-15 1200

35

Hartikka, J., Sawdey, M., Cornefert-Jensen, F., Margalith, M., Barnhart, K., Nolasco, M., Vahlsing, H. 1201

L., Meek, J., Marquet, M., Hobart, P., Norman, J., & Manthorpe, M. (1996). An improved plasmid 1202

DNA expression vector for direct injection into skeletal muscle. Human Gene Therapy, 7(10), 1203

1205–1217. https://doi.org/10.1089/hum.1996.7.10-1205 1204

Hattori, N., Michaels, F., Fargnoli, K., Marcon, L., Gallo, R. C., & Franchini, G. (1990). The human 1205

immunodeficiency virus type 2 vpr gene is essential for productive infection of human 1206

macrophages. Proceedings of the National Academy of Sciences, 87(20), 8080–8084. 1207

https://doi.org/10.1073/pnas.87.20.8080 1208

Heiber, J. F., & Barber, G. N. (2012). Evaluation of innate immune signaling pathways in transformed 1209

cells. Methods in Molecular Biology (Clifton, N.J.), 797, 217–238. https://doi.org/10.1007/978-1-1210

61779-340-0_15 1211

Heinzinger, N. K., Bukrinsky, M. I., Haggerty, S. A., Ragland, A. M., Kewalramani, V., Lee, M.-A., 1212

Gendelman, H. E., Ratner, L., Stevenson, M., & Emerman, M. (1994). The Vpr protein of human 1213

immunodeficiency virus type 1 influences nuclear localization of viral nucleic acids in nondividing 1214

host cells. Proceedings of the National Academy of Sciences of the United States of America, 1215

91(15), 7311–7315. https://doi.org/10.1073/pnas.91.15.7311 1216

Hotter, D., Krabbe, T., Reith, E., Gawanbacht, A., Rahm, N., Ayouba, A., Van Driessche, B., Van Lint, 1217

C., Peeters, M., Kirchhoff, F., & Sauter, D. (2017). Primate lentiviruses use at least three 1218

alternative strategies to suppress NF-κB-mediated immune activation. PLoS Pathogens, 13(8), 1219

e1006598. https://doi.org/10.1371/journal.ppat.1006598 1220

Iyer, S. S., Bibollet-Ruche, F., Sherrill-Mix, S., Learn, G. H., Plenderleith, L., Smith, A. G., Barbian, H. 1221

J., Russell, R. M., Gondim, M. V. P., Bahari, C. Y., Shaw, C. M., Li, Y., Decker, T., Haynes, B. 1222

F., Shaw, G. M., Sharp, P. M., Borrow, P., & Hahn, B. H. (2017). Resistance to type 1 1223

interferons is a major determinant of HIV-1 transmission fitness. Proceedings of the National 1224

Academy of Sciences of the United States of America, 114(4), E590–E599. 1225

https://doi.org/10.1073/pnas.1620144114 1226

Jacques, D. A., McEwan, W. A., Hilditch, L., Price, A. J., Towers, G. J., & James, L. C. (2016). HIV-1 1227

uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis. Nature, 1228

536(7616), 349–353. https://doi.org/10.1038/nature19098 1229

Jacquot, G, Le Rouzic, E., David, A., Mazzolini, J., Bouchet, J., Bouaziz, S., Niedergang, F., Pancino, 1230

G., & Benichou, S. (2007). Localization of HIV-1 Vpr to the nuclear envelope: Impact on Vpr 1231

functions and virus replication in macrophages. Retrovirology, 4(84), 123–176. 1232

https://doi.org/10.1186/1742-4690-4-84 1233

Jacquot, Guillaume, Le Rouzic, E., David, A., Mazzolini, J., Bouchet, J., Bouaziz, S., Niedergang, F., 1234

Pancino, G., & Benichou, S. (2007). Localization of HIV-1 Vpr to the nuclear envelope: Impact 1235

on Vpr functions and virus replication in macrophages. Retrovirology, 4(84), 123–176. 1236

https://doi.org/10.1186/1742-4690-4-84 1237

Jimenez-Guardeño, J. M., Apolonia, L., Betancor, G., & Malim, M. H. (2019). Immunoproteasome 1238

activation enables human TRIM5α restriction of HIV-1. Nature Microbiology, 4(6), 933–940. 1239

https://doi.org/10.1038/s41564-019-0402-0 1240

36

Jones, R. B., Song, H., Xu, Y., Garrison, K. E., Buzdin, A. A., Anwar, N., Hunter, D. V, Mujib, S., 1241

Mihajlovic, V., Martin, E., Lee, E., Kuciak, M., Raposo, R. A. S., Bozorgzad, A., Meiklejohn, D. 1242

A., Ndhlovu, L. C., Nixon, D. F., & Ostrowski, M. A. (2013). LINE-1 retrotransposable element 1243

DNA accumulates in HIV-1-infected cells. Journal of Virology, 87(24), 13307–13320. 1244

https://doi.org/10.1128/JVI.02257-13 1245

Kane, M., Yadav, S. S., Bitzegeio, J., Kutluay, S. B., Zang, T., Wilson, S. J., Schoggins, J. W., Rice, 1246

C. M., Yamashita, M., Hatziioannou, T., & Bieniasz, P. D. (2013). MX2 is an interferon-induced 1247

inhibitor of HIV-1 infection. Nature, 502(7472), 563–566. https://doi.org/10.1038/nature12653 1248

Kogan, M., Deshmane, S., Sawaya, B. E., Gracely, E. J., Khalili, K., & Rappaport, J. (2013). Inhibition 1249

of NF-κB activity by HIV-1 Vpr is dependent on Vpr binding protein. Journal of Cellular 1250

Physiology, 228(4), 781–790. https://doi.org/10.1002/jcp.24226 1251

Kumar, K. P., McBride, K. M., Weaver, B. K., Dingwall, C., & Reich, N. C. (2002). Regulated Nuclear-1252

Cytoplasmic Localization of Interferon Regulatory Factor 3, a Subunit of Double-Stranded RNA-1253

Activated Factor 1. Molecular and Cellular Biology, 20(11), 4159–4168. 1254

https://doi.org/10.1128/mcb.20.11.4159-4168.2000 1255

Laguette, N., Brégnard, C., Hue, P., Basbous, J., Yatim, A., Larroque, M., Kirchhoff, F., Constantinou, 1256

A., Sobhian, B., & Benkirane, M. (2014). Premature activation of the slx4 complex by vpr 1257

promotes g2/m arrest and escape from innate immune sensing. Cell, 156(1–2), 134–145. 1258

https://doi.org/10.1016/j.cell.2013.12.011 1259

Lahaye, X., Satoh, T., Gentili, M., Cerboni, S., Conrad, C., Hurbain, I., ElMarjou, A., Lacabaratz, C., 1260

Lelièvre, J.-D., & Manel, N. (2013). The Capsids of HIV-1 and HIV-2 Determine Immune 1261

Detection of the Viral cDNA by the Innate Sensor cGAS in Dendritic Cells. Immunity, 39(6), 1262

1132–1142. https://doi.org/10.1016/j.immuni.2013.11.002 1263

Lahouassa, H., Blondot, M.-L., Chauveau, L., Chougui, G., Morel, M., Leduc, M., Guillonneau, F., 1264

Ramirez, B. C., Schwartz, O., & Margottin-Goguet, F. (2016). HIV-1 Vpr degrades the HLTF 1265

DNA translocase in T cells and macrophages. Proceedings of the National Academy of 1266

Sciences of the United States of America, 113(19), 5311–5316. 1267


Le Rouzic, E., Mousnier, A., Rustum, C., Stutz, F., Hallberg, E., Dargemont, C., & Benichou, S. 1269

(2002). Docking of HIV-1 vpr to the nuclear envelope is mediated by the interaction with the 1270

nucleoporin hCG1. Journal of Biological Chemistry, 277(47), 45091–45098. 1271

https://doi.org/10.1074/jbc.M207439200 1272

Liang, P., Zhang, H., Wang, G., Li, S., Cong, S., Luo, Y., & Zhang, B. (2013). KPNB1, XPO7 and 1273

IPO8 mediate the translocation ofNF-kappaB/p65 into the nucleus. Traffic (Copenhagen, 1274

Denmark), 14(11), 1132–1143. https://doi.org/10.1111/tra.12097 1275

Lin, R., Mamane, Y., & Hiscott, J. (1999). Structural and functional analysis of interferon regulatory 1276

factor 3: localization of the transactivation and autoinhibitory domains. Molecular and Cellular 1277

Biology, 19(4), 2465–2474. 1278

Liu, R, Lin, Y., Jia, R., Geng, Y., Liang, C., Tan, J., & Qiao, W. (2014). HIV-1 Vpr stimulates NF-κB 1279

and AP-1 signaling by activating TAK1. Retrovirology, 11(1). https://doi.org/10.1186/1742-4690-1280

37

11-45 1281

Liu, Ruikang, Tan, J., Lin, Y., Jia, R., Yang, W., Liang, C., Geng, Y., & Qiao, W. (2013). HIV-1 Vpr 1282

activates both canonical and noncanonical NF-κB pathway by enhancing the phosphorylation of 1283

IKKα/Β. Virology. https://doi.org/10.1016/j.virol.2013.01.020 1284

Liu, S., Cai, X., Wu, J., Cong, Q., Chen, X., Li, T., Du, F., Ren, J., Wu, Y. T., Grishin, N. V., & Chen, 1285

Z. J. (2015). Phosphorylation of innate immune adaptor proteins MAVS, STING, and TRIF 1286

induces IRF3 activation. Science, 13(347), 2630. https://doi.org/10.1126/science.aaa2630 1287

Liu, X., Guo, H., Wang, H., Markham, R., Wei, W., & Yu, X. F. (2015). HIV-1 Vpr suppresses the 1288

cytomegalovirus promoter in a CRL4(DCAF1) E3 ligase independent manner. Biochemical and 1289

Biophysical Research Communications, 459(2), 214–219. 1290

https://doi.org/10.1016/j.bbrc.2015.02.060 1291

Mankan, A. K., Schmidt, T., Chauhan, D., Goldeck, M., Höning, K., Gaidt, M., Kubarenko, A. V, 1292

Andreeva, L., Hopfner, K., & Hornung, V. (2014). Cytosolic RNA:DNA hybrids activate the cGAS 1293

–STING axis. The EMBO Journal, 33(24), 2937–2946. https://doi.org/10.15252/embj.201488726 1294

Mashiba, M., Collins, D. R., Terry, V. H., & Collins, K. L. (2014). Vpr overcomes macrophage-specific 1295

restriction of HIV-1 Env expression and virion production. Cell Host and Microbe, 17(3), 414. 1296


Matikainen, S., Pirhonen, J., Miettinen, M., Lehtonen, A., Govenius-Vintola, C., Sareneva, T., & 1298

Julkunen, I. (2000). Influenza A and sendai viruses induce differential chemokine gene 1299

expression and transcription factor activation in human macrophages. Virology, 276(1), 138–1300

147. https://doi.org/10.1006/viro.2000.0542 1301

Matsuda, T., & Cepko, C. L. (2004). Electroporation and RNA interference in the rodent retina in vivo 1302

and in vitro. Proceedings of the National Academy of Sciences of the United States of America, 1303

101(1), 16–22. https://doi.org/10.1073/pnas.2235688100 1304

Mesika, A., Grigoreva, I., Zohar, M., & Reich, Z. (2001). A regulated, NFκB-assisted import of plasmid 1305

DNA into mammalian cell nuclei. Molecular Therapy, 3(5Pt1), 653–657. 1306

https://doi.org/10.1006/mthe.2001.0312 1307

Miller, C. M., Akiyama, H., Agosto, L. M., Emery, A., Ettinger, C. R., Swanstrom, R. I., Henderson, A. 1308

J., & Gummuluru, S. (2017). Virion-Associated Vpr Alleviates a Postintegration Block to HIV-1 1309

Infection of Dendritic Cells. Journal of Virology, 91(13). https://doi.org/10.1128/JVI.00051-17 1310

Miyatake, H., Sanjoh, A., Murakami, T., Murakami, H., Matsuda, G., Hagiwara, K., Yokoyama, M., 1311

Sato, H., Miyamoto, Y., Dohmae, N., & Aida, Y. (2016). Molecular Mechanism of HIV-1 Vpr for 1312

Binding to Importin-α. Journal of Molecular Biology, 428(13), 2744–2757. 1313

https://doi.org/10.1016/j.jmb.2016.05.003 1314

Morellet, N., Bouaziz, S., Petitjean, P., & Roques, B. P. (2003). NMR structure of the HIV-1 regulatory 1315

protein VPR. Journal of Molecular Biology, 285(5), 2105–2117. https://doi.org/10.1016/S0022-1316

2836(03)00060-3 1317

Mori, M., Yoneyama, M., Ito, T., Takahashi, K., Inagaki, F., & Fujita, T. (2004). Identification of Ser-1318

386 of Interferon Regulatory Factor 3 as Critical Target for Inducible Phosphorylation That 1319

Determines Activation. Journal of Biological Chemistry, 279(11), 9698–9702. 1320

38

https://doi.org/10.1074/jbc.M310616200 1321

Nitahara-Kasahara, Y., Kamata, M., Yamamoto, T., Zhang, X., Miyamoto, Y., Muneta, K., Iijima, S., 1322

Yoneda, Y., Tsunetsugu-Yokota, Y., & Aida, Y. (2007). Novel nuclear import of Vpr promoted by 1323

importin a is crucial for human immunodeficiency virus type 1 replication in macrophages. 1324

Journal of Virology, 81(10), 5284–5293. https://doi.org/10.1128/JVI.01928-06 1325

Okumura, A., Alce, T., Lubyova, B., Ezelle, H., Strebel, K., & Pitha, P. M. (2008). HIV-1 accessory 1326

proteins VPR and Vif modulate antiviral response by targeting IRF-3 for degradation. Virology, 1327

373(1), 85–97. https://doi.org/10.1016/j.virol.2007.10.042 1328

Pallett, M. A., Ren, H., Zhang, R.-Y., Scutts, S. R., Gonzalez, L., Zhu, Z., Maluquer de Motes, C., & 1329

Smith, G. L. (2019). Vaccinia Virus BBK E3 Ligase Adaptor A55 Targets Importin-Dependent 1330

NF-κB Activation and Inhibits CD8 + T-Cell Memory. Journal of Virology, 93(10), e00051-19. 1331

https://doi.org/10.1128/jvi.00051-19 1332

Popov, S., Rexach, M., Zybarth, G., Railing, N., Lee, M.-A., Ratner, L., Lane, C. M., Moore, M. S., 1333

Blobel, G., & Bukrinsky, M. (1998). Viral protein R regulates nuclear import of the HIV-1 pre-1334

integration complex. EMBO Journal, 17(4), 909–917. https://doi.org/10.1093/emboj/17.4.909 1335

Rasaiyaah, J., Tan, C. P., Fletcher, A. J., Price, A. J., Blondeau, C., Hilditch, L., Jacques, D. A., 1336

Selwood, D. L., James, L. C., Noursadeghi, M., & Towers, G. J. (2013). HIV-1 evades innate 1337

immune recognition through specific cofactor recruitment. Nature, 503(7476), 402–405. 1338

https://doi.org/10.1038/nature12769 1339

Rehwinkel, J., Tan, C. P., Goubau, D., Schulz, O., Pichlmair, A., Bier, K., Robb, N., Vreede, F., 1340

Barclay, W., Fodor, E., & Reis e Sousa, C. (2010). RIG-I Detects Viral Genomic RNA during 1341

Negative-Strand RNA Virus Infection. Cell, 140(3), 397–408. 1342

https://doi.org/10.1016/j.cell.2010.01.020 1343

Rossenkhan, R., MacLeod, I. J., Brumme, Z. L., Magaret, C. A., Sebunya, T. K., Musonda, R., Gashe, 1344

B. A., Edlefsen, P. T., Novitsky, V., & Essex, M. (2016). Transmitted/Founder HIV-1 Subtype C 1345

Viruses Show Distinctive Signature Patterns in Vif, Vpr, and Vpu That Are Under Subsequent 1346

Immune Pressure During Early Infection. AIDS Research and Human Retroviruses, 32(10–11), 1347

1031–1045. https://doi.org/10.1089/AID.2015.0330 1348

Schaller, T., Ocwieja, K. E., Rasaiyaah, J., Price, A. J., Brady, T. L., Roth, S. L., Hué, S., Fletcher, A. 1349

J., Lee, K., KewalRamani, V. N., Noursadeghi, M., Jenner, R. G., James, L. C., Bushman, F. D., 1350

& Towers, G. J. (2011). HIV-1 capsid-cyclophilin interactions determine nuclear import pathway, 1351

integration targeting and replication efficiency. PLoS Pathogens, 7(12). 1352


Schirrmacher, V. (2015). Signaling through RIG-I and type I interferon receptor: Immune activation by 1354

Newcastle disease virus in man versus immune evasion by Ebola virus (Review). International 1355

Journal of Molecular Medicine, 36(1), 3–10. https://doi.org/10.3892/ijmm.2015.2213 1356

Schmidt, N., Domingues, P., Golebiowski, F., Patzina, C., Tatham, M. H., Hay, R. T., & Hale, B. G. 1357

(2019). An influenza virus-triggered SUMO switch orchestrates co-opted endogenous 1358

retroviruses to stimulate host antiviral immunity. Proceedings of the National Academy of 1359


39


Schwefel, D., Groom, H. C. T., Boucherit, V. C., Christodoulou, E., Walker, P. A., Stoye, J. P., Bishop, 1362

K. N., & Taylor, I. A. (2014). Structural basis of lentiviral subversion of a cellular protein 1363

degradation pathway. Nature. https://doi.org/10.1038/nature12815 1364

Servant, M. J., Grandvaux, N., TenOever, B. R., Duguay, D., Lin, R., & Hiscott, J. (2003). 1365

Identification of the minimal phosphoacceptor site required for in vivo activation of interferon 1366

regulatory factor 3 in response to virus and double-stranded RNA. Journal of Biological 1367

Chemistry, 278(11), 9441–9447. https://doi.org/10.1074/jbc.M209851200 1368

Shaw, G. M., & Hunter, E. (2012). HIV transmission. Cold Spring Harbor Perspectives in Medicine, 1369

2(11), a006965. https://doi.org/10.1101/cshperspect.a006965 1370

Stacey, A. R., Norris, P. J., Qin, L., Haygreen, E. A., Taylor, E., Heitman, J., Lebedeva, M., DeCamp, 1371

A., Li, D., Grove, D., Self, S. G., & Borrow, P. (2009). Induction of a Striking Systemic Cytokine 1372

Cascade prior to Peak Viremia in Acute Human Immunodeficiency Virus Type 1 Infection, in 1373

Contrast to More Modest and Delayed Responses in Acute Hepatitis B and C Virus Infections. 1374

Journal of Virology, 83(8), 3719–3733. https://doi.org/10.1128/jvi.01844-08 1375

Su, J., Rui, Y., Lou, M., Yin, L., Xiong, H., Zhou, Z., Shen, S., Chen, T., Zhang, Z., Zhao, N., Zhang, 1376

W., Cai, Y., Markham, R., Zheng, S., Xu, R., Wei, W., & Yu, X.-F. (2019). HIV-2/SIV Vpx targets 1377

a novel functional domain of STING to selectively inhibit cGAS–STING-mediated NF-κB 1378

signalling. Nature Microbiology, 4(12), 2552–2564. https://doi.org/10.1038/s41564-019-0585-4 1379

Suhara, W., Yoneyama, M., Iwamura, T., Yoshimura, S., Tamura, K., Namiki, H., Aimoto, S., & Fujita, 1380

T. (2000). Analyses of virus-induced homomeric and heteromeric protein associations between 1381

IRF-3 and coactivator CBP/p300. Journal of Biochemistry, 128(2), 301–307. 1382

https://doi.org/10.1093/oxfordjournals.jbchem.a022753 1383

Sumner, R.P., Thorne, L. G., Fink, D. L., Khan, H., Milne, R. S., & Towers, G. J. (2017). Are evolution 1384

and the intracellular innate immune system key determinants in HIV transmission? Frontiers in 1385

Immunology, 8(OCT). https://doi.org/10.3389/fimmu.2017.01246 1386

Sumner, Rebecca P, Harrison, L., Touizer, E., Peacock, T. P., Spencer, M., Zuliani-Alvarez, L., & 1387

Towers, G. J. (2019). Disrupting HIV-1 capsid formation causes cGAS sensing of viral DNA. 1388

BioRxiv, 838011. https://doi.org/10.1101/838011 1389

Taylor, S. L., Frias-Staheli, N., Garcia-Sastre, A., & Schmaljohn, C. S. (2009). Hantaan Virus 1390

Nucleocapsid Protein Binds to Importin Proteins and Inhibits Tumor Necrosis Factor Alpha-1391

Induced Activation of Nuclear Factor Kappa B. Journal of Virology, 83(3), 1271–1279. 1392

https://doi.org/10.1128/jvi.00986-08 1393

Towers, G. J., & Noursadeghi, M. (2014). Interactions between HIV-1 and the cell-autonomous innate 1394

immune system. Cell Host and Microbe, 16(1), 10–18. 1395


Trotard, M., Tsopoulidis, N., Tibroni, N., Willemsen, J., Binder, M., Ruggieri, A., & Fackler, O. T. 1397

(2016). Sensing of HIV-1 Infection in Tzm-bl Cells with Reconstituted Expression of STING. 1398

Journal of Virology. https://doi.org/10.1128/jvi.02966-15 1399

Vermeire, J, Roesch, F., Sauter, D., Rua, R., Hotter, D., Van Nuffel, A., Vanderstraeten, H., 1400

40

Naessens, E., Iannucci, V., Landi, A., Witkowski, W., Baeyens, A., Kirchhoff, F., & Verhasselt, B. 1401

(2016). HIV Triggers a cGAS-Dependent, Vpu- and Vpr-Regulated Type I Interferon Response 1402

in CD4+ T Cells. Cell Reports, 17(2), 413–424. https://doi.org/10.1016/j.celrep.2016.09.023 1403

Vermeire, Jolien, Naessens, E., Vanderstraeten, H., Landi, A., Iannucci, V., van Nuffel, A., Taghon, 1404

T., Pizzato, M., & Verhasselt, B. (2012). Quantification of Reverse Transcriptase Activity by 1405

Real-Time PCR as a Fast and Accurate Method for Titration of HIV, Lenti- and Retroviral 1406

Vectors. PLoS ONE, 7(12), e50859. https://doi.org/10.1371/journal.pone.0050859 1407

Vodicka, M. A., Koepp, D. M., Silver, P. A., & Emerman, M. (1998). HIV-1 Vpr interacts with the 1408

nuclear transport pathway to promote macrophage infection. Genes and Development, 12(2), 1409

175–185. https://doi.org/10.1101/gad.12.2.175 1410

Wu, Y., Zhou, X., Barnes, C. O., DeLucia, M., Cohen, A. E., Gronenborn, A. M., Ahn, J., & Calero, G. 1411

(2016). The DDB1-DCAF1-Vpr-UNG2 crystal structure reveals how HIV-1 Vpr steers human 1412

UNG2 toward destruction. Nature Structural and Molecular Biology, 23(10), 933–939. 1413

https://doi.org/10.1038/nsmb.3284 1414

Xia, T., Konno, H., Ahn, J., & Barber, G. N. (2016). Deregulation of STING Signaling in Colorectal 1415

Carcinoma Constrains DNA Damage Responses and Correlates With Tumorigenesis. Cell 1416

Reports, 14(2), 282–297. https://doi.org/10.1016/j.celrep.2015.12.029 1417

Xia, T., Konno, H., & Barber, G. N. (2016). Recurrent Loss of STING Signaling in Melanoma 1418

Correlates with Susceptibility to Viral Oncolysis. Cancer Research, 76(22), 6747–6759. 1419

https://doi.org/10.1158/0008-5472.CAN-16-1404 1420

Xu, S., Ducroux, A., Ponnurangam, A., Vieyres, G., Franz, S., Müsken, M., Zillinger, T., Malassa, A., 1421

Ewald, E., Hornung, V., Barchet, W., Häussler, S., Pietschmann, T., & Goffinet, C. (2016). 1422

cGAS-Mediated Innate Immunity Spreads Intercellularly through HIV-1 Env-Induced Membrane 1423

Fusion Sites. Cell Host and Microbe, 20(4), 443–457. 1424


Xu, W., Edwards, M. R., Borek, D. M., Feagins, A. R., Mittal, A., Alinger, J. B., Berry, K. N., Yen, B., 1426

Hamilton, J., Brett, T. J., Pappu, R. V., Leung, D. W., Basler, C. F., & Amarasinghe, G. K. 1427

(2014). Ebola virus VP24 targets a unique NLS binding site on karyopherin alpha 5 to selectively 1428

compete with nuclear import of phosphorylated STAT1. Cell Host and Microbe, 16(2), 187–200. 1429


Yamashita, M., & Emerman, M. (2005). The cell cycle independence of HIV infections is not 1431

determined by known karyophilic viral elements. PLoS Pathogens, 1(3), e18–e18. 1432


Yan, J., Shun, M. C., Zhang, Y., Hao, C., & Skowronski, J. (2019). HIV-1 Vpr counteracts HLTF-1434

mediated restriction of HIV-1 infection in T cells. Proceedings of the National Academy of 1435



Yan, N., Regalado-Magdos, A. D., Stiggelbout, B., Lee-Kirsch, M. A., & Lieberman, J. (2010). The 1438

cytosolic exonuclease TREX1 inhibits the innate immune response to human immunodeficiency 1439

virus type 1. Nature Immunology, 11(11), 1005–1013. https://doi.org/10.1038/ni.1941 1440

41

Ye, J., Chen, Z., Li, Y., Zhao, Z., He, W., Zohaib, A., Song, Y., Deng, C., Zhang, B., Chen, H., & Cao, 1441

S. (2017). Japanese Encephalitis Virus NS5 Inhibits Type I Interferon (IFN) Production by 1442

Blocking the Nuclear Translocation of IFN Regulatory Factor 3 and NF-κB. Journal of Virology, 1443

91(8), e00039-17. https://doi.org/10.1128/jvi.00039-17 1444

Yoneyama, M., Suhara, W., Fukuhara, Y., Fukuda, M., Nishida, E., & Fujita, T. (1998). Direct 1445

triggering of the type I interferon system by virus infection: Activation of a transcription factor 1446

complex containing IRF-3 and CBP/p300. EMBO Journal, 17(4), 1087–1095. 1447

https://doi.org/10.1093/emboj/17.4.1087 1448

Zander, K., Sherman, M. P., Tessmer, U., Bruns, K., Wray, V., Prechtel, A. T., Schubert, E., Henklein, 1449

P., Luban, J., Neidleman, J., Greene, W. C., & Schubert, U. (2003). Cyclophilin A Interacts with 1450

HIV-1 Vpr and Is Required for Its Functional Expression. Journal of Biological Chemistry, 1451

278(44), 43202–43213. https://doi.org/10.1074/jbc.M305414200 1452

Zhang, C., Shang, G., Gui, X., Zhang, X., Bai, X. chen, & Chen, Z. J. (2019). Structural basis of 1453

STING binding with and phosphorylation by TBK1. In Nature (pp. 567(7748):394-398). 1454

https://doi.org/10.1038/s41586-019-1000-2 1455

Zhang, F., & Bieniasz, P. D. (2019). HIV-1 Vpr induces cell cycle arrest and enhances viral gene 1456

expression by depleting CCDC137. BioRxiv, 2019.12.24.888230. 1457

https://doi.org/10.1101/2019.12.24.888230 1458

Zhang, S., Feng, Y., Narayan, O., & Zhao, L. J. (2001). Cytoplasmic retention of HIV-1 regulatory 1459

protein V pr by protein-protein interaction with a novel human cytoplasmic protein VprBP. Gene, 1460

263(1–2), 131–140. https://doi.org/10.1016/S0378-1119(00)00583-7 1461

Zhao, Y., Ye, X., Dunker, W., Song, Y., & Karijolich, J. (2018). RIG-I like receptor sensing of host 1462

RNAs facilitates the cell-intrinsic immune response to KSHV infection. Nature Communications, 1463

9(1), 4841. https://doi.org/10.1038/s41467-018-07314-7 1464

Zhu, H., Jian, H., & Zhao, L. J. (2004). Identification of the 15FRFG domain in HIV-1 Gag p6 essential 1465

for Vpr packaging into the virion. Retrovirology, 1(26), 343–398. https://doi.org/10.1186/1742-1466

4690-1-26 1467

Zila, V., Müller, T. G., Laketa, V., Müller, B., & Kräusslich, H. G. (2019). Analysis of CA content and 1468

CPSF6 dependence of early HIV-1 replication complexes in SupT1-R5 cells. MBio, 10(6), 1469

e02501-19. https://doi.org/10.1128/mBio.02501-19 1470

Zufferey, R., Nagy, D., Mandel, R. J., Naldini, L., & Trono, D. (1997). Multiply attenuated lentiviral 1471

vector achieves efficient gene delivery in vivo. Nature Biotechnology, 15(9), 871–875. 1472

https://doi.org/10.1038/nbt0997-871 1473

1474

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25

30

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Figure 1 HIV-1 replication in cGAMP stimulatedMDMs requires Vpr(A) Replication of WT Yu2 HIV-1 or Yu2 HIV-1ΔVpr in MDMs stimulated with 1 μg/ml, 2 μg/ml or 4 μg/ml cGAMP or leftunstimulated, infection measured by counting Gag positive cells stained with anti-p24. Mean+/-SEM n=3 1 and 2 μg/ml cGAMP; n=24 μg/ml cGAMP. *** = 2 way ANOVA p value <0.001, * = p<0.05. (B) Fold induction of IFIT1-Luc after activation of STING by cGAMP(5 μg/ml) and infection with HIV-1 virus like particles (VLP) lacking genome and bearing Vpr (+Vpr) or lacking Vpr (-Vpr) (1 RT U/ml)in IFIT1-Luc reporter THP-1 cells. cGAMP and virus were added to cells at the same time. (C) Fold induction of CXCL10 after infectionof THP-1 cells with HIV-GFP -Vpr or HIV-GFP +Vpr at the indicated MOI. (D) Percentage of THP-1 cells infected by HIV-GFP -Vpr orHIV-GFP +Vpr in (C). (E) Fold induction of CXCL10 after infection of THP-1 cells with HIV-GFP -Vpr, HIV-GFP +Vpr or HIV-1 particleslacking Vpr and genome, at indicated doses measured by reverse transcriptase SG-PERT assay. (F) Percentage of THP-1 cells infectedby HIV-GFP viruses in (E). (G) Fold induction of CXCL10 after infection of unmodified control, cGAS-/- or MAVS-/- THP-1 knock outcells with HIV-GFP lacking Vpr (0.3 RT U/ml). (H) Percentage infection of control, cGAS-/- or MAVS-/- THP-1 knockout cells infectedwith HIV-GFP at indicated doses of RT (SG-PERT). (B-H) Data are expressed as means ± SD (n = 3) with two-way ANOVA * (p<0.05), **(p<0.01), *** (p<0.001), **** (p<0.0001) compared to virus without genome (B), HIV GFP+Vpr (C, E) and control (G).

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Figure 1 figure supplement 1 HIV-1 replication in cGAMP stimulated MDMs requires Vpr and Vpr suppresses HIV-1 innateimmune sensing by cGAS(A) Replication of wild type (WT) NL4-3 HIV-1, or NL4-3 HIV-1ΔVpr, in activated primary human CD4+ T cells stimulated with 1, 2 or 4μg/ml cGAMP or left unstimulated as a control. Two representative examples of three are shown with virus replication measured bypercentage T-cell p24 positivity, measured by flow, (top panels) or supernatant RT activity (lower panels). This experiment was alsoperformed twice in Jurkat cells with virus replication measured by percentage T-cell p24 positivity, measured by flow, giving similarresults as shown.” Replication of WT NL4-3 HIV-1 or NL4-3 HIV-1ΔVpr in activated CD4+ T cells stimulated with 1 μg/ml, 2 μg/ml or 4μg/ml cGAMP or left unstimulated, measured by flow cytometry staining infected cells with anti-p24 antibody. (B) HIV-GFP titre incontrol, cGAS-/- or MAVS-/- THP-1 cells used in Figure 1 (G). (C) Immunoblot detecting cGAS, MAVS, or actin as a loading control,from extracted cGAS-/- or MAVS-/- knock out THP-1 cells or their CRISPR/Cas control cells. Size marker positions are shown on theright (kDa).

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A BLUC reporter

LUC reporter

CXCL10LUC reporter

LUC reporter

ISG induction

Figure 2 HIV-1 Vpr expression inhibits interferon stimulated gene expression after stimulation with various innate immunestimuli(A) Fold induction of IFIT1-Luc, after activation of STING by cGAMP (5 μg/ml), in IFIT1-Luc reporter THP-1 cells expressing Vpr from alentiviral vector delivered at MOIs of 0.25, 0.5, 1, or after empty vector transduction (MOI 1) or in untransduced cells. (B) Foldinduction of ISGs MxA, CXCL10, IFIT2 and Viperin after activation of STING by cGAMP (5 μg/ml) in cells expressing Vpr from alentiviral vector (MOI 1), or after empty vector transduction (MOI 1) or in untransduced THP-1 cells. (C) Secreted CXCL10 (ELISA)after activation of STING by cGAMP (5 μg/ml) in cells expressing Vpr from a lentiviral vector (MOI 0.5, 1), or after transduction withempty vector (MOI 0.5, 1) or in untransduced THP-1 cells. Dotted line shows limit of detection. (D) Fold induction of IFIT1-Luc afterHT-DNA transfection (5 μg/ml) of cells expressing Vpr from a lentiviral vector (MOI 0.5, 1), or empty vector (MOI 0.5, 1) or inuntransduced IFIT1-Luc reporter THP-1 cells. (E) Fold induction of IFIT1-Luc, after Sendai virus infection, of cells expressing Vpr froma lentiviral vector (MOI 0.5, 1), or after transduction by empty vector (MOI 0.5, 1) or in untransduced IFIT1-Luc reporter THP-1 cells.(F) Fold induction of IFIT1-Luc, after LPS treatment (1 μg/ml), of cells expressing Vpr from a lentiviral vector (MOI 0.25, 0.5, 1), aftertransduction by empty vector (MOI 1) or in untransduced IFIT1-Luc reporter THP-1 cells. Data are expressed as mean ± SD (n = 3)analysed using two-way ANOVA * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001) compared to data for empty vector. n= 3(A, D-F) or 2 (B-C) independent experiments.

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Figure 2 figure supplement 1 HIV-1 Vpr expression inhibits interferon stimulated gene expression after stimulation with variousinnate immune stimuli(A) Vpr encoding lentiviral expression construct (pCSVIG) contained self-inactivating Long terminal repeat (SIN LTR), Rev responseelement (RRE), Central polypurine tract (cPPT), Spleen focus-forming virus promoter (SFFV), internal ribosome entry site (IRES),green fluorescent protein (GFP) and Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). (B) Immunoblotdetecting VSV-G envelope, capsid (p24) and Vpr in vector supernatant and Vpr additionally in target cell lysate. Size markers in kDaare indicated on the right. (C) Percentage of THP-1 cells in Figure 2A transduced by the vector encoding Vpr and GFP (MOI 0.25, 0.5,1) or empty vector encoding GFP alone (MOI 1) and treated with cGAMP (5 μg/ml) or left untreated as a control. (D) Percentage ofTHP-1 cells in Figure 2B transduced by the vector encoding Vpr and GFP (MOI 1) or empty vector encoding GFP alone (MOI 1) andtreated with cGAMP (5 μg/ml) or left untreated as a control. (E) Percentage of THP-1 cells in Figure 2C transduced by the vectorencoding Vpr and GFP (MOI 0.5, 1) or empty vector expressing GFP alone (MOI 0.5, 1) and treated with cGAMP (5 μg/ml) or leftuntreated as a control. (F) Percentage of THP-1 cells in Figure 2D transduced by the vector encoding Vpr and GFP (MOI 0.5, 1) orempty vector encoding GFP alone (MOI 0.5, 1) and stimulated with HT-DNA transfection (5 μg/ml) or left untreated as a control. (G)Percentage of THP-1 cells in Figure 2E transduced by the vector encoding Vpr and GFP (MOI 0.5, 1) or empty vector expressing GFPalone (MOI 0.5, 1) and stimulated with Sendai virus infection or left untreated as a control. (H) Percentage of THP-1 cells in Figure 2Ftransduced by the vector encoding Vpr and GFP (MOI 0.25, 0.5, 1) or empty vector encoding GFP alone (MOI 1) and stimulated withLPS treatment (1 μg/ml) or left untreated as a control Data are expressed as means ± SD (n = 3). Data are representative of three (C,F-H) or two (B, D, E) independent experiments.

GFP

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no vector Control vector WT Vpr

VprF34I+P35N VprQ65R VprR80A

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Figure 3 Vpr inhibition of innate immune activation is dependent on DCAF1 but independent of cell cycle arrest(A) Immunoblot detecting p24 (capsid) and Vpr in pelleted VSV-G pseudotyped VLP lacking genome used in (B). Size markers in kDaare indicated on the right. (B) Fold induction of IFIT1-Luc after activation of STING by cGAMP (5 μg/ml) and infection with VLPbearing WT or mutant Vpr, or lacking Vpr (1 RT U/ml) in IFIT1-Luc reporter THP-1 cells. Cells were infected at the same time ascGAMP treatment. (C) Flow cytometry plots showing cell cycle phases of THP-1 cells transduced with an empty vector, WT Vpr, ormutant Vpr, encoding vector (MOI 1) or left untransduced as a control and stained with propidium iodide to label DNA. Percentagecells in each cell cycle stage are shown. (D) Fold induction of IFIT1-Luc after activation of STING by cGAMP (5 μg/ml) in cellsexpressing Vpr from a lentiviral vector, or expressing empty vector, or in untransduced IFIT1-Luc reporter THP-1 cells expressing acontrol, or a DCAF1 targeting shRNA. Mean +/-SEM n=3 independent experiments. (E) Immunoblot detecting DCAF1, or actin as aloading control, from extracted THP-1 cells expressing a non-targeting, or DCAF1-targeting, shRNA. Size markers are shown in kDaon the right. (F) Fold induction of IFIT1-Luc after activation of STING by cGAMP (5 μg/ml) in cells expressing WT, or mutant, Vprfrom a lentiviral vector (MOI 1), or empty vector (MOI 1) or in untransduced IFIT1-Luc reporter THP-1 cells. (G) Fold induction ofMxAmRNA after activation of STING by cGAMP (5 μg/ml) in cells expressing WT, or mutant, Vpr from a lentiviral vector (MOI 1), orafter transduction by empty vector (MOI 1) or in untransduced THP-1 cells. Data are mean ± SD (n = 3). Two-way ANOVA test: *(p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001) compared to no Vpr or empty vector controls. Data are representative ofthree (B-D, F) or two (A, E, G) independent experiments.

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Figure 3 figure supplement 1 Vpr inhibition of innate immune activation is dependent on DCAF1 but independent of cell cyclearrest(A) Percentage of THP-1 cells in Figure 3C transduced by the vector encoding Vpr and GFP, or empty vector encoding GFP alone, atthe indicatedMOI and treated with cGAMP (5 μg/ml) or left untreated. (B) NMR structure of full length Vpr showing position of Vprmutants (PDB: 1M8L). White region (c-terminus) of Vpr shown in (B) is unresolved in the crystal structure (C). (C) Crystal structureof Vpr (orange) with its target protein UNG2 (blue) and cofactors DCAF1(pink) and DDB1 (green) showing position of Vpr mutations(PDB: 5JK7). (D) Percentage of THP-1 cells in Figure 3F transduced by the vector encoding WT, or mutant, Vpr and GFP (MOI 1), orempty vector encoding GFP alone (MOI 1), and treated with cGAMP (5 μg/ml), or left untreated as a control. (E) Fold induction ofIFIT1-Luc after HT-DNA (5 μg/ml) transfection in cells expressing WT, or mutant, Vpr from a lentiviral vector (MOI 1), or emptyvector (MOI 1), or in untransduced IFIT1-Luc reporter THP-1 cells. (F) Percentage of THP-1 cells in Figure S3E transduced with HIV-1vector encoding WT, or mutant, Vpr and GFP (MOI 1), or empty vector encoding GFP alone (MOI 1), and transfected with HT-DNA(5 μg/ml) or left untransfected as a control. (G) Percentage of THP-1 cells in G2/M phase of cell cycle after transduction with anempty vector (MOI), or vector encoding WT Vpr, or mutant Vpr, (MOI 1) or left untransduced as a control. Mean+/- SEM n=2.Unless stated data are expressed as means ± SD (n = 3). Data is analysed using two-way ANOVA test. * (p<0.05), ** (p<0.01), ***(p<0.001), **** (p<0.0001) compared to empty vector. Data are representative of three (A), (D) or two (E-G) independentexperiments.

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Figure 4Wild Type Vpr, but not sensing antagonism inactive Vpr mutants, localise to nuclear pores(A) Immunofluorescence confocal projections of HeLa cells transfected with Flag-taggedWT, or mutant, Vpr encoded by pcDNA3.1plasmid (50 ng) and stained using antibodies detecting the Flag-tag (green) or nuclear pore complex (mab414) (red). 4ʹ,6-Diamidine-2ʹ-phenylindole dihydrochloride (DAPI) stains nuclear DNA (Blue). (B) Selected confocal images (z-section) of cells in (A)showing effect of Vpr mutation on Vpr colocalization with mab414 nuclear pore staining. (C) Assessment of colocalization of Vprwith mab414 nuclear pore staining. Scale bars represent 10 𝜇m.

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Control shRNA shNup358

C

Figure 4 figure supplement 1 Nup358 is not required for Vpr colocalization withmab414 nuclear pore staining(A) Immunofluorescence images of HeLa cells expressing a control, or Nup358 targeting, shRNA transfected with empty vector orFlag-tagged Vpr encoding pcDNA3.1 plasmid (50 ng) using antibodies detecting the Flag-tag (green) or the nuclear pore complex(mab414) (red). 4ʹ,6-Diamidine-2ʹ-phenylindole dihydrochloride (DAPI) stains nuclear DNA (Blue). (B) Selected confocal images (z-section) of cells in (A) showing effect of Nup358 depletion on colocalization of Vpr with mab414 nuclear pore staining (C)Immunoblot detecting Nup358, or actin as a loading control, from extracted Hela cells expressing a control, or Nup358 targeting,shRNA in cells from A. Size markers are shown (kDa). (D) Assessment of colocalization of Flag-tagged Vpr and mab414 stainednuclear pores in cells expressing a control, or Nup358 targeting, shRNA. Scale bars represent 10 𝜇m.

IRF3

Tra

nslo

catio

n co

effic

ient

1.0

0.5

0.0

-0.5

-1.0

no

Vpr -

WT

Vpr

Vpr

F34I

+P35

N

VprQ

65R

VprR

80A

HT-DNA: _ + _ + _ + _ + _ + _ + _

Lipof

ecta

min

e

**** ****

E

G

A B

F

CV

pr-V

LPV

pr+ VLP

unstimulated HT-DNA cGAMP

FSC

p-S396-IRF3

No Vpr - Vpr + No Vpr - Vpr +0

5

10

15

20

25

IFIT

1 lu

cife

rase

repo

rter

fold

act

ivat

ion - HT-DNA

+ HT-DNA

*

Luc reporter

unsti

mulated

HT-DNA

cGAMP

Lipofe

ctamine

only

0

10

20

30

p-S3

96-IR

F3 +

ve c

ells

(%)

Vpr- VLPVpr+ VLP

********

unsti

mulated

HT-DNA

cGAMP

Lipofe

ctamine

only

0

10

20

30

p-S3

96-IR

F3 +

ve c

ells

(%)

Vpr- VLPVpr+ VLP

********

Figure 5 Vpr inhibits IRF3 nuclear translocation(A) Immunoblot detecting Phospho-STING (Ser366), total STING, phospho-TBK1 (Ser172), total TBK1, phospho-IRF3 (Ser386), totalIRF3, or actin as a loading control, from extracted THP-1 cells expressing Vpr from a lentiviral vector (MOI 1), expressing emptyvector, or THP-1 left untransduced as a control and transfected with HT-DNA (5 μg/ml) or left untransfected as a control. Sizemarkers are shown in kDa. (B) Mean fold induction of IFIT1-Luc in cells from Figure 5A and Figure S5B (C) Flow cytometry plot(forward scatter vs pIRF3-S396 fluorescence) of THP-1 cells infected with Vpr bearing virus-like particles (VLP) lacking genome (1 RTU/ml), or Vpr free VLP, stimulated with cGAMP (5 μg/ml) or HT-DNA transfection (5 μg/ml). Lower panel shows the flow cytometrydata as a bar graph, plotting pIRF3-S396 positive cells. (D) Single cell immunofluorescence measurement of IRF3 nucleartranslocation in PMA differentiated THP-1 cells treated with cGAMP, or left untreated, and infected with HIV-1 GFP bearing Vpr,lacking Vpr or left untransduced. Cells were fixed and stained three hours after infection/transfection. Red line shows thetranslocation coefficient threshold. Blue lines represent mean translocation coefficient. (E) Percentage of cells in Figure 5Dwith IRF3translocation coefficient greater than 0.5 (above red line). (F) Single cell immunofluorescence measurement of IRF3 nucleartranslocation in PMA differentiated THP-1 cells stimulated with cGAMP (5 μg/ml), or left unstimulated, and infected with HIV-1 GFPlacking Vpr or bearing WT Vpr or Vpr mutants as shown (1 RT U/ml) or left uninfected. (G) Single cell immunofluorescencemeasurement of IRF3 nuclear translocation in PMA differentiated THP-1 cells transfected with HT-DNA (5 μg/ml), or leftuntransfected, and infected with HIV-1 GFP lacking Vpr, or bearing WT or mutant Vpr (1 RT U/ml) or left uninfected. Data in B isexpressed as means ± SEM (n = 2). Data is analysed using two-way ANOVA: * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001)compared to data from infection with HIV-1 lacking Vpr. Data are representative of three (C–G) or two (A, B) independentexperiments.

D

no Vpr

- Vpr

+ no

0

10

20

30

40

50

IRF3

pos

itive

nuc

lei (

%)

Vpr - Vpr +

+_ _ _ ++ ++ ++++cGAMP:

IRF3 nuclear translocation

no

Vpr -

WT

Vpr

Vpr

F34I

+P35

N

VprQ

65R

VprR

80A

IRF3

Tra

nslo

catio

n co

effic

ient

1.0

0.5

0.0

-0.5

-1.0cGAMP: _ + _ + _ + _ + _ + _ +

**** ****

cGAMP: _ + ++ + + + + + +

Vpr - Vpr +NoNo Vpr -

Vpr +

_ _

IRF3

Tra

nslo

catio

n co

effic

ient

1.0

0.5

0.0

-0.5

-1.0

****

-HT DNA +HT DNA

-42

-42

-84

-84

-50

-50

-42actin

STING

IRF3

pSTING

pIRF3

pTBK1

TBK1

No Vpr -

Vpr +

No Vpr -

Vpr +

IRF3

Tran

sloca

tion

coef

ficie

nt

1.0

0.5

0.0

-0.5

-1.0

No Vpr -

Vpr +

- LPS + LPS

No Vpr -

Vpr +

****

no in

fectio

nVpr

-

WT V

pr

VprF34

I+P35

N

VprQ65

R

VprR80

A0

10

20

30

IRF3

pos

tive

nucl

ei (%

) - cGAMP

+ cGAMP


no in

fectio

nVpr

-

WT V

pr

VprF34

I+P35

N

VprQ65

R

VprR80

A0

10

20

30

IRF3

pos

tive

nucl

ei (%

) - cGAMP

+ cGAMP

- HT DNA +HT DNA0

20

40

60

80

Tran

sduc

ed c

ells

(%) no infection

Vpr -

Vpr +

Infection

- HT DNA +HT DNA0

5

10

15

20

25

IFIT

1 lu

cife

rase

repo

rter

fold

act

ivat

ion

no infection

Vpr -

Vpr +

LUC reporter

- LPS + LPS0

2

4

6

8

10

IRF3

pos

tive

nucl

ei (%

) no infection

Vpr -

Vpr +

CA B

D E- HT DNA +HT DNA

0

20

40

60

80

100

Tran

sduc

ed c

ells

(%G

FP+v

e)

untransduced

empty vector

Vpr vector

Infection

- HT DNA +HT DNA0

20

40

60

80

Tran

sduc

ed c

ells

(%) no infection

Vpr -

Vpr +

- HT DNA +HT DNA0

5

10

15

IFIT

1 lu

cife

rase

repo

rter

fold

act

ivat

ion

untransduced

empty vector

Vpr vector

LUC reporter

- HT DNA +HT DNA0

20

40

60

80

Tran

sduc

ed c

ells

(%) no infection

Vpr -

Vpr +

F

G

no in

fectio

nVpr

-

WT V

pr

Vpr F34

I+P35

N

Vpr Q65

R

Vpr R80

A

Lipofe

ctamine

0

5

10

15

20

IRF3

pos

tive

nucl

ei (%

) - HT-DNA

+ HT-DNA

IRF3 nuclear translocationH

no in

fectio

nVpr

-

WT V

pr

Vpr F34

I+P35

N

Vpr Q65

R

Vpr R80

A

Lipofe

ctamine

0

5

10

15

20

IRF3

pos

tive

nucl

ei (%

) - HT-DNA

+ HT-DNA

I J

IRF3

Tra

nslo

catio

n co

effic

ient

1.0

0.5

0.0

-0.5

-1.0

no

Vpr -

WT

Vpr

Vpr

F34I

+P35

N

VprQ

65R

VprR

80A

Poly I:C: _ + _ + _ + _ + _ + _ + _

Lipof

ecta

min

e

**** ****

no in

fectio

nVpr

-

WT V

pr

Vpr F

34I+P

35N

Vpr Q

65R

Vpr R

80A

Lipofe

ctamine

0

5

10

15

20

25

IRF3

pos

tive

nucl

ei (%

) - polyI:C

+ polyI:C


no in

fectio

nVpr

-

WT V

pr

Vpr F

34I+P

35N

Vpr Q

65R

Vpr R

80A

Lipofe

ctamine

0

5

10

15

20

25

IRF3

pos

tive

nucl

ei (%

) - polyI:C

+ polyI:C

STING

IRF3

pSTING

pIRF3

pTBK1

TBK1

-42

-42

-84

-84

-50

-50

-HT DNA +HT DNA

No Vpr -

Vpr +

No Vpr -

Vpr +

- LPS + LPS0

2

4

6

8

10IR

F3 p

ostiv

e nu

clei

(%) no infection

Vpr -

Vpr +

- HT DNA +HT DNA0

20

40

60

80

Tran

sduc

ed c

ells

(%) no infection

Vpr -

Vpr +


Figure 5 figure supplement 1 Vpr inhibits IRF3 nuclear translocation(A) Percentage of THP-1 cells in Figure 5A transduced by HIV-1 GFP vector bearing Vpr , or HIV-1 GFP lacking Vpr, transfected withHT-DNA (5 μg/ml) or left untransfected as a control. (B) Immunoblot detecting Phosho-STING (Ser366), total STING, phospho-TBK1(Ser172), total TBK1, phospho-IRF3 (Ser386) or total IRF3 from extracted THP-1 cells expressing Vpr, empty vector or leftuntransduced as a control, and transfected with HT-DNA (5 μg/ml), or left untransfected as a control. Size markers are shown(kDa). (C) Fold induction of IFIT1-Luc in cells from gel in Figure 5A, expressing Vpr, or empty vector, and transfected with HT-DNA (5μg/ml) or left untransfected as a control. (D) Percentage of THP-1 cells from Figure S5B transduced by HIV-1 GFP bearing Vpr, orlacking Vpr, transfected with HT-DNA (5 μg/ml) or left untransfected as a control. (E) Fold induction of IFIT1-Luc in cells from secondexperiment (gel presented in Figure S5B) expressing Vpr, or empty vector, and transfected with HT-DNA (5 μg/ml) or leftuntransfected as a control. (F) Single cell measurement of IRF3 nuclear translocation in PMA differentiated THP-1 cells stimulatedwith LPS, or left unstimulated, and infected with HIV-1 GFP lacking Vpr or bearing Vpr (1 RT U/ml), or left uninfected (top panel).Percentage of cells with IRF3 translocation coefficient greater than 0.5 plotted as a percentage (bottom panel). (G) Percentage ofcells with IRF3 translocation coefficient greater than 0.5 plotted as a percentage from Figure 5F. (H) Percentage of cells with IRF3translocation coefficient greater than 0.5 plotted as a percentage from Figure 5G. (I) Single cell measurement of IRF3 nucleartranslocation in PMA differentiated THP-1 cells transfected with poly I:C, or left untransfected, and infected with HIV-1 GFP lackingVpr or bearing WT or mutant Vpr (1 RT U/ml), or left uninfected. (J) Percentage of cells with IRF3 translocation coefficient greaterthan 0.5 plotted as a percentage from Figure S5I.Data is analysed using two-way ANOVA test: * (p<0.05), ** (p<0.01), *** (p<0.001),**** (p<0.0001) compared to empty vector. Data are representative of three (F-K) or two (A-E) independent experiments.

A

Figure 5 figure supplement 2 Nuclear translocation of IRF3 after cGAMP stimulation in the presence or absence of Vpr(A) Representative immunofluorescence images showing IRF3 (red) nuclear translocation in PMA differentiated THP-1 cells treatedwith cGAMP, or left untreated, and infected with HIV-1 GFP bearing Vpr, or lacking Vpr, or left uninfected. 4ʹ,6-Diamidine-2ʹ-phenylindole dihydrochloride (DAPI) stains nuclear DNA (Blue). Scale bars represent 20 𝜇m.

actin

GFP

- 42

- 26

TNFα:

CMV-GFP Ub-GFP EF1α-GFP

___ +++ ___ +++ ___ +++

1 2 3

0

5

10

15

20

RT U/ml

NFk

B lu

cife

rase

repo

rter

fold

act

ivat

ion

0.03 0.1 0.3

HIV GFP -VprHIV GFP +Vpr HIV GFP -Vpr -genome

- + - + - + - + - + - +PolyI:C

WT Vpr

VprF34I+P35N

VprQ65R

VprR80A

No infection

Vpr -

0 5010020

0 0 5010020

0 0 5010020

0

0.01

0.1

1

GFP

exp

ress

ion

norm

alis

ed to

act

in (

AU

) CMV-GFP

EF1α-GFP

Ub-GFP

Vpr

untransduced empty vector WT Vpr0

5

10

15

20

IL-6

fold

indu

ctio

nno

rmal

ised

to G

APD

H

- cGAMP

+ cGAMP

****

1 2 3

1

10

100

RT U/ml

Infe

ctio

n (%

)


0.03 0.1 0.3

1 2 3

0

5

10

15

20

RT U/ml

NFk

B lu

cife

rase

repo

rter

fold

act

ivat

ion

0.03 0.1 0.3


E

F G

A C

D

Actin

GFP

Flag(Vpr)

Vpr: ---CMV-GFP EF1!-GFP Ub-GFP

- 42

- 27

- 11

emptyvector

GFP:

WTVp

r

VprF

34I+P35N

VprQ

65R

VprR

80A

+++++

GFP

Flag(Vpr)

Actin

- 11

- 27

- 42

10 2 0.4 10 2 0.4 10 2 0.40

2

4

6

8

Fold

GFP

exp

ress

ion

norm

alis

ed to

act

in (A

U) -TNFα

+TNFα

CMV-GFP(ng)

Ub-GFP(ng)

EF1α-GFP(ng)

Control cGAS KO

MAVS KO

0.1

1

10

100

CXC

L10

fold

indu

ctio

n no

rmal

ised

to G

APD


****

Control cGAS KO

MAVS KO

0.1

1

10

100

CXC

L10

fold

indu

ctio

n no

rmal

ised

to G

APD


****

BLUC reporter %GFP +ve cells IL-6

**** ****

empty

vecto

r

WT V

pr

VprF34

I+P35

N

VprQ65

R

VprR80

A0

50

100

GFP

exp

ress

ion

norm

alis

ed to

act

in (%

)

*

**

untransduced empty vector WT Vpr0

5

10

15

20

IL-6

exp

ress

ion

norm

alis

ed to

GAP

DH

- cGAMP

+ cGAMP

****

Figure 6 Vpr inhibits NF-κB p65 nuclear translocation and NF-κB sensitive plasmid expression(A) Fold induction of NF-kB-Luc after infection of THP-1 cells with HIV-GFP lacking Vpr, HIV-GFP bearing Vpr, or HIV-GFP lacking Vprand genome, at the indicated doses. (B) Percentage of THP-1 cells in (A). (C) Fold induction of IL-6 after activation of STING bycGAMP (5 μg/ml) in cells expressing empty vector or Vpr encoding vector (MOI 1), or in untransduced THP-1 cells. (D) Single cellimmunofluorescence measurement of NF-kB (p65) nuclear translocation in PMA differentiated THP-1 cells transfected with Poly I:C(50 ng/ml), or left untreated, and infected with HIV-1 GFP lacking Vpr, HIV-1 GFP bearing Vpr (1 RT U/ml) or left uninfected. Cellswere stained three hours after transfection and infection. (E) Immunoblot detecting Flag-Vpr, GFP, or actin as a loading control,from HEK293T cells transfected with 50 ng of empty vector, Flag-tagged WT Vpr vector, or Flag-tagged mutant Vpr vector, andCMV-GFP vector (50 ng). Size markers are shown in kDa. GFP expression from two independent immunoblots was quantified bydensitometry and is shown in the lower panel. (F) Immunoblot detecting Flag-Vpr, GFP, or actin as a loading control, from HEK293Tcells transfected with empty vector (200 ng) or Vpr vector (50ng, 100ng, 200ng) and CMV-GFP, EF1α-GFP or Ub-GFP plasmids (50ng). Size markers are shown in kDa. GFP expression quantified by densitometry is shown in the lower panel. (G) Immunoblotdetecting GFP, or actin as a loading control, from HEK293T cells transfected with CMV-GFP, EF1α-GFP or Ub-GFP plasmids (10 ng, 2ng, 0.4 ng) and stimulated with TNFα (200 ng/ml) or left unstimulated. Size markers are shown in kDa. GFP expression, from twoindependent immunoblots, quantified by densitometry, is shown in the lower panel. Data in (A, B, C) is expressed as mean ± SD (n =3). Data in (E, F, G) is expressed as mean ± SD (n=2). Two-way ANOVA: * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001)compared to empty vector or HIV GFP+Vpr.

Noin

fect

ion

Vpr -

Vpr +

- LPS + LPS

Noin

fect

ion

Vpr -

Vpr +

****

Plasmid Promoter

CSLW SFFV

CMV-Luc CMVimmediateearly

TK-Luc Thymidinekinase

M5P-Luc MLVLTR

CSLW CMV-Luc TK-Luc M5P-Luc105

106

107

108

109

Luci

fera

se e

xpre

ssio

n (R

LU)

Vpr 0ngVpr 50ngVpr 100ngVpr 200ng

A

G

C

- LPS + LPS0

20

40

60

80

p65

post

ive

nucl

ei (%

) no infection

Vpr -

Vpr +

- LPS + LPS0

20

40

60

80

p65

post

ive

nucl

ei (%

) no infection

Vpr -

Vpr +

p65 nuclear translocation

actin

GFP

- 42

- 26

TNFα:

CMV-GFP Ub-GFP EF1α-GFP

___ +++ ___ +++ ___ +++

10 2 0.4 10 2 0.4 10 2 0.40

2

4

6

Fold

GFP

exp

ress

ion

norm

alis

ed to

act

in (A

U) -TNFα

+TNFα

CMV-GFP(ng)

Ub-GFP(ng)

EF1α-GFP(ng)

10 2 0.4 10 2 0.4 10 2 0.40

2

4

6

Fold

GFP

exp

ress

ion

norm

alis

ed to

act

in (A

U) -TNFα

+TNFα

CMV-GFP(ng)

Ub-GFP(ng)

EF1α-GFP(ng)

F

Figure 6 figure supplement 1 Vpr inhibits NF-κB p65 nuclear translocation and NF-κB sensitive plasmid expression(A) Induction of luciferase reporter in HEK293T cells transfected with CSLW, CMV-Luc, TK-Luc or M5P-Luc (10ng), and empty vector,or Vpr encoding vector (50 ng, 100 ng, 200 ng). Table shows the promoters driving the luciferase reporter in each plasmid. (B)Percentage of cells in Figure 6D with translocation coefficient greater than 0.5. (C) Single cell measurement of NF-kB nucleartranslocation in PMA differentiated THP-1 cells stimulated with LPS, or left unstimulated, and infected with HIV-1 GFP lacking Vpr orbearing Vpr (1 RT U/ml), or left uninfected (top panel). Percentage of cells with NF-kB translocation coefficient greater than 0.5plotted as a percentage (bottom panel). Data is analysed using two-way ANOVA: * (p<0.05), ** (p<0.01), *** (p<0.001), ****(p<0.0001) compared to data from infection with HIV-1 lacking Vpr. (D) Quantification of GFP expression by densitometry for theimmunoblot in Figure 6E. (E) Immunoblot detecting flag-Vpr, GFP or actin as a loading control from HEK293T cells transfected withempty vector, flag-tagged WT Vpr encoding vector or flag-tagged mutant Vpr encoding vector and CMV-GFP vector or leftuntransfected. Size markers are shown in kDa. Quantification of GFP expression by densitometry for the immunoblot is shown below.(F) Quantification of GFP expression by densitometry for the immunoblot in Figure 6G. (G) Immunoblot detecting GFP, or actin as aloading control, from HEK293T cells transfected with CMV-GFP, EF1α-GFP or Ub-GFP plasmids (10 ng, 2 ng, 0.4 ng) and stimulatedwith TNFα (200 ng/ml) or left unstimulated. Size markers are shown in kDa. Quantification of GFP expression by densitometry for theimmunoblot is shown below.

D

empty

vecto

r

WT V

pr

VprF34

I+P35N

VprQ65

R

VprR80

A0

50

100

GFP

exp

ress

ion

norm

alis

ed to

act

in (%

)

empty

vecto

r

WT V

pr

VprF34

I+P35N

VprQ65

R

VprR80

A0

50

100

GFP

exp

ress

ion

norm

alis

ed to

act

in (%

)

Actin

GFP

Flag(Vpr)

-42

-27

-11

GFP: +++++

Eno

infec

tion

Vpr -

WT V

pr

Vpr F

34I+P

35N

Vpr Q

65R

Vpr R

80A

0

5

10

15

20

25

p65

post

ive

nucl

ei (%

) - poly (I:C)

+ poly (I:C)

B p65 nuclear translocation

no in

fectio

nVpr

-

WT V

pr

Vpr F

34I+P

35N

Vpr Q

65R

Vpr R

80A

0

5

10

15

20

25

p65

post

ive

nucl

ei (%

) - poly (I:C)

+ poly (I:C)

55 FLAG

70

35 25

GFP

KPNA1

KP

NA2

KPNA3

FLAG:

INPUT

GFP

KPNA1

KP

NA2

KPNA3

IP: HA

15 HA (Vpr)

Tubulin 55FLAG (KPNA1)

Vpr

cypB

HA (IRF3)

FLAG (KPNA1)

IP: F

LAG

WCL

HA (IRF3)

EV WT

Vpr

Vpr

F34I

+P35

N

HA-IRF3 +

FLAG-KPNA1

- 60

- 50

- 50

- 60

- 11- 22

- 60FLAG (KPNA1)(low exposure)

KPNA1

KPNA2

KPNA3

KPNA4

KPNA5

KPNA6

KPNB1

-60

-57

-57

-57

-60

-60

-90

Tubulin -50

B

C

A

Figure 7 HIV-1 Vpr interacts with karyopherins and inhibits IRF3/NF-κB(p65) recruitment to KPNA1(A) Immunoblot detecting KPNA1-6 or KPNB1 from extracted HEK293T cells infected with empty vector, or Vpr encoding vector at adose of 0.05 RT U/ml (MOI=2). Size markers are shown in kDa. Percentage infection by HIV-1 GFP bearing Vpr encoding or emptyvector is shown on the right. (B) Co-immunoprecipitation of Flag-KPNA1-3 and HA-Vpr. Input shows immunoblot detectingextracted HEK293T whole cell lysates expressing flag-KPNA1-3, flag-GFP and HA-Vpr before immunoprecipitation. Co-immunoprecipitation precipitates Vpr with HA-beads and detects Flag-KPNA1-3. (C) Co-immunoprecipitation of Flag-KPNA1-3 andWT HA-Vpr or HA-Vpr F34I+P35N. Input shows immunoblots detecting HA-Vpr or Flag-KPNA1-3 in extracted HEK293T whole celllysates (WCL) before immunoprecipitation. β-Actin is detected as a loading control. Co-immunoprecipitation precipitates Vpr withHA-beads and detects Flag-KPNA1-3. (D) Co-immunoprecipitation of HA-IRF3 and Flag-KPNA1 in the presence and absence of WTVpr or Vpr F34I+P35N to detect competition between Vpr and IRF3 for KPNA1. Input shows immunoblots detecting HA-IRF3 or Flag-KPNA1 or Vpr in extracted HEK293T whole cell lysates (WCL) before immunoprecipitation. CypB is detected as a loading control. Co-immunoprecipitation precipitates KPNA1 with Flag-beads and detects HA-IRF3 in the presence and absence of WT Vpr or inactiveVpr F34I+P35N. (E) Co-immunoprecipitation of HA-p65 and Flag-KPNA1 in the presence and absence of WT Vpr or Vpr F34I+P35N todetect competition between Vpr and p65 for KPNA1. Input shows immunoblots detecting HA-p65 or Flag-KPNA1 or Vpr in extractedHEK293T whole cell lysates (WCL) before immunoprecipitation. CypB is detected as a loading control. Co-immunoprecipitationprecipitates KPNA1 with Flag-beads and detects HA-p65 in the presence and absence of WT Vpr or Vpr F34I+P35N.

FLAG

HA (Vpr)

β-actin

FLAG

HA (Vpr)IP: HA

Input

EV KPNA

1

KPNA

2

KPNA

3

EV KPNA

1

KPNA

2

KPNA

3FLAG:

HA-WT VprHA-Vpr

F34I+P35N

- 60

- 14

- 60

- 14

- 42

FLAG

HA (Vpr)

β-actin

FLAG

HA (Vpr)IP: HA

Input

EV KPNA

1

KPNA

2

KPNA

3

EV KPNA

1

KPNA

2

KPNA

3

FLAG:

HA-WT VprHA-Vpr

F34I+P35N

- 60

- 14

- 60

- 14

- 42 FLAG (KPNA1)

Vpr

cypB

HA (p65)

FLAG (KPNA1)IP

: FLA

GW

CL

HA (p65)

EV

HA-p65+

FLAG-KPNA1

WT

Vpr

Vpr

F34I

+P35

N

- 60

- 65

- 65

- 60

- 11

- 22

FLAG (KPNA1)(low exposure)

- 60

empty vector

Vpr vector

0

20

40

60

80

100

GFP

exp

ress

ing

cells

(%) Infection

D

E

Figure 7 figure supplement 1 A unifying model of Vpr function(1) Stimulation of various PRRs results in activation of transcription factors such as IRF3 and NF-kB. To activate ISGs orproinflammatory genes expression, NF-kB and IRF3 translocate to the nucleus via the classical Karyopherin-a/b dependent nuclearimport pathway. (2) Nuclear import of a plasmid transfected into cellular cytoplasm is essential for gene expression. Transcriptionfactors such as IRF3 and NF-kB bind to their cognate response elements present in the promoter of the plasmid and allow nuclearimport via the classical karyopherin-a/b dependent pathway (Mesika et al., 2001) as well as transcription. (3) HIV-1 based vectorsdeliver genes to the nucleus in a karyopherin-a/b independent manner. Vpr localises to the nuclear pores and targets karyopherin-a dependent nuclear import in a DCAF1 E3 ubiquitin ligase dependent manner. This inhibits nuclear translocation of transcriptionfactors such as IRF3 and NF-kB and subsequent antiviral ISG expression. This also inhibits IRF3 and NF-kB dependent plasmidexpression or nuclear import but does not impact lentiviral gene delivery.

HIV-1 Vpr antagonizes innate immune activation by targeting ...

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