1

Activation of NFκB via Endosomal Toll-like Receptors 7 or 9 1

Contributes to Limiting Murine Herpes Virus 68 Reactivation 2

3

4

Florian Haasa,b,c, Kazuma Yamauchia,b,c,d, Monika Murata,b,c, Michele Bernasconia,b,c, 5

Noboru Yamanakad, Roberto F. Specke, and David Nadal a,b,c # 6

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a Experimental Infectious Diseases and Cancer Research, b Division of Infectious 8

Diseases and Hospital Epidemiology, c Children’s Research Center, University 9

Children’s Hospital of Zurich, University of Zurich, Zurich, Switzerland, 10

d Department of Otolaryngology-Head and Neck Surgery, Wakayama Medical 11

University, Wakayama, Japan, 12

e Division of Infectious Diseases and Hospital Epidemiology, University Hospital 13

Zurich, University of Zurich, Zurich, Switzerland. 14

15

Running title: TLR7 and TLR9 signaling limits lytic MHV-68 16

17

# Address correspondence to Dr. David Nadal, Division of Infectious Diseases and 18

Hospital Epidemiology, University Children's Hospital of Zurich, Steinwiesstrasse 75, 19

CH-8032 Zürich, Switzerland. Phone +41 44 266 7562; FAX +41 44 266 8072 20

e-mail: david.nadal@kispi.uzh.ch 21

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Word count: 23

Abstract: 249 24

Text: 5695 25

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JVI Accepts, published online ahead of print on 18 June 2014J. Virol. doi:10.1128/JVI.01486-14Copyright © 2014, American Society for Microbiology. All Rights Reserved.

2

ABSTRACT 27

In order to understand and possibly treat B-cell malignancies associated with latent 28

gamma-herpesvirus infection, it is vital to understand the factors that control the 29

balance between the two transcriptional states of gamma-herpesviruses: latency and 30

lytic replication. We used murine gamma-herpesvirus (MHV)-68 as a model system 31

to investigate how engagement of endosomal Toll-like receptors (TLRs) impacts on 32

reactivation from latency in vitro and on establishment of latent infection in vivo. We 33

found that treatment with TLR7 ligand R848 or TLR9 ligand CpG ODN suppresses 34

reactivation of MHV-68 in vitro. These suppressive effects correlated with the ability 35

to activate cellular transcription factor NFκB. Downregulation of TLR9 by RNA 36

interference in vitro led to a reduction of nuclear levels of NFκB p65 and 37

consequently to an increase of spontaneous reactivation in MHV-68 latently infected 38

cells, indicating that the TLR9 pathway contributes to limiting spontaneous 39

reactivation events. In vivo, sustained stimulation of TLR7 by repeated R848 40

treatment led to an increased frequency of infected splenocytes compared to mock-41

treated control. Frequencies of infected splenic B-cells in tlr7-/- or tlr9-/- mice after 42

establishment of latency did not differ from their wild-type counterpart. Nevertheless, 43

MHV-68-infected B-cells from tlr9-/- mice showed a higher frequency of reactivation 44

compared to B-cells from wild-type or tlr7-/- mice in ex vivo reactivation assays. Thus, 45

we show a suppressive effect of TLR7 or TLR9 triggering on MHV-68 reactivation 46

that correlates with NFκB activation and that the mere presence of functional TLR9 47

signaling pathway contributes to dampen lytic gamma-herpesvirus reactivation in 48

infected cells. 49

50

IMPORTANCE 51

3

A hallmark of gamma-herpesviruses is their establishment of latency in B-cells that is 52

reversible through lytic reactivation. Latency can result in B-cell malignancies. 53

Activation of the innate immune system is thought to contribute to controlling the 54

switch between the transcriptional states of latency and reactivation. Nevertheless, 55

the mechanisms involved are not clear. Here, we show that engagement of Toll-like 56

receptors (TLRs)7 and 9 suppress reactivation of murine gamma-herpesvirus MHV-57

68 in vitro and that stimulation of TLR7 in vivo increases the frequency of infected 58

cells. TLRs 7 and 9 are innate immunity sensors of nucleic acids localized in 59

endosomes. Additionally, we demonstrate that impairment of TLR9 signaling in 60

latently infected B-cells leads to increased reactivation. Thus, activated endosomal 61

TLR7 and TLR9 pathways play an important role in promoting establishment of latent 62

gamma-herpesvirus infection. Counteracting signaling of these pathways allows for 63

reactivation and could represent treatment targets in gamma-herpesvirus-associated 64

malignancies. 65

66

4

INTRODUCTION 67

Gamma-herpesviruses are double-stranded DNA B-lymphotropic viruses capable of 68

establishing life-long latent infections. Epstein-Barr virus (EBV) is the most prominent 69

human gamma-herpesvirus with a seroprevalence in the adult population of over 70

90%. Even though latent infection with EBV is usually asymptomatic, it is associated 71

with several B-cell malignancies including endemic Burkitt’s lymphoma (BL) (1). 72

Notably, the incidence of endemic BL is restricted to areas with holoendemic chronic 73

infection with the malaria parasite Plasmodium falciparum, suggesting a role for co-74

infections in the etiology of this particular EBV-associated tumor entity (2, 3). The 75

mechanism of how chronic malaria impacts on usually asymptomatic gamma-herpes-76

virus infection is not known. 77

An important hallmark of gamma-herpesviruses is sporadic reactivation from 78

latent infection with subsequent production and release of infectious viral particles 79

(4). Sporadic reactivation is essential to ensure transmission to a new host but needs 80

to be tightly controlled since viral gene products represent targets for host immunity 81

(5). Intrinsic and extrinsic factors are thought to control lytic reactivation of gamma-82

herpesviruses. Activation of the innate immune system, in particular signaling via 83

Toll-like receptors (TLRs) has been shown to be an important factor in the balance 84

between latency and lytic reactivation (6-9). TLRs are a family of pattern-recognition 85

receptors that play a central role in innate immunity (10). How TLR signaling impacts 86

on gamma-herpesvirus infection is particularly relevant to understanding the potential 87

role of P. falciparum malaria in endemic BL since chronic P. falciparum infection 88

provides constant stimulation of endosomal TLRs (11, 12). 89

In mammals, four members of the TLR family (TLR3, TLR7, TLR8, and TLR9) 90

are expressed almost exclusively in intracellular compartments (10), where they 91

function as sensors for microbial nucleic acids (13). The natural ligand of TLR3 is 92

5

double-stranded RNA while TLR7 senses single-stranded RNA, and TLR9 detects 93

unmethylated DNA containing CpG motifs. TLR8 is structurally closely related to 94

TLR7 and independently recognizes single-stranded RNA (14) but is thought to be 95

biologically inactive in mice, having instead a regulatory function in modifying 96

expression and signaling of TLR7 (15). Subsequent to ligation of the receptor, 97

signaling is forwarded via the recruitment of specific Toll/IL-1 receptor (TIR)-domain 98

containing adaptor proteins, myeloid differentiation primary response protein 88 99

(MyD88) in the case of TLR7 as well as TLR9 (16) and TIR-domain containing 100

adaptor inducing IFN-β (TRIF) in the case of TLR3 (17). Eventually, signaling via 101

TLRs 3, 7, and 9 leads to activation of the nuclear factor-κB (NFκB) axis which 102

triggers pro- and anti-inflammatory cytokines (18). Importantly, activation of NFκB 103

has been demonstrated to be crucial for the establishment and maintenance of latent 104

gamma-herpesvirus infection in distinct ways. First, high levels of NFκB subunit p65 105

inhibit activation of lytic gene promoters of several gamma-herpesviruses (19, 20) 106

and second, recombinant murine gamma-herpesvirus 68 (MHV-68) expressing the 107

constitutively active form of the NFκB-inhibitor IκBα is impaired in its ability to 108

establish latent infection in vivo (21). Controversially, triggering of TLR3 or TLR9 was 109

found to induce reactivation of MHV-68 but not triggering of TLR7 (6). This is 110

remarkable since TLR7 and TLR9 share the signaling pathway and TLR3, TLR7, and 111

TLR9 are canonical activators of NFκB. Thus, additional investigation of this issue 112

are required to precisely assess how activation of TLR signaling pathways impacts 113

gamma-herpesvirus reactivation. 114

Here, we used MHV-68 to study the effect of endosomal TLR triggering and 115

activation of NFκB on the expression of lytic viral genes and shedding of viral 116

particles in vitro and on the establishment of latent infection in vivo. MHV-68 is 117

commonly accepted as a model system to investigate gamma-herpesvirus infection 118

6

in vivo, since it is highly homologous to the human gamma-herpesviruses EBV and 119

Kaposi sarcoma-associated herpesvirus (KSHV) (22). We aimed at advancing the 120

mechanistic understanding of the impact of TLR stimulation on lytic reactivation from 121

latent infection using a cell line harboring latent MHV-68. Further, we tested whether 122

TLR7 or TLR9 signaling contributes to NFκB activation in latently MHV-68-infected 123

cells and if it impacts on viral behavior in vitro and in vivo. 124

125

MATERIAL AND METHODS 126

Ethics Statement. All animal experiments were done according to the guidelines of 127

the Animal Welfare Act provided by the Swiss Federal Veterinary Office 128

(www.bvet.admin.ch), and the veterinarian authorities of the Canton of Zurich, 129

Switzerland approved all procedures (License Nr. 100/2012). 130

131

Cell culture and MHV-68 viral production. The MHV-68-infected murine B-cell line 132

S11 (courtesy of Prof. Ren Sun, UCLA, CA) was cultured in RPMI 1640 medium 133

supplemented with 10% heat-inactivated fetal calf serum (FCS), 1% penicillin-134

streptomycin and 2mM L-glutamine; baby hamster kidney cells (BHK-21) (23) 135

(courtesy of Prof. A. Nash, University of Edinburgh, UK) were cultured in Glasgow 136

minimal essential medium (GMEM) supplemented with 10% Tryptose Phosphate 137

Broth, 10% heat-inactivated FCS and 1% penicillin-streptomycin; murine fibroblast 138

cells NIH3T12 (courtesy of Prof. S. Speck, Emory Vaccine Center, GA) were cultured 139

in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-140

inactivated FCS, 1% penicillin-streptomycin and 2mM L-glutamine (all reagents from 141

Invitrogen, Basel, Switzerland). 142

Wild type MHV-68 clone g2.4 (24) was grown in BHK-21 cells (25). Cells and 143

supernatant were harvested at 7 dpi, centrifuged at 1,400 rpm for 5 min, and the 144

7

supernatant was passed through a TPP PES 0.22μm filter (Omnilab, Mettmenstetten, 145

Switzerland) and stored at -80°C. MHV-68 titers were determined by plaque assay on 146

BHK-21 cells. 147

148

Mice. C57BL/6 mice were purchased from Harlan (The Netherlands). Tlr7-/- and tlr9-/- 149

mice on a C57BL/6 background were obtained from the Swiss Immunological Mutant 150

Mouse Repository (SwiMMR, Zurich, Switzerland). All animals were kept in a specific 151

pathogen free environment. 152

153

Detection of tlr gene expression. Gene expression of tlrs was determined by 154

reverse transcription PCR using gene specific primers as described in (26) and 155

gapdh gene expression was used as a loading control. 156

157

TLR triggering. S11 cells were resuspended at 1×106 cells/ml of supplemented 158

RPMI and stimulated with 25 μg/ml Poly(I:C), 3 μM of R848 or 0.5 μM of CpG ODN 159

1826 (Invivogen, San Diego, CA), 2 hours prior to stimulation with 10 ng/ml of 12-O-160

tetradecanoylphorbol-13-acetate (TPA; Sigma-Aldrich, Buchs, Switzerland). 24 hours 161

after treatment, cell pellets were assayed for MHV-68 lytic gene expression by qPCR 162

and supernatants were tested for MHV-68 production by plaque assay (see below). 163

164

Reverse transcription and quantitative PCR. Expression of MHV-68 lytic gene 165

ORF50, was determined by quantitative polymerase chain reaction (qPCR) using ABI 166

7900HT Fast Real-Time PCR System (Applied Biosystems, Rotkreuz, Switzerland). 167

Total RNA was extracted from cells using RNeasy Mini Kit (Qiagen, Hombrechtikon, 168

Switzerland) and contaminating DNA was removed by DNAse treatment (DNA-free 169

Ambion; Applied Biosystems, Rotkreuz, Switzerland). cDNA was synthesized using 170

8

High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Amplification of 171

synthesized cDNA was performed using TaqMan® Gene Expression Master Mix 172

(Applied Biosystems). The primers and probe used for MHV-68 ORF50 were as 173

follows: ORF50_fw 5’-CCAACGTGTTCCCAGAAC-3’; ORF50_rev 5’-174

CGATGAACGCGTCCTCAG-3’; ORF50_probe FAM-175

TACTCAGGAAGCGTGTCCCGGATCA-BHQ-1. Primers and probes for tlr7 176

(Mm00446590_m1), tlr9 (Mm00446193_m1), ifnb1 (Mm00439552_s1), il-6 177

(Mm99999064_m1), il-10 (Mm00439614_m1) and gapdh (Mm99999915_g1) were all 178

from Applied Biosystems. 179

180

Plaque assay. MHV-68 titers in supernatant of S11 cells were determined by plaque 181

assay as described (25). BHK-21 cells were plated onto 6-well plates at 2×105 182

cells/well one day prior to infection. Serially diluted viral supernatants were placed 183

onto monolayers and incubated for 1h at 37oC in 5% CO2. Supernatants were then 184

removed and replaced with complete GMEM containing 1% methyl cellulose (Sigma-185

Aldrich). After 3-4 days, the monolayers were fixed with methanol, stained with 186

neutral red solution (Sigma-Aldrich) and the numbers of plaques were counted. 187

188

Preparation of nuclear extracts. 2×106 cells were washed with Tris-buffered saline 189

(TBS) and cell pellet was resuspended in 800 μl cold cell lysis buffer (10 mM HEPES 190

pH 7.9; 10 mM KCl; 0.1 mM EDTA; 0.1 mM EGTA; 1 mM DTT; 0.5 mM PMSF), kept 191

at 4oC for 15 min, and vortexed for 10 seconds after 50 μl of a 10% solution of 192

Nonidet P-40 (Roche, Mannheim, Germany) was added. The homogenate was 193

centrifuged at 10'000×g for 30 seconds; the nuclear pellet was re-suspended in 100 194

μl ice-cold nuclear lysis buffer (20 mM HEPES pH 7.9; 0.4 M NaCl; 0.1 mM EDTA; 195

0.1 mM EGTA; 1 mM DTT; 0.5 mM PMSF) and rocked at 4oC for 15 min on a 196

9

shaking platform. The nuclear extract was centrifuged at 10'000×g for 5 min at 4oC 197

and the supernatant was stored at -80oC. 198

199

Western blot analysis. Nuclear extracts were analyzed by SDS-PAGE on a 200

NuPAGE 10% Bis-Tris gel (Invitrogen). The proteins were transferred onto 201

PROTRAN Nitrocellulose Transfer Membrane (Whatman, Kent, UK) and probed with 202

specific antibodies against NFκB p65 (sc-8008, Santa Cruz Biotechnology, Inc., 203

Santa Cruz, CA), PCNA (sc-25280; Santa Cruz) and beta-actin (Cat. No. 4967, Cell 204

Signaling Technology, Beverly, MA). 205

206

Detection of spliced variant of ORF73. RNA isolation and cDNA synthesis were 207

performed as described above. 2 μl of each cDNA reaction was used as template in 208

a nested PCR reaction specific for ORF73 spliced transcripts as described (27), and 209

products of the reaction were separated on a 1.5% agarose gel. cDNA from S11 cells 210

was used as a positive control, and cDNA generated from uninfected splenocytes 211

was used as a negative control. 212

213

Mice infection, R848 treatment, and limiting-dilution nested PCR. Mice were 214

infected by injecting 1×105 pfu of MHV-68 i.p. in 100μl PBS. Daily treatment was 215

done with 1mg/kg body weight R848 in 100μl PBS based on previous work (28), or 216

PBS alone by i.p. injection for 19 days. At 20 dpi, mice were sacrificed by inhalation 217

of CO2. Whole spleens were harvested, transferred to ice-cold PBS and passed 218

through a 70 μm nylon mesh cell strainer (BD Falcon, BD Biosciences, Chicago, IL). 219

Erythrocytes were removed by ammonium chloride lysis solution (Stemcell 220

Technologies, Grenoble, France). The frequency of MHV-68 genome-positive cells 221

was determined by a previously published method (29). Nonlinear regression curve 222

10

was fit using Prism 6.0b software (GraphPad Software, La Jolla, CA). Frequencies 223

were calculated as the splenocyte number at which 63.2% of reactions were positive 224

based on Poisson distribution. 225

226

TLR downregulation by RNA interference. S11 cells were transfected with 227

psiRNA-h7SK plasmids expressing shRNA targeting tlr7, tlr9, or Luciferase gene, 228

respectively, as well as green-fluorescent protein (GFP) and Zeocin resistance from 229

a separate promoter (Invivogen). Transfection was performed using NEON® 230

Transfection System (Invitrogen) according to the manufacturers instructions. 231

Transfected cells were selected in complete RPMI containing 200 μg/ml Zeocin 232

(Invivogen) and successful selection was confirmed by flow cytometry. 233

234

Ex vivo MHV-68 reactivation assay. Mice were sacrificed using a CO2 chamber 235

and splenocytes were recovered as described above. B-cells were isolated by 236

negative selection using a murine B-cell isolation Kit (Miltenyi Biotec, Bergisch 237

Gladbach, Germany). Purified B-cells were counted and resuspended in a two-fold 238

dilution series starting at 1×106 cells/ml. Dilutions were plated on monolayers of 239

NIH3T12 murine fibroblasts in a 96-well plate, 100 μl/well and 12 wells per dilution. 240

As a control, uninfected B-cells were plated in 12 wells at 1×105 cells per well. To 241

control for preformed virus particles, infected B-cells were flash-frozen in liquid 242

nitrogen and thawed in a 37°C water bath to disrupt cells. Serial dilutions of disrupted 243

cells were plated on NIH3T12 monolayers analogous to intact cells. 14 days later, 244

wells were inspected for cytopathic effect by microscopy and the frequency of 245

reactivation was calculated based on Poisson distribution. 246

247

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Statistical analysis. If not stated otherwise, data were statistically analyzed with the 248

unpaired t-test using Prism 6.0b software (GraphPad Software). P values below 0.05 249

were considered statistically significant. 250

251

RESULTS 252

Stimulation of endosomal TLR7 and TLR9 but not of TLR3 causes nuclear 253

accumulation of NFκB p65 in S11 cells 254

To investigate the effects of TLR triggering on latent gamma-herpesvirus infection in 255

vitro, we used the latently MHV-68-infected murine B-cell line S11. S11 cells 256

originate from a lymphoma harboring MHV-68 in a BALB/c mouse. Each S11 cell 257

carries reactivation competent MHV-68 (30). 258

First, we verified that S11 cells express TLR3, TLR7, and TLR9 using reverse 259

transcription PCR (Fig. 1A). To confirm activation of NFκB upon TLR triggering, we 260

treated S11 cells with ligands to TLR3, TLR7, TLR9 and NFκB inhibitor Bay 11-7082, 261

respectively, and assessed subsequent nuclear accumulation of NFκB subunit p65 at 262

different time points by Western blotting (Fig. 1B). To ensure that Bay 11-7082 is 263

able to inhibit activation of NFκB, we pretreated cells with the inhibitor 2 hours prior to 264

stimulation with R848 or CpG ODN. 265

Poly(I:C) did not lead to any accumulation of NFκB p65 in the nucleus over the 266

course of 48h. Conversely, R848 that triggers TLR7 and CpG ODN 1826, which 267

triggers TLR9 led to activation of NFκB already 1h after stimulation. S11 cells treated 268

with R848 or CpG ODN 1826 showed stronger nuclear accumulation of NFκB p65 269

compared to untreated cells for at least 12h after treatment. By contrast, NFκB 270

inhibitor Bay 11-7082 reduced nuclear NFκB p65 to minimal levels 1h after treatment 271

(Fig. 1B) and for at least 12h. Bay treatment did not fully inhibit nuclear translocation 272

of NFκB upon TLR7 or TLR9 triggering (Fig. 1B, lanes 6 and 7) but markedly reduced 273

12

nuclear NFκB p65 compared to stimulated cells (Fig. 1B, lanes 4 and 5). 24h after 274

treatment, nuclear levels of NFκB seemed to have normalized; the week bands in the 275

first 2 lanes are likely due to suboptimal blotting rather than reduced activity. 276

To verify signaling competence, we measured the induction of cytokine 277

expression in response to TLR ligands by qPCR (Fig. 1C). TLR3 ligand Poly(I:C) 278

robustly induced expression of IFN-β1 (Fig. 1C, top panel), consistent with activation 279

of Interferon regulatory factor 3 (IRF3) pathway (17) while R848 and CpG ODN 1826 280

did not. On the other hand, R848 and CpG ODN 1826 induced expression of the 281

cytokines IL-6 (Fig. 1C, middle panel) and IL-10 (Fig. 1C, bottom panel) while 282

Poly(I:C) did not, consistent with the observed differences in NFκB activation. 283

These results demonstrate that endosomal TLRs activate distinct signaling 284

pathways resulting in differential induction of transcription factors in the S11 model 285

cells. While the stimulation of TLR7 and TLR9 leads to activation of NFκB and 286

subsequent induction of IL-6 and IL-10, stimulation of TLR3 does not activate NFκB 287

but rather induces expression of IFN-β, probably via IRF3 transcription factor. 288

289

Stimulation of TLR7 and TLR9 but not TLR3 suppresses both spontaneous and 290

induced MHV-68 lytic reactivation in S11 cells 291

A fraction of MHV-68 latently infected S11 cells constantly undergoes spontaneous 292

lytic reactivation as evidenced by expression of ORF50, the initiator of lytic replication 293

(31), as well as by shedding of infectious MHV-68 particles into the supernatant. The 294

frequency of reactivation of MHV-68 can be increased by treatment with 12-O-295

tetradecanoyl-13-phorbolacetate (TPA) (30). 296

To monitor the impact of endosomal TLR triggering on MHV-68 reactivation, 297

S11 cells were treated with Poly(I:C), R848, and CpG ODN 1826, respectively, for 298

24h before analyzing mRNA expression levels of immediate-early lytic gene ORF50 299

13

by qPCR (Fig. 2A, black bars) as well as measuring infectious MHV-68 particles in 300

culture supernatants by plaque assay (Fig. 2A, white bars). Treatment with 25 µg/ml 301

Poly(I:C) did not change expression of ORF50 nor the release of infectious particles. 302

Treatment with 3 µM R848 or 0.5 µM CpG ODN 1826, however, decreased ORF50 303

expression compared to the mock-treated control. To ensure suppression of the 304

entire MHV-68 lytic transcription program, we also analyzed expression of early lytic 305

gene ORF21 and late lytic gene M7 (32) by qPCR, which followed the pattern of 306

ORF50 expression in all samples (data not shown). Consequently, the titer of 307

infectious MHV-68 in the supernatant of cells treated with R848 or CpG ODN was 308

reduced by about 50% compared to the mock-treated control. Even though the 309

magnitude of the observed effects was modest, they were reproducible in multiple 310

independent experiments and statistically significant. 311

Addition of 10 ng/µl TPA to culture medium for 24h increased mRNA levels of 312

ORF50 about 4-fold and MHV-68 particle production about 2-fold compared to 313

untreated control. When the cells were treated with R848 or CpG ODN 1826 2h 314

before addition of TPA, the pro-lytic effect was abolished. These results indicate that 315

signaling by TLR7 and TLR9 suppresses both spontaneous as well as induced 316

reactivation of MHV-68 while TLR3 signaling does not. 317

Next, we wanted to see whether the suppressive effect of TLR7 and TLR9 318

stimulation correlates with their ability to activate NFκB. To this end, we treated S11 319

cells with 1 µM NFκB inhibitor Bay 11-7082 2h before stimulation with TLR ligands 320

and analyzed lytic reactivation 24h later (Fig. 2B). We found that pre-treatment with 321

Bay 11-7082 completely abolished the suppressive effect of CpG ODN 1826 and 322

partially abolished the effect of R848, indicating that the impact of TLR triggering on 323

lytic replication is at least partially dependent on NFκB activation. In the absence of 324

TLR stimulation, the treatment with Bay 11-7082 led to a slight increase in lytic 325

14

reactivation compared to mock-treated control, in agreement with earlier reports that 326

Bay 11-7082 provokes reactivation of EBV or KSHV from latently infected B-cells in 327

vitro (19, 33). Treatment with TPA increased both lytic gene expression as well as 328

MHV-68 particle production as seen before. 329

330

Stimulation of TLR7 in vivo leads to increased frequency of infected 331

splenocytes after intraperitoneal injection of MHV-68 332

Based on our in vitro data, and since NFκB activation was shown to be important for 333

the establishment of latent MHV-68 infection in vivo (21), we hypothesized that 334

TLR7-mediated activation of NFκB would promote the establishment of MHV-68 335

latency in vivo in a similar fashion as shown for TLR9 (6). 336

To test this hypothesis, we infected 10 C57BL/6 mice intraperitoneally (i.p.) 337

with 1×105 pfu MHV-68. We chose i.p. infection since this route seeds MHV-68 338

directly to the spleen without the need of amplification through lytic replication in the 339

respiratory tract (34), therefore allowing the virus to reach the main site of latent 340

infection directly. One day post inoculation (dpi) the mice were segregated into two 341

groups and treated daily with either 1 mg/kg R848 in 100 μl PBS or PBS alone 342

(mock) i.p. for 19 days. Mice were sacrificed at 20 dpi, a time when lytic MHV-68 343

infection has been cleared from the spleen and predominantly latent infection can be 344

expected (34). 345

To investigate whether treatment with R848 following inoculation with MHV-68 346

facilitates establishment of latent infection we isolated splenocytes from mice at 20 347

dpi and subjected them to analysis by limiting dilution nested PCR targeting MHV-68 348

gene v-cyclin (ORF72). We found that splenocytes from mice treated daily with R848 349

showed a 7-fold higher frequency of cells positive for MHV-68 genome than 350

splenocytes from mice mock-treated with PBS (1 in 100 vs. 1 in 750; Fig 3A). 351

15

To confirm latent MHV-68 infection, we tested the splenocytes for expression 352

of spliced variant of ORF73 (mLANA), a transcript associated with MHV-68 latency 353

(27), by reverse transcription PCR (Fig. 3B). At 20 dpi, ORF73 spliced transcripts 354

were detected in all samples from mice treated with R848 but inconsistently in 355

samples from mock treated mice, reflecting the lower frequency of splenocytes 356

harboring latent MHV-68. To ensure that the higher frequency of MHV-68 DNA 357

copies detected by nested PCR (Fig. 3A) represent latent infection and not persistent 358

or secondary lytic replication, we assayed homogenates of disrupted splenocytes 359

from infected mice for the presence of infectious MHV-68. To disrupt splenocytes, the 360

cells were flash-frozen in liquid nitrogen and thawed in a 37°C water bath, a process 361

that kills >99% of cells as judged by Trypan Blue exclusion assay. Disrupted 362

splenocytes were plated onto susceptible epithelial cell line NIH3T12 in a limiting 363

dilution assay and the plates were scored for cytopathic effect after 14 days. The 364

amount of cytopathic effect observed was negligible in both treated and mock-treated 365

cells (Fig. 3C). To test whether the process of flash-freezing used for cell disruption 366

might impact on the infectiousness of viral particles, we tested the method with two 367

separate stocks of cell-free virus and analyzed viral titers before and after freezing by 368

plaque assay (Fig. 3D). Flash-freezing in liquid nitrogen slightly reduced the mean 369

titer of cell-free virus stocks (by 27% and 23%, respectively), but did not abolish 370

infectiousness completely. We therefore conclude that lytic replication is absent in 371

splenocytes of both R848-treated and control samples and that viral genome 372

amplified by nested PCR in Fig. 3A stems from latent infection. Thus, TLR7 triggering 373

during primary infection promotes the establishment of latency and increases the 374

reservoir size of latent MHV-68. 375

376

16

Silencing of TLR9 but not of TLR7 leads to decreased levels of nuclear NFκB 377

and enhanced lytic reactivation in S11 cells 378

Latently MHV-68-infected S11 cells show a basal level of NFκB activity (Fig. 1B). 379

Since nucleic-acid binding endosomal TLRs have been implicated in the sensing of 380

various viral infections and TLR7 and TLR9 both have the ability to activate NFκB, 381

we aimed at investigating whether the basal activity of NFκB is mediated by TLR7 or 382

TLR9 signaling in latently MHV-68-infected cells. 383

To assess the roles of TLR7 and TLR9 in this context, S11 cells were 384

transfected with plasmids expressing shRNA against TLR7, TLR9, or luciferase as 385

control. Antibiotic selection yielded bulk stable cell lines, and >95% of the cells 386

expressed the GFP cassette included in the vector containing the shRNA as 387

measured by flow cytometry (data not shown). Stably transfected cells showed a 388

reduction in TLR7 mRNA of 70% and a reduction of TLR9 mRNA of 55%, 389

respectively, as measured by qPCR (Fig. 4A). S11 cells genetically complemented 390

with shRNA against luciferase (shLuc) did not show any effect on TLR expression 391

and served as control. 392

Levels of NFkB p65 were similar in cells with reduced expression of TLR7 as 393

in parental S11 cells or the control cell line shLuc (Fig. 4B). In cells with reduced 394

TLR9 expression, however, lower levels of nuclear NFκB p65 were detected. We 395

therefore hypothesized that latent MHV-68 infection causes a constant low-level 396

activation of TLR9 but not TLR7 signaling pathway culminating in the nuclear 397

translocation of NFκB p65, thereby contributing to a basal NFκB activation. 398

Since in previous experiments a reduction in nuclear NFκB p65 by the inhibitor 399

Bay 11-7082 resulted in increased reactivation and MHV-68 production (Fig. 2B), we 400

tested whether the bulk stable cell lines silenced for TLR7 or TLR9 differed in their 401

propensity to spontaneous reactivation compared to the parental cell line. 402

17

Quantification of lytic viral gene expression by qPCR and of infectious MHV-68 403

production by plaque assay revealed a 2-fold increase in expression of ORF50 (Fig. 404

4C, black bars) and a 2-fold increase in the concentration of viral particles in the 405

supernatant (Fig. 4C, white bars) of cell lines silenced for TLR9 but not of cell lines 406

silenced for TLR7 or of the control cells expressing shRNA against luciferase. 407

These results suggest a contribution of continuous TLR9 signaling to basal 408

NFκB activation levels and suppression of spontaneous reactivation. 409

410

Lack of TLR9 does not impact on establishment of latent infection in vivo but 411

favors lytic reactivation of primary B-cells ex vivo 412

Since activation of the TLR7 or TLR9 pathways support the establishment and 413

maintenance of latent MHV-68 infection in splenocytes and since MHV-68 infection 414

itself might promote latency by triggering TLR9, we hypothesized that lack of TLR9 415

would result in a reduced latent reservoir of MHV-68. Thus, we infected i.p. wild-type 416

(wt), tlr7-/-, and tlr9-/- mice (n=15 for each genotype) with MHV-68 as in experiments 417

mentioned above. None of the mice showed signs of distress or illness throughout 418

the experiment. All animals were sacrificed at 34 dpi. 419

We did not find significant differences in the frequency of infected B-cells 420

between any of the three groups (wt: 1 in 2,600; tlr7-/-: 1 in 2,300; tlr9-/-: 1 in 1,900) 421

performing limiting-dilution nested PCR on purified B-cells (Fig 5A), indicating that 422

TLR signaling is dispensable for the establishment of latency. Next, we assessed ex 423

vivo reactivation of B-cells by limiting-dilution reactivation assay on NIH3T12 424

fibroblasts and in parallel we tested for the presence of preformed infectious virus by 425

plating disrupted cells (Fig. 5B). After 14 days, the numbers of reactivation events 426

were minimal in cells from both wt and tlr7-/- mice, indicating poor reactivation 427

capability of latently infected B-cells. In contrast, reactivation was markedly increased 428

18

in cells from tlr9-/- mice, in line with both our in vitro results from silencing TLR7 or 429

TLR9 in S11 cells as well as with the findings reported by Guggemoos et al. (35), 430

who found higher titers of lytic MHV-68 in tlr9-/- mice than in wt mice after i.p. 431

infection. Preformed infectious virus was absent in both wt and tlr7-/- cells and barely 432

detectable in tlr9-/- cells. Thus, it seems that impaired TLR9 signaling renders MHV-433

68-infected B-cells more likely to reactivate whereas impaired TLR7 signaling does 434

not. 435

436

DISCUSSION 437

Activation of the innate immune system via TLRs largely impacts on gamma-438

herpesvirus latency and reactivation. We here studied the effect of endosomal TLR 439

stimulation on latently MHV-68-infected cells in vitro and on the establishment of 440

gamma-herpesvirus infection in vivo using the MHV-68 mouse model. We found that 441

(i) signaling of TLR7 or TLR9 but not of TLR3 inhibits both spontaneous and induced 442

reactivation in the latently MHV-68-infected S11 cells and that this effect is, at least 443

partially, dependent on the induction of NFκB; (ii) stimulation of TLR7 during infection 444

increases the number of latently infected splenocytes; (iii) downregulation of TLR9 445

but not of TLR7 reduces basal NFκB activity and increases spontaneous MHV-68 446

reactivation in latently infected cells; and (iv) while the lack of TLR7 or TLR9 does not 447

impact on the frequency of latently infected B-cells in vivo, MHV-68-infected B-cells 448

from tlr9-/- mice show increased propensity to reactivate ex vivo. Our results 449

unprecedentedly show that activation of NFκB via TLR7 signaling profoundly impacts 450

the establishment and maintenance of latent MHV-68 infection, as is the case for 451

TLR9 signaling. In the absence of external ligands, constant activation of TLR9 452

caused by persistent MHV-68 infection might contribute to robust activation of NFκB, 453

sufficient to enable gamma-herpesviral latency. 454

19

Our in vitro experiments using S11 cells as a model for established MHV-68 455

latency in B-cells invariably showed that activation of the TLR9 pathway suppresses 456

both spontaneous and induced MHV-68 reactivation (Fig. 2A). It seems likely that this 457

feature is shared by other gamma-herpesviruses, as previous studies in our lab 458

demonstrated that stimulation of TLR9 similarly leads to a suppression of EBV’s 459

master regulator lytic gene BZLF1 in Burkitt’s lymphoma cells (8, 9). Our finding that 460

signaling via TLR7 suppresses MHV-68 reactivation is unprecedented. It seems 461

plausible that TLR7 and TLR9 would have a comparable effect on MHV68 when 462

stimulated, since they share a signaling pathway via the common adaptor molecule 463

MyD88. Upon stimulation of either receptor we observed comparable activation of 464

NFκB (Fig. 1B). NFκB was shown to suppress the lytic gene promoters of MHV-68 as 465

well as of the human gamma-herpesviruses EBV or KSHV (19, 20), which supports 466

the hypothesis that downregulation of lytic MHV-68 upon TLR7 or TLR9 stimulation in 467

S11 cells is due to activation of NFκB. Indeed, in our experiments NFκB inhibitor Bay 468

counteracts the suppression of viral reactivation by TLR ligands (Fig. 2B). 469

Nevertheless, we cannot exclude that triggering of endosomal TLRs might activate 470

cellular signaling pathways apart from NFκB that might impact on MHV-68 gene 471

expression. In vitro data of Gargano et al. (6) showed that TLR3 or TLR9 triggering 472

provoked lytic MHV-68 reactivation in the cell lines A20HE1 and A20HE2 while TLR7 473

triggering had no effect. The activation of NFκB was not tested. Thus, differences in 474

endogenous levels of NFκB activation or TLR signaling competence between these 475

cell lines might account for the divergent observations. Alternatively, the reason for 476

the apparently contradictory observations may lie in the distinct origin of persistent 477

MHV-68 infection. While S11 cells originate from a naturally MHV-68-associated 478

lymphoproliferation, A20HE1 and A20HE2 cell lines derive from murine lymphoma 479

cells secondarily infected in vitro with a recombinant strain of MHV-68 (36). In a 480

20

different study, increased reactivation of KSHV from primary effusion lymphoma cells 481

was shown upon treatment with ligands to TLR7/8 (7). The critical signaling pathway 482

in these cells was shown to be dependent on IRF7, however, further demonstrating 483

that the impact of TLR signaling on gamma-herpesvirus reactivation may depend on 484

cellular context. 485

In vivo, recurrent activation of TLR7 signaling by treatment with R848 following 486

de novo infection of wt mice led to a higher frequency of latently MHV-68 infected 487

splenocytes compared to mock treatment (Fig. 3A). This observation is comparable 488

with the findings of Gargano et al. (6) who reported increased frequency of infected 489

splenocytes in vivo upon stimulation of TLR9 by administration of CpG ODN. A 490

higher frequency of cells positive for MHV-68 DNA indicates an increase in the pool 491

of latently MHV-68-infected cells and this is supported by the detection of MHV-68 492

latency-associated mLANA transcripts in our experiments (Fig. 3B). Failure to detect 493

mLANA transcripts in two samples from untreated controls reflects the limit of 494

detection of the nested PCR rather than absent infection, since splenocytes from all 495

mice were positive for MHV-68 DNA (Fig. 3A) but only 5-10% of latently infected cells 496

are expected to express mLANA (27). Based on our observations in vitro, we 497

hypothesize that both TLR7- and TLR9-induced activation of NFκB promotes 498

maintenance and expansion of the latently infected B-cell pool. Notably, stimulation 499

with TLR ligands has been shown to induce proliferation of murine B-cells (37) and is 500

known to synergize with EBV in driving proliferation of EBV-infected human B-cells 501

(38), which could explain elevated frequencies of infected cells without the need for 502

lytic replication. The absence of preformed virus in our samples from R848-treated 503

mice (Fig. 3C) points towards a reactivation-independent mechanism for the 504

expansion of the latent MHV-68 reservoir rather than, e.g., through de novo infection 505

21

of B-cells by MHV-68 released upon TLR stimulation, as suggested by in vitro 506

experiments by Gargano and colleagues (6). 507

We observed a constant activation of the NFκB pathway in the model cell line 508

S11, as evidenced by high basal levels of NFκB subunit p65 in the nucleus (Fig. 4B), 509

which drives survival and proliferation (39). NFκB levels in the nucleus were reduced 510

by stable downregulation of TLR9 but not of TLR7 expression by RNA interference, 511

which consequently led to an increased rate of spontaneous reactivation (Fig. 4C), 512

implying a constant stimulation of TLR9 in infected S11 cells. Unlike TLR9, TLR7 did 513

not contribute to NFκB activation in S11 cells in the absence of external stimulation 514

(Fig. 3B). It has been shown by various groups that TLR9 is triggered upon infection 515

by herpesviruses (40, 41), including the gamma-herpesviruses EBV (42) and KSHV 516

(43). Many viruses have evolved strategies to benefit from NFκB activation either 517

directly by having NFκB binding sites in their promoters or indirectly by using the 518

enhanced proliferation and survival of the host cell due to NFκB signaling to their 519

advantage (44). For MHV-68, suppression of lytic viral genes mediated by NFκB 520

induction upon infection might serve as a negative feedback loop with the potential to 521

limit productive replication and initiate the latent state during primary infection in vivo. 522

The importance of functional NFκB for the establishment of MHV-68 latency in vivo 523

has been elegantly demonstrated by Krug et al. (21) who reported severe impairment 524

in establishment of splenic latency by a recombinant strain of MHV-68 that expresses 525

a constitutively active form of the NFκB inhibitor IκBαM. Furthermore, NFκB signaling 526

has been shown to be an important factor for the survival of lymphoma cells latently 527

infected with KSHV (45) and EBV-transformed B-cells in vitro (46). 528

In the context of de novo infection in vivo, our experiments with mice lacking 529

either TLR7 or TLR9 did not show a significant difference in the frequency of infected 530

splenic B-cells following i.p. infection. It is possible that more severe deficiency in 531

22

TLR signaling impact on the number of latently infected cells since a ten-fold 532

decrease in infected splenocytes was reported in myd88-/- mice that lack functional 533

signaling of most TLRs (47). While the frequencies of infected B-cells in our 534

experiments were comparable between mice of different genotypes (Fig. 5A), B-cells 535

from tlr9-/- mice showed a higher propensity to reactivate ex vivo while the tlr7-/- B-536

cells did not (Fig. 5B). This is in agreement with our results obtained with S11 cells. 537

Taken together, our results point towards an important role of TLR7- and 538

TLR9-mediated activation of NFκB in promoting persistent infection of MHV-68 in B-539

cells and show that continuous TLR9 signaling, possibly caused by persistent MHV-540

68 infection, might contribute to maintaining NFκB activation during latency. 541

Considering that we did not see a significant difference in the frequency of infected 542

B-cells in vivo, it is conceivable that there are other systems in place contributing to 543

NFκB activation, for example the retinoic-acid-inducible-gene (RIG)-I system that 544

synergizes with TLR9 to induce the immune response against herpes simplex virus 545

(40). Other gamma-herpesviruses use activation of NFκB in the host cell for benefit 546

as evidenced by EBV’s binding to the cellular receptor CD21, which induces NFκB 547

that in turn mediates activation of the viral latent gene promoter (48) or the sustained 548

moderate activation of NFκB reported upon infection of endothelial cells by KSHV 549

that regulates viral gene expression (49). 550

Finally, the latency-promoting effect of endosomal TLR-mediated NFκB 551

activation reported here suggests a possible mechanism for the role of co-infections 552

that are implicated in the pathogenesis of certain gamma-herpesvirus-associated 553

malignancies. Indeed, with respect to EBV-associated B-cell malignancies, P. 554

falciparum malaria is epidemiologically associated with endemic BL, and the parasite 555

was reported to directly stimulate TLR9 (11, 12). Earlier we showed that P. 556

falciparum hemozoin suppresses lytic reactivation of EBV in BL cells (9), further 557

23

underscoring activation of TLR signaling pathways as a possible mechanistic link 558

between chronic malaria infection and the incidence of endemic BL. The situation in 559

KSHV-associated primary effusion lymphoma, however, may be distinct as 560

suggested by the lytic reactivation of KSHV following triggering of TLR7/8 (7). 561

Nevertheless, our results are important in the light of TLR ligands being increasingly 562

used as adjuvants in the treatment of infectious diseases, cancer, or autoimmunity 563

because of their potential to promote gamma-herpesvirus latency. 564

565

ACKNOWLEDGMENTS 566

This work was supported by grants from the Swiss National Foundation (#310040-567

114118), Edoardo R., Giovanni, Giuseppe and Chiarina Sassella Foundation, OPO 568

Foundation, Wolfermann-Nägeli Foundation, the Forschungskredit of the University 569

of Zurich, an unrestricted grant from AstraZeneca, a UBS donation by a client, and a 570

grant from GSK International Award for Research of Japanese Society of 571

Immunology & Allergology in Otorhinolaryngology and Grants-in-Aid for Scientific 572

Research from the Ministry of Education, Culture, Sports, Science, and Technology 573

and the Japan Society for Promotion of Science (Research Project Number 574

22591915). 575

576

577

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579

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45. Keller SA, Schattner EJ, Cesarman E. 2000. Inhibition of NF-kappaB induces 703 apoptosis of KSHV-infected primary effusion lymphoma cells. Blood 96:2537-2542. 704

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47. Gargano LM, Moser JM, Speck SH. 2008. Role for MyD88 signaling in murine 708 gammaherpesvirus 68 latency. J. Virol. 82:3853-3863. 709

48. Sugano N, Chen W, Roberts ML, Cooper NR. 1997. Epstein-Barr virus binding to 710 CD21 activates the initial viral promoter via NF-kappaB induction. J. Exp. Med. 711 186:731-737. 712

49. Sadagopan S, Sharma-Walia N, Veettil MV, Raghu H, Sivakumar R, Bottero V, 713 Chandran B. 2007. Kaposi's sarcoma-associated herpesvirus induces sustained NF-714 kappaB activation during de novo infection of primary human dermal microvascular 715 endothelial cells that is essential for viral gene expression. J. Virol. 81:3949-3968. 716

717 718

27

FIGURE LEGENDS 719

Figure 1 720

Triggering of endosomal TLR7 and TLR9 but not TLR3 activates NFκB in S11 cells. 721

(A) Expression of TLR3, TLR7 and TLR9 mRNA was detected in S11 cells by 722

reverse transcription PCR. (B) Triggering of TLR7 and TLR9 but not TLR3 activates 723

NFκB in S11 cells. NFκB subunit p65 was detected in nuclear extracts of S11 cells at 724

different timepoints after the treatment of mock, 25 µg/ml Poly(I:C), 3 µM R848, 0.5 725

µM CpG ODN 1826 or 1 µM NFκB inhibitor Bay 11-7082 by Western Blot. PCNA was 726

used as a loading control. (C) Triggering of TLR3 induces a different pattern of 727

cytokine expression than TLR7 or TLR9. S11 cells were stimulated with TLR ligands 728

as done in (B) and harvested at different timepoints. Expression of IFN-beta, IL-6 and 729

IL-10 was analyzed by qPCR, normalized to gapdh, and plotted relative to mock 730

treated controls. Data shown in (C) are mean ± SD of three independent 731

experiments. Some of the error bars are covered by the symbols. 732

733

Figure 2 734

Triggering of endosomal TLR7 and TLR9 but not TLR3 inhibits expression of lytic 735

MHV-68 gene ORF50 and reduces viral particle shedding while TPA or NFκB 736

inhibitor Bay 11-7082 increase spontaneous reactivation. (A) S11 cells were mock 737

treated or treated with TLR3 ligand Poly(I:C), TLR7 ligand R848, TLR9 ligand CpG 738

ODN 1826, TPA or a combination of TLR ligand followed by TPA 2h later. 24h after 739

the begin of treatment, expression levels of MHV-68 lytic gene ORF50 were analyzed 740

by qPCR, normalized to gapdh, and plotted relative to untreated controls (black 741

bars). Viral titers in the supernatants of treated cells were measured by plaque assay 742

(white bars). (B) S11 cells were mock treated or treated with TPA, R848, CpG ODN 743

1826, NFκB inhibitor Bay 11-7082 or a combination thereof. 24h after treatment, 744

28

expression levels of MHV-68 lytic gene ORF50 were analyzed by qPCR (black bars). 745

Viral titers in the supernatants of the treated cells were measured by plaque assay 746

(white bars). Data shown in (A) and (B) are mean ±SD of three independent 747

experiments. Statistics were calculated using unpaired t test (*, p<0.05; **, p<0.01). 748

749

Figure 3 750

Treatment with TLR7 ligand R848 promotes establishment of latent MHV-68 infection 751

in vivo. C57BL/6 mice were infected with MHV-68 by i.p. injection and treated daily 752

i.p. with either 1mg/kg R848 in 100μl of PBS or PBS alone (mock) for 19 days. 753

Treated and mock-treated groups contained 5 mice each. (A) Splenocytes isolated 754

from infected mice at 20dpi were analyzed by limiting-dilution nested PCR targeting 755

ORF72. Data are expressed as mean percentages of positive reactions ± SD. 756

Sigmoidal dose-response curve was fit by nonlinear regression analysis using 757

GraphPad software and the frequency of MHV-68 positive splenocytes was 758

calculated based on Poisson distribution. The resulting curves were compared by 759

Mann-Whitney test (mock-treated vs. R848-treated p=0.016). (B) Primary 760

splenocytes from in vivo MHV-68 infected mice express virus latency-associated 761

gene ORF73 (mLANA). Total RNA isolated from bulk splenocytes was reverse 762

transcribed and analyzed for spliced ORF73 transcripts by nested PCR. Amplification 763

of the housekeeping gene gapdh served as control for successful reverse 764

transcription. (C) Preformed virus is not detected in disrupted splenocytes from in 765

vivo infection. Splenocytes isolated from in vivo MHV-68 infected mice were flash-766

frozen in liquid nitrogen to disrupt cells and plated on NIH3T12 fibroblasts in a limiting 767

dilution assay. Wells were scored for cytopathic effect 14 days after plating. (D) 768

Flash-freezing slightly reduces the infectious titer of cell-free virus. Plaque assays 769

29

were performed with cell-free virus stocks before and after freeze-thaw cycle. Data 770

shown are mean ±SD of three independent experiments. 771

772

Figure 4 773

Downregulation of TLR9 but not of TLR7 leads to a decrease in nuclear NFκB p65 774

and increased spontaneous reactivation in S11 cells. (A) Expression of TLR7 (left 775

side) and TLR9 (right side) was analyzed by qPCR in parental S11 cells and stable 776

cell lines expressing shRNA against Luciferase (shLuc), TLR7 (shTLR7) or TLR9 777

(shTLR9). Expression levels were normalized to gapdh and plotted relative to control 778

cells shLuc. (B) NFκB subunit p65, PCNA and beta-actin was detected in nuclear and 779

cytoplasmic protein fractions from parental S11 and bulk stably transfected cell lines 780

by Western Blot. (C) Expression of MHV-68 lytic gene ORF50 was analyzed by 781

qPCR in parental S11 cells and stable cell lines shLuc, shTLR7 and shTLR9 (black 782

bars). Expression levels were normalized to gapdh and plotted relative to control 783

cells shLuc. Titers of infectious viral particles in the supernatant after 24h were 784

quantified by plaque assay (white bars). Data shown in (A) and (C) are mean ± SD of 785

three independent experiments. Statistics were calculated using unpaired t test (*, 786

p<0.05; **, p<0.01; n.s., p>0.05). 787

788

Figure 5 789

Lack of TLR7 or TLR9 does not impact establishment of infection in B-cells upon i.p. 790

injection in vivo but tlr9-/- B-cells show higher reactivation ex vivo. 15 mice each of the 791

genotypes wt, tlr7-/- and tlr9-/- were infected with MHV-68 and sacrificed on day 34 792

post infection. Splenic B-cells were isolated by negative selection. (A) Splenic B-cells 793

from three mice of the same genotype were pooled and analyzed by limiting-dilution 794

nPCR as in Fig.3. The resulting curves (5 per genotype) were compared by Mann-795

30

Whitney test (wt vs. tlr7-/- p=0.89; wt vs. tlr9-/- p=0.1; tlr7-/- vs. tlr9-/- p=0.1). (B) Splenic 796

B-cells were plated in two-fold dilution series on monolayers of susceptible 797

fibroblasts. In parallel, disrupted samples were plated as controls. 14 days after 798

seeding, the percentage of wells showing cytopathic effect was determined 799

microscopically and plotted against the number of B-cells seeded. Non-linear 800

regression curve was fit using GraphPad software. 801