1

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Title: Activation of NFκB by human papillomavirus 16 E1 limits E1-dependent viral replication 2

through degradation of E1. 3

4

Running title: A possible negative feedback loop between E1 and NFκB 5

6

Tomomi Nakahara, Katsuyuki Tanaka, Shin-ichi Ohno*, Nagayasu Egawa**, Takashi Yugawa, 7

Tohru Kiyono#. 8

Division of Carcinogenesis and Cancer Prevention, National Cancer Center Research Institute, 5-9

1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. 10

#Address correspondence to Tohru Kiyono: tkiyono@ncc.go.jp 11

*Present address: Department of Reproduction, National Research Institute for Child Health and 12

Development, 2-10-1 Okura, Setagaya-ku, Tokyo, 157-8535, Japan 13

**Present address: Division of Virology, National Institute for Medical Research, The Ridgeway, 14

Mill Hill, London, NW7 1AA, United Kingdom 15

The abstract: 211 words, Importance: 116 words. 16

The text: 12,398 words 17

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JVI Accepted Manuscript Posted Online 25 February 2015J. Virol. doi:10.1128/JVI.00389-15Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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Abstract 20

NFκB is a family of transcription factors that regulate gene expression involved in many 21

processes such as the inflammatory response and cancer progression. Little is known about 22

associations of NFκB with the HPV life cycle. We have developed a tissue culture system to 23

conditionally induce E1-dependent replication of HPV16 genome in human cervical 24

keratinocytes, and found that expression of HPV16 E1, a viral helicase, results in reduction of 25

IκBα and subsequent activation of NFκB in a manner dependent on helicase activity. Exogenous 26

expression of a degradation-resistant mutant of IκBα, which inhibits the activation of NFκB, 27

enhanced E1-dependent replication of the viral genome. Wortmannin, a broad inhibitor of 28

phosphoinositide 3-kinases (PI3Ks), and to a lesser extent, VE-822, an ATR kinase inhibitor, but 29

not KU55933, an ATM kinase inhibitor, suppressed the activation of NFκB and augmented E1-30

dependent replication of HPV16 genome. Interestingly, the enhancement of E1-dependent 31

replication of the viral genome was associated with increased stability of E1 in the presence of 32

Wortmannin as well as the IκBα mutant. Collectively, we propose that expression of E1 induces 33

NFκB activation at least in part through the ATR-dependent DNA damage response and NFκB in 34

turn limits E1-dependent replication of HPV16 through degradation of E1, so that E1 and NFκB 35

may constitute a negative feedback loop. 36

37

3

Importance 38

A major risk factor in HPV-associated cancers is persistent infection with high-risk HPVs. To 39

eradicate viruses from infected tissue, it is important to understand molecular mechanisms 40

underlying the establishment and maintenance of persistent infection. In this study, we obtained 41

evidence that HPV16 E1, a viral DNA helicase essential for amplification of the viral genomes, 42

induces NFκB activation and that this limits E1-dependent genome replication of HPV16. These 43

results suggest that NFκB mediates a negative feedback loop to regulate HPV replication and this 44

feedback loop could be associated with control of the viral copy numbers. We could thus show 45

for the first time that NFκB activity is involved in the establishment and maintenance of 46

persistent HPV infection. 47

48

4

Introduction 49

Human papillomaviruses (HPVs) are a large family of non-enveloped, small DNA viruses 50

with an approximately 8-kbp double stranded circular genome that infect stratified squamous 51

epithelium in various anatomical sites such as skin, the anogenital tract and the oral cavity. Virus 52

infection can cause hyperproliferative lesions, ranging from verrucas to cancer. To date, at least 53

180 types of HPVs have been cloned from clinical lesions and classified as either cutaneous or 54

mucosal types based on their predilection for different sites of infection. A subset of mucosal 55

HPVs, high risk (HR)-HPVs such as HPV16, is strongly associated with anogenital cancers 56

including nearly 100% of cervical cancers and some proportion of head and neck cancers. 57

Altogether, it is estimated that HR-HPV infections are responsible for more than 5% of all 58

cancers worldwide. A major risk factor for HR-HPV associated carcinogenesis is persistent 59

infection of HR-HPVs. Therefore, it is important to elucidate molecular mechanisms underlying 60

the establishment and maintenance of persistent infection of HR-HPVs in order to allow 61

eradication of the viruses from infected tissue (1) . 62

The life cycle of HPVs is characterized by tight associations with differentiation processes in 63

the stratified epithelium. The production of progeny virions is exclusively restricted to upper 64

differentiated layers of stratified epithelium. The viruses enter nuclei of basal, mitotic cells and 65

establish their genomes as nuclear episomes at 50 - 100 copies per cell following transient initial 66

amplification. In basal cells, viral early genes are expressed at minimal levels and the copy 67

number is maintained constant by limiting replication of the viral genome on average once in S 68

phase, termed maintenance replication. As the infected cells initiate differentiation, levels of the 69

viral early genes are increased and the expression of late viral genes including major and minor 70

5

capsid proteins, L1 and L2, respectively, is induced. The viral genomes are drastically amplified 71

to many thousands by continued replication at a high level, termed productive amplification, and 72

eventually encapsidated into progeny virions (2). Two viral early proteins, E1 and E2 are 73

necessary for HPV genome replication along with the cellular replication machinery. A basic 74

mechanism of E1-dependent replication of papillomavirus genomes is well elucidated in which 75

E1 binds to the replication origin of the viral genome in cooperation with E2, forming a 76

hexameric DNA helicase and unwinding the viral DNA by power of ATP hydrolysis. E1 also 77

recruits host cell replication factors such as DNA polymerase α, topoisomerase I and replication 78

protein A (RPA). However, it is poorly understood how this E1-dependent replication is 79

regulated during the three phases of viral genome replication, initial amplification, maintenance 80

replication and productive amplification in the viral life cycle (3, 4). 81

Recently, several studies have indicated that DNA damage response (DDR) pathways are 82

involved in HPV genome replication. Key regulators of DDR are phosphoinositide 3-kinase like 83

kinases (PI3KKs), ataxia telangiectasia mutated (ATM), ATM and Rad3-related (ATR) and 84

DNA-dependent protein kinase (DNA-PKcs). Upon sensing DNA damage, these PIKKs initiate 85

signaling inducing the cell cycle checkpoints and DNA repair machinery or apoptosis (5-7). 86

Moody and Laimins reported a series of studies demonstrating that activation of ATM is required 87

for productive amplification but not for stable maintenance of HPV31 in CIN612 cells isolated 88

from a cervical intraepithelial neoplasia grade I (CIN) biopsy. HPV31 E7, an oncogene of HR-89

HPV, was shown to activate ATM and its downstream mediator checkpoint kinase 2 (CHK2). (8-90

10). Sakakibara et al. and Fradet-Turcotte et al. reported that E1 proteins of various HPVs, 91

including HPV16 and 31 and Bos taurus papillomavirus 1 (BPV1), also cause activation of ATM 92

6

(11, 12). In addition, it is reported that transient replication of HPV18 genomes in U2OS cells 93

induces activation of ATR mediated DDRs which depend on the expression of E1 and E2 (13). 94

Nuclear factor κ-light-chain-enhancer of activated B cells (NFκB) comprises a family of 95

transcription factors that activate or repress expression of a large number of genes in a myriad of 96

physiological and pathological processes contributing to immune and inflammatory responses, 97

cellular stress responses, differentiation, cell proliferation and apoptosis. Currently, five 98

members of the family are known in mammals, RELA/p65, p50/p105, p52/p100, RELB and c-99

REL that form homo-or hetero-dimers. The activity of NFκB is negatively regulated primarily 100

through its cytoplasmic retention by binding to inhibitors of NFκB (IκBs) including IκBα. Upon 101

stimulation, phosphorylation dependent degradation of IκBs by an ubiquitin proteasome pathway 102

liberates NFκBs into the nucleus (14, 15). Such activation of NFκB is a fundamental immediate 103

early step of immune activation, inducing expression of interferons (IFNs) as well as 104

inflammatory cytokines. Many viruses have been shown to target NFκB pathways to evade 105

innate immune responses. On the other hand, accumulated studies have revealed that activation 106

of NFκB signaling is induced and incorporated in the viral life cycle and is important for 107

pathogenicity of oncogenic viruses such as Epstein Barr virus (EBV), Kaposi sarcoma herpes 108

virus (KSHV) and human adult T leukemia virus -1 (HTLV-1)(16). 109

Increased expression of NFκB has been described for HPV associated lesions such as cervical 110

cancers as well as their precursor lesions, and laryngeal papillomas and cancers (17-22). Several 111

studies have shown that E6 and E7 proteins of HPVs modulate NFκB pathways (23-30). 112

However, the results of these studies have been contradictory as to whether E6 and/or E7 activate 113

or suppress. NFκB p65 (RELA) has been shown to bind to long control region (LCR) of HPV16 114

7

and suppress its promoter, p97, activity (31). Recently, it was also reported that E2 proteins of 115

various HPVs enhance tumor necrosis factor (TNF) α induced activation of NFκB in HaCaT 116

cells (32). NFκB plays an important role in maintaining homeostasis of epithelium and cellular 117

differentiation of keratinocytes (33, 34). Altogether, the available data suggests involvement of 118

NFκB in the pathogenesis and the life cycle of HPVs. In the present study, we investigated the 119

relationship between NFκB activity and replication of HPV16 genomes. As the outcomes and 120

pathways of NFκB signaling are known to vary dependent on the cell type, we developed a tissue 121

culture system to efficiently induce E1-dependent replication of the HPV16 genome in human 122

cervical keratinocytes (HCKs), a natural host of HPV replication. We found that NFκB is 123

activated upon E1 expression, which in turn limits E1-dependent replication of HPV16 genome 124

in mitotic, undifferentiated HCKs. The activation of NFκB was induced through DDR upon E1 125

expression. Implications of the NFκB-mediated suppression of E1-dependent replication for the 126

three phases of viral genome replication in the viral life cycle will be discussed. 127

128

8

Materials and Methods 129

Cells and tissue culture 130

Human cervical keratinocytes immortalized with TERT (HCK1T) were described previously 131

(35). HCK1T-HPV16 cells were generated by co-transfecting 1.25μg of pAd/HPV16/neo with 132

1.25μg of pxCANCre to HCK1T using lipofectamine 2000 (Life Technologies, Grand Island, 133

NY) according to the manufacturer’s protocol. Cells were selected with 150 μg/ml of G418 for 2 134

days starting from 2 days post-transfection and then maintained until G418 resistant colonies 135

became visible. These colonies were isolated and the presence of HPV16 genomes as episomes 136

was confirmed by Southern blotting. Oligo-clonal HCK1T that contained HPV16 genomes as 137

episomes was named HCK1T-HPV16 and used in this study. HCK1T tetON E1, tetON E1+E2 138

and tetON E2 were generated by retrovirus gene transfer and subsequent drug selection as 139

indicated below. HCK1T-HPV16 tetON E1+E2, tetON E1K483A+E2, tetON E1 and tetON E2 140

were generated by lentivirus infection. HCK1T-HPV16 tetON E1+E2 clone A5 and B2 141

introduced with expression of IκBα wild type, the IκBα mutant and the multi cloning site (MCS) 142

control were generated by retrovirus infection and subsequent drug selection. HCK1T-HPV16 143

and its derivatives were maintained in Keratinocyte-Serum Free medium (K-SFM) at 37°C in 5% 144

CO2 incubator. HCK1T and its derivatives not containing an HPV16 genome were maintained in 145

EpiLife medium (Life Technologies, Grand Island, NY). CIN612-9E cells were maintained on 146

feeder cells in E medium as described previously (36). Inhibitors and doxycycline were dissolved 147

in Dexamethasone (DMSO) and stored as high concentration stocks at -20°C until used at the 148

indicated concentrations. The concentrations of the stock solutions were as follows: Doxycycline 149

(Cat. Z1311N, TAKARA BIO Inc. Shiga, Japan) at 1 mg/ml, KU55933 (Cat. 118500, Merk 150

9

Millipore, Billerica, MA, USA), Wortmannin (Cat. 230-02341, WAKO Pure Chemical Industries, 151

Ltd., Osaka, Japan), SB203580 (Cat. 20-2173, Funakoshi Co., Ltd., Tokyo, Japan), VE-822 (Cat. 152

S7102, Selleck Chemicals, Houston, TX, USA) and Nu7026 (Cat. 2828, Tocris Bioscience, 153

Minneapolice, MN, USA) at 10 mM. Human TNFα (Cat. 203-15263, WAKO Pure Chemical 154

Industries, Ltd.) was dissolved and stored in phosphate buffered saline (PBS) at 10 μg/ml. 155

Plasmids 156

pAd/HPV16/neo encoding the full length of HPV16 genome flanked with two loxP sites was 157

constructed by replacing an XbaI-NotI fragment encoding EGFP of the original vector, 158

pAd/HPV16/Cre (37), with an XbaI-NotI fragment coding a neomycin resistant gene of pMSCV-159

neo (TAKARA BIO INC, Shiga, Japan). pxCANCre was obtained from the RIKEN DNA bank 160

(RDB10748, RIKEN Bioresource center, Tsukuba, Japan). Lentivirus vectors, CSII-CMV-161

tetON-ADV and CSII-TRE-Tight-HA16E1, were described previously (38). The codon 162

optimized HPV16 E2 cDNA (GeneScript, Piscataway, NJ) was used to construct CSII-TRE-163

Tight-HA16E2, in which the N-terminal hemaglutinin (HA)-tagged HPV16 E2 was inserted 164

under a tetracycline responsible promoter. CSII-TRE-Tight-HA16E1K483A was generated by site-165

directed mutagenesis (QuickChange Site-Directed Mutagenesis kit, Agilent Technologies, Santa 166

Clara, CA). CSII-TRE-Tight-HA16E1-PB and CSII-TRE-Tight-3xFLAG16E2-PB were 167

generated by inserting an expression cassette of the blastcidin (PB) resistant gene under the PGK 168

promoter into downstream of HA16E1 or 3xFLAG16E2. pQCXIZeo-tetON was generated by 169

inserting rtTA (tetON) from pRevTet-ON (Code 631007, BIO INC, Shiga, Japan) under CMV 170

promoter of pQCXIZeo, in which a puromycin resistant gene downstream of IRES of the 171

retroviral vector pQCXIP (Code.631516, TAKARA BIO INC, Shiga, Japan) was replaced by a 172

10

zeocin resistant gene from pCXbleo (Gene accession No. AB086388, a kind gift from Dr. Akagi, 173

Osaka Bioscience Institute). pQCXIZeo- IkBαWT and IkBαMT (Ser32/36Ala) were generated 174

by inserting cDNAs of IkBα and IkBαMT (Ser32/36Ala) derived from pCMV-IkBα and pCMV-175

IkBαMT (Ser32/36Ala) (Cat. 631923, TAKARA BIO INC, Shiga, Japan) into pQCXIZeo using 176

Gateway recombination (Life Technologies, Grand Island, NY). pCMV-6xHis-Ub expressing 177

histidine-tagged ubiquitin (His-Ub) was a kind gift from Dr. Bohmann (University of Rochester, 178

USA). 179

Retrovirus gene transduction 180

The production and infection of recombinant lentiviruses and retroviruses were accomplished 181

as previously described (39). To generate populations of HCK1T-HPV16 tetON E1, E1K483A 182

and/or E2, HCK1T-HPV16 cells were seeded at one day before and inoculated with CSII-CMV-183

tetON-ADV and one or corresponding combinations of lentiviruses, CSII-TRE-Tight-HA16E1, 184

CSII-TRE-Tight-HA16E1K483A and CSII-TRE-Tight-HA16E2 at multiplicity of infection (MOI) 185

20. For generation of HCK1T-HPV16 tetON E1 and FLAG-E2, a pooled population of HCK1T-186

HPV16 tetON E1 was infected with lentiviruses containing CSII-TRE-Tight-3xFLAG16E2 at 187

MOI 20. To generate HCK1T tetON E1, tetON E1+E2 and tetON E2, HCK1T cells were first 188

infected with pQCXIZeo-tetON at MOI 3 followed by zeocin selection (2 μg/ml). The generated 189

HCK1T-tetON cells were then infected with lentiviruses, CSII-TRE-Tight-HA-HPV16E1-PB 190

and/or CSII-TRE-Tight-3XFLAG-HPV16 E2-PB, at MOI 3 and selected with blastcidin (2 μg 191

/ml). Parental HCK1T-HPV16 and HCK1T-HPV16 tetON E1+E2 clones A5 and B2 were 192

infected with retroviruses, pQCXIZeo-MCS, IkBαWT or IkBαMT, and selected with zeocin (10 193

μg/ml). 194

11

Cell collection 195

The cultural supernatant was harvested and “detached cells” were collected by centrifugation at 196

2,000 x g for 5 minutes at 4˚C. The cells staying attached were washed with PBS twice and 197

removed from a tissue culture plate by incubation with trypsin. The “attached cells” were then 198

incubated with a soybean trypsin inhibitor (0.25 mg/ml in PBS, Life Technologies, Grand Island, 199

NY) and collected by centrifugation at 2,000 x g for 5 minutes at 4˚C. When necessary, 200

“attached cells” were counted using a Coulter Counter (Beckman Coulter, Inc. Brea, CA, USA). 201

The “attached cells” and “detached cells” were combined and the mixtures were washed with 202

PBS twice. The combined cells were lysed with a buffer specific to each assay. 203

Southern blotting and quantitative real-time PCR (qPCR) 204

Total genomic DNA for Southern blotting was isolated after several passages and at least one 205

freezing and thawing from transfection as described previously (38). For qPCR, total genomic 206

DNA was isolated using a Wizard SV Genomic Purification System following the 207

manufacturer’s protocol (Cat. A2361, Promega, Madison, WI, USA). For Southern blotting, 10 208

μg of total genomic DNA was digested either with BamHI or XhoI overnight and 209

electrophoresed through a 0.7% agarose gel. The gel was alkali transferred onto a nylon 210

membrane using a TurboBlotter (Cat. 10416336, GE healthcare, Fairfield, Connecticut, USA). 211

The probe used for Southern blotting was randomly labeled with biotin by using a NEBlot 212

Phototope kit (New England BioLabs. Inc., Ipswich, MA, USA). The linearized plasmid 213

encoding the full length of HPV16 was used as a template. Hybridization and chemiluminescent 214

detection was performed as described previously. The LAS3000 charge-coupled device (CCD) 215

imaging system (Fujifilm Co. Ltd., Japan) was employed for detection. Quantification of images 216

12

was conducted using software NIH image J and Multi Gauge (Fujifilm, Co. Ltd.). Realtime PCR 217

to quantify the viral genome copy number was performed as described previously. In brief, 10-218

100 ng of total genomic DNA was mixed with a mastermix of KAPA SYBR FAST qPCR kits 219

(Kapa Biosystems, Woburn, MA) and 300 nM of each primer and then subjected to a real time 220

PCR reaction using StepOnePlus (Life Technologies, Grand Island, NY). Serial dilutions of 221

linearized HPV16 genome from pUC-HPV16 plasmid DNA by BamHI digestion were used as 222

standards to measure the amount of HPV16 DNA. All real-time PCRs were run in triplicate and 223

at least three independent experiments were performed. The copy number of HPV16 per cell was 224

calculated based on the assumption that total human genomic DNA is 6.6 pg/diploid cell. P-225

values were determined by Sudent’s t- test. Two sets of primer pairs were used for qPCR to 226

confirm that the viral copy numbers estimated by each pair were consistent. The pairs of primers 227

used were as follows: E6 pairs (HPV16-97F, 5’-GAACTGCAATGTTTCAGGACCC-3’and 228

HPV16-174R, 5’-TGTATAGTTGTTTGCAGCTCTGTGC-3) and L2 pairs (HPV16-4481F, 5’-229

ACAGATACACTTGCTCCTGTAAGACC-3’ and HPV16-4665R, 5’- 230

GCAGGTGTGGTATCAGTTGAAGTAGT-3’). 231

RNA extraction and reverse transcription (RT)-qPCR 232

Total RNA was isolated using an RNeasy Plus Mini kit (Cat. 74136, Qiagen, Venlo, 233

Netherlands) following the manufacturer’s instructions. 1 μg of total RNA was subjected to 10 μl 234

RT reaction using a PrimeScript RT reagent kit (Cat. RR037A, TAKARA BIO INC, Shiga, 235

Japan) and 1 μl of the RT products was used for real time PCR reaction to measure mRNAs of 236

the interest. In the case of β-actin mRNA, 10 time-diluted RT products were used due to 237

abundance of the mRNA. The PCR mixtures were prepared using KAPA SYBR FAST qPCR 238

13

kits and the PCR run was achieved with StepOnePlus. The relative mRNA levels of the interest 239

were calculated by a delta-delta CT method using β-actin mRNA as an internal control. All PCRs 240

were run in triplicate. All experiments were repeated at least three times. P-values were 241

determined by Student’s t- test. The following primers were used. β-actin: Forward 5’-242

ACCAACTGGGACGACATGGAGAAA-3’ and Reverse 5’-243

TAGCACAGCCTGGATAGCAACGTA-3’, TNFα: Forward, 5’- 244

CGAGTGACAAGCCTGTAGC-3’ and Reverse, 5’-GGTGTGGGTGAGGAGCACAT-3’, IL-6: 245

Forward, 5’-AAATTCGGTACATCCTCGACGGCA-3’ and Reverse, 5’-246

AGTGCCTCTTTGCTGCTTTCACAC-3’, IL-8: Forward, 5’- 247

AGCCTTCCTGATTTCTGCAGCTCT-3’, and Reverse, 5’-248

AATTTCTGTGTTGGCGCAGTGTGG-3’, E1^E4: Forward, 5’-249

GCTGATCCTGCAAGCAACGAAGTATC-3’, and Reverse, 5’-250

GGATTGGAGCACTGTCCACTGAG-3’, HA-E1: Forward, 5’-251

CCTTATGACGTGCCAGATTACGC-3’, and Reverse, 5’-252

GTCATTTTCGTTCTCATCGTCTGAGATG-3’. These primers were designed based on the 253

published data (40). 254

Western blotting 255

Cells were collected as described above and lysed in lysis buffer (50 mM Tris-HCl, 250 mM 256

NaCl, 5 mM EDTA, 1% NP-40, 20% glycerol, 0.1% SDS, 1% Deoxycholate) containing a 257

proteinase inhibitor cocktail (Nacalai tesque, Kyoto, Japan) and phosphatase inhibitors (500 μM 258

sodium orthovanadate, 100 mM sodium fluoride, 10 mM sodium pyrophosphate) and then 259

subjected to brief sonication in an ice-cold water bath. A protein concentration of cell lysates was 260

14

measured by using a DC Protein Assay kit (BIO-RAD, Hercules, CA, USA) after centrifugation 261

at 20,000g for 15 minutes. When necessary, an insoluble fraction was lysed in an SDS sample 262

buffer (50mM Tris-HCl, 2%SDS and 10% glycerol). The equivalent protein amounts of cell 263

lysates were subjected to SDS-polyaclylamide gel electrophoresis (SDS-PAGE) and Western 264

blot analysis was conducted as described previously (38). The primary antibodies used in this 265

study were follows. Mouse monoclonal antibodies: HA (16B12; Covance, Princeton, NJ), FLAG 266

(M2; Sigma-Aldrich, St.Louis, MO, USA ), Vinculin (hVIN-1; Sigma-Aldrich), pATM(S1981) 267

(10H11E12; EMD Millipore, Billerica, MA, USA), CHK2 (clone 7; EMD Millipore), ATM 268

(2C1: Santa Cruz Biotechnology, Santa Cruz, CA, USA), CHK1 (G-4; Santa Cruz 269

Biotechnology), IκBα (L35A5; Cell Signaling Technology, Danvers, MA, USA), His tag (Cat. 270

70796; EMD Millipore), Histone H3 (Ab10799, Abcam, Chambridge, UK). Rabbit monoclonal 271

antibodies: pCHK1(S345) (133D3), RELA (D14E12) and rabbit polyclonal antibodies: Caspase-272

3 (Cat. 9662), PARP-1 (Cat. 9542), pCHK2(T68) (Cat. 2661), α-Tubulin (Cat.2144), 273

p38MAPK(T180/Y182) (Cat. 9211) and p38MAPK (Cat. 9212), all purchased from Cell 274

Signaling Technology. Rabbit polyclonal antibodies: NBS1 (Cat. NB100-143, Novus 275

Biologicals, Littleton, CO, USA), GFP (A6455, Life Technologies, Grand Island, NY), pDNA-276

PKcs(S2056) (Ab18192, Abcam) and DNA-PKcs (Ab70250, Abcam). A goat polyclonal MCM2 277

antibody was also purchased from Santa Cruz Biotechnology (sc-9839). Secondary antibodies 278

used were horseradish peroxidase (HRP) -conjugated anti-mouse, anti-rabbit (Jackson 279

ImmunoResearch Laboratories, West Grove, PA) and anti-goat antibodies (sc2020, Santa Cruz 280

Biotechnology). 281

Indirect immunofluorescence analysis 282

15

The cells were first rinsed with PBS twice and fixed in 4% paraformaldehyde (4% PFA) in PBS 283

for 15 minutes at room temperature. The fixed cells were incubated with 0.01% of Triton-X in 284

PBS for 10 minutes after rinsing twice with PBS, and then incubated with the blocking buffer 285

(2% Bovine Serum Albumin, BSA, in PBS) for 30 minutes at room temperature. Incubation with 286

the primary antibody diluted in the blocking buffer was done overnight at 4˚C and the samples 287

were then washed with PBS three times each for 5 minutes. Incubation with secondary 288

antibodies was done for 30 minutes at room temperature and the samples were then sealed with a 289

mounting medium containing 4', 6-diamidino-2-phenylindole (DAPI) (Prolong Gold, Life 290

technologies, Grand Island, NY) to counterstain nucleus. Fluorescence images were examined 291

using a Leica FW4000 microscope (Leica Microsystems, Wetzlar, Germany). 292

Ubiquitin conjugation assays 293

293FT cells were seeded at 1 x 106 cells in 6-cm tissue culture dishes on the day before 294

transfection. 3.2 μg of pCMV-6xHis-Ub, 0.8 μg of CSII-CMV-HA16E1 or CSII-CMV-MCS, 1 295

μg of pCMV-IκBαMT or an empty vector and 0.2 μg of pEGFP-C3 (TAKARA BIO INC., Shiga, 296

Japan) were mixed and incubated with 18 μl of polyethylenimine (PEI) max solution (1 mg/ml) 297

in 800 μl of serum-free medium for 15 minutes at room temperature. The DNA-PEI max solution 298

was then added to the cells followed by further incubation. At 18 hours post-transfection, the 299

cells were removed by trypsin and divided into three 6-cm tissue culture dishes and incubated 300

with DMSO, 10 μM of Wortmannin or 20 ng/ml of TNFα from 20 hours post-transfection. Cells 301

transfected with pCMV-IκBαMT were also divided and incubated with a vehicle or TNFα. At 48 302

hours post-transfection, the cells were harvested according to a published protocol (41). Briefly, 303

the cells were lysed with the lysis buffer [2% SDS, 150 mM NaCl, 10 mM Tris-HCl pH8.0, 304

16

proteinase inhibitors, phosphatase inhibitors and 2 mM N-ethylmaleimide (NEM)] and 305

immediately boiled for 10 minutes. The cell lysates were subjected to sonication, then diluted in 306

the dilution buffer (10 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton-X100, 2 307

mM NEM) and rotated at 4˚C for 30 minutes. The equivalent protein amounts of the lysates were 308

incubated with anti-HA tag mAb-magnetic beads (Cat. M132-9; Medical and Biological 309

Laboratories Co., Ltd. Nayoga, Japan) overnight with rotation at 4˚C. The immunoprecipitates 310

were collected using MagneSphere Magnetic Separation Stands (Promega, Madison, WI, USA) 311

and extensively washed. The immunoprecipitates were suspended in 2x Laemmli sample buffer 312

and subjected to Western blotting. 313

Cell cycle analysis 314

The HCK1T-HPV16 tetON E1+E2 clone B2 and A5 were collected as described above and 315

fixed in chilled 70% EtOH for at least 1 hour at -20˚C. The fixed cells were then rinsed with PBS 316

twice, resuspended in PBS containing 0.05% Triton X and 0.5mg/ml of RNase A (Nacalai tesque, 317

Kyoto, Japan) and incubated for 30 minutes at 37˚C. Propidium iodide (PI) was added to a final 318

concentration of 0.05 μg/ml and the cells were subjected to flow cytometry using a cell analyzer 319

EC800 (SONY, Tokyo, Japan). 320

321

17

Results 322

Conditional expression of E1 and E2 induces amplification of the HPV16 genome as 323

episomes in human cervical keratinocytes (HCKs). 324

In order to study E1-dependent replication of HPV16 genome in human cervical keratinocytes, 325

we first established immortal human cervical keratinocytes stably maintaining HPV16 genomes 326

as episomes as described in the Materials and Methods. We confirmed that HCK1T-HPV16 cells 327

were able to maintain HPV16 genomes as episomes at a constant copy number (approximately 328

30 copies/cell) for at least 3 months and several freeze and thaw cycles (Fig.1A, lanes 4, 6, 8 and 329

10). They were co-infected with lentivirus vectors expressing the reverse tetracyclin-regulated 330

transactivator (rtTA; tetON) together with those expressing HA-tagged HPV16 E1 (HA16E1), a 331

helicase defective mutant of E1 (HA16E1K484A), or HA-tagged HPV16 E2 under the control of 332

tetracyclin-responsive promoter or combinations of HA16E2 and HA16E1 or HA16E1K484A. The 333

same numbers of those cells were seeded the day before and then incubated with either vehicle or 334

1 μg per ml of doxycycline (DOX) for 24 hours. The total genomic DNA was isolated and the 335

copy number of HPV16 genomes was measured by Southern blotting and quantitative real time 336

PCR (qPCR) (Fig 1A and B). Expression levels of HA16-E1 and -E2 were also analyzed by 337

Western blotting (Fig 1F). Episomal copies of HPV16 genomes increased by approximately 100 338

fold in HCK1T-HPV16 cells designed to express both E1 and E2 (tetON E1+E2) upon 339

incubation with DOX (Fig.1A, lanes 4, 5, 8 and 9 and Fig.1B), whereas no increase was 340

detectable in those designed to express E1K483A and E2 (tetON E1K483A+E2) with the DOX 341

treatment (Fig.1A, lanes 6, 7, 10 and 11 and Fig.1B) even though the level of E1K483A appeared 342

higher than that of the wild type E1 (Fig.1F, lanes 2 and 4). The copy number of HPV16 343

18

genomes increased slightly (by approximately 3 fold) in cells designed to express E1 alone 344

(tetON E1) upon DOX treatment, while no increase was detected in those designed to express E2 345

alone (tetON E2). The basal copy number of HPV16 genomes was comparable among those 346

cells in the absence of DOX. These results clearly indicated that exogenous E2 expression 347

enhanced E1-dependent replication of the HPV16 genome though we failed to detect HA-tagged 348

E2 by Western blotting. The slight increase in the copy number of HPV16 genomes in the tetON 349

E1 cells could depend on E2 derived from episomal HPV16 genomes. The expression level of E1 350

from episomal genomes appeared to be too low to support detectable replication even when 351

excess amounts of E2 were provided exogenously. In the tetON E1+E2 cells, the copy number of 352

the viral genomes increased in a time dependent manner (Fig.1C); increase was detected as early 353

as at 4 hours and continued up to 24 hours after DOX addition whereas no further increase was 354

detected thereafter. The maximal level of E1 as well as the copy number of HPV16 genomes 355

were observed at the highest concentration of DOX tested (1 μg/mL) (data not shown). Therefore, 356

E1-dependent replication of the HPV16 genome was evaluated at 24 hours after 1 μg per ml of 357

DOX addition in subsequent analyses unless otherwise indicated. As the estimated copy number 358

of the viral genomes by qPCR was well correlated with that obtained by Southern blotting, the 359

copy number of HPV16 genomes was measured by qPCR in the subsequent experiments. 360

The intracellular localization of HA-tagged E1 and E2 was examined by immunofluorescence 361

analysis (IFA) using an anti-HA tag antibody at 24 hours after addition of vehicle or DOX 362

(Fig.1D). In the presence of DOX, HA positive signals were observed in roughly 40% of cells, 363

indicating heterogeneity in expression levels of E1 and E2 in the population. In a fraction of the 364

tetON E1+E2 cells, positive signals with anti-HA antibody were found accumulated to nuclear 365

foci (Fig.1D, E1+E2 DOX+). In contrast, no obvious accumulation of HA positive signals to 366

19

nuclear foci was observed in the tetON E1K483A+E2 cells. Instead, strong HA-positive signals 367

were diffusely distributed throughout nuclei and weaker signals were also detected in the 368

cytoplasm. In the tetON E1 cells, a diffuse pattern of HA-positive signals was detected in nuclei 369

and weaker signals were also detected in cytoplasm in a majority of the positive cells. On the 370

other hand, homogeneous HA positive signals were found mostly confined to nucleus and no 371

obvious accumulation to foci was detected in the tetON E2 cells. Our results coincided with the 372

previous reports that E1 and E2 co-localize to nuclear foci when co-expressed and a helicase 373

defective mutant of E1 does not form nuclear foci with E2 (11, 42). Although we could not 374

distinguish E1 and E2 due to the same tag, taking into account the previous report and the fact 375

that the HA positive nuclear foci were detectable only in cells in which expression of HA-E1 and 376

HA-E2 was expected, it is likely that those nuclear foci represent co-localization of E1 and E2. 377

The nuclear foci formation required helicase activity of E1 and well corresponded with the copy 378

number increase of HPV16 genomes, reinforcing the notion that E1 and E2 form nuclear foci to 379

support viral genome replication. No signals were detected in any of those cells when incubated 380

with a vehicle control, suggesting basal expression levels of exogenous E1 and E2 are very low 381

without DOX treatment (Fig. 1D and F). 382

Sakakibara et al. and Fradet-Turcotte et al. reported that E1 expression suppresses cell 383

proliferation (11, 12). Consistent with their results, we noted that the tetON E1+E2 cells and the 384

tetON E1 cells but not the tetON E1K483A+E2 cells and the tetON E2 cells appeared to be not 385

proliferating very well and some of those cells were detached from bottom of tissue culture 386

plates by 24 hours when incubated with DOX. We counted the cells which stayed attached to the 387

culture plates and found that the number of the tetON E1+E2 cells and the tetON E1 cells grown 388

in the presence of DOX for 24 hours was significantly less than those grown in the presence of a 389

20

vehicle control (Fig.1E, data not shown for the tetON E1 cells). On the other hand, no such 390

difference in the cell numbers was observed in the tetON E1K483A+E2 or the tetON E2 cells 391

(Fig.1E, data not shown for E2 alone). To investigate whether the apparent inhibition of cell 392

proliferation was caused by induction of apoptosis, markers of apoptosis such as caspase-3 and 393

poly (ADP ribose) polymerase-1 (PARP-1) were analyzed by Western blotting. The cells 394

detached from a tissue culture plate as well as the cells remaining attached were collected and 395

equivalent protein amounts of cell lysates were subjected to SDS-PAGE and subsequent Western 396

blot analysis (Fig.1F). The cleaved forms of PARP-1 and caspase-3 were detected in the cells 397

expressing E1 and E2 or E1 alone but not in the cells expressing E1K483A and E2 or E2 alone, 398

suggesting that E1 expression induces apoptosis depending on its helicase activity. 399

HPV16 E1 induces activation of NFκB in human cervical keratinocytes containing 400

episomal HPV16 genomes. 401

To explore associations of NFκB with viral genome replication, we first studied the status of 402

NFκB p65 (RELA) and IκBα in HCK1T-HPV16 cells after induction of E1, E1K483A and/or E2 403

expression. We found that the steady state level of IκBα was reduced upon induction of E1 and 404

E2 or E1 alone but not E1K483A and E2 or E2 alone (Fig.2A, lanes 2 and 8 compared to lanes 1 405

and 7, respectively). On the other hand, the levels of RELA remained comparable. As expected, 406

nuclear translocation of RELA was detected in the cells containing HA positive nuclear foci 407

upon induction of E1 and E2 expression, while RELA was localized to cytoplasm in a majority 408

of the cells incubated with a vehicle control (Fig.2B). These results indicated that NFκB is 409

activated upon induction of E1 and E2 expression. Then transcriptional activation of several 410

NFκB target genes was examined (Fig.2C). Total RNA was isolated following incubation of 411

21

DOX or vehicle and subjected to reverse transcription (RT)-qPCR. All the NFκB target genes 412

examined were up-regulated upon induction of E1 and E2 expression. The mRNA levels of 413

TNF-α, interleukin (IL) 6 and IL8 were increased by 2.5 +/- 0.11, 4.9+/- 1.17 and 14.2 +/- 3.46 414

fold, respectively, whereas no significant increase of these mRNA levels was detected upon 415

induction of E1K483A and E2 expression. Taken together, these results indicate that the expression 416

of E1 and E2 induces activation of NFκB most likely through promoting degradation of IκBα. 417

Viral early transcripts containing spliced E1^E4 ORF were slightly reduced in the tetON E1+E2 418

cells (0.88+/- 0.12) even though the viral copy number increased (Fig.1A). It is possible that 419

exogenous E2 expression suppressed the viral early promoter, p97. However, in the 420

E1K483A+E2 cells, similar reduction of the viral early transcripts was observed (0.73+/-0.12) 421

when neither NFκB activation nor viral genome replication was induced. Therefore, no increase 422

of viral early transcripts despite the increased viral genome copies was likely associated with 423

suppression of p97 by NFκB activation in the tetON E1+E2 cells (31). 424

HPV16 E1 induces activation of NFκB in human cervical keratinocytes in the absence of 425

an episomal HPV genome. 426

We next examined whether E1 can reduce the IκBα level in the absence of an episomal HPV16 427

genome. Parental HCK1T cells were used to establish new cell lines expressing HA-tagged E1 428

and/or 3xFLAG-tagged E2 of HPV16 upon DOX administration. The steady state level of IκBα 429

was reduced in the parental HCK1T cells upon expression of E1 and E2 as well as E1 alone but 430

not E2 alone (Fig.3A, lanes 4 and 6 compared to lanes 3 and 5, respectively), indicating that 431

replication of the viral genomes is not necessary for NFκB activation, E1 expression per se rather 432

causing the activation. Reduction of the IκBα level appeared more prominent in the cells 433

22

expressing only E1 compared to E1 and E2, most likely due to higher expression of E1 (Fig.3A, 434

lanes 4 and 6). By switching the tag from HA to 3xFLAG, we were able to detect E2 by Western 435

blotting. Consistent with reduction of the IκBα level, NFκB target genes were up-regulated upon 436

expression of E1 and E2 as well as E1 alone in the absence of an HPV16 genome (Fig.3B). We 437

noted that endogenous mRNA levels of NFκB target genes were lower in parental HCK1T cells 438

than in HCK1T-HPV16. In addition, endogenous levels of the NFκB target genes were also 439

higher in CIN612-9E cells harboring episomal HPV31 genomes, compared to HCK1T, 440

suggesting constitutive activation of NFκB in the presence of episomal HPV genomes and this 441

was not specific to HCK1T-HPV16 (Fig.3C). Similar to HCK1T-HPV16, cell proliferation of 442

HCK1T was also inhibited and cleaved forms of caspase-3 and PARP-1 were detected upon 443

expression of E1 alone or E1 and E2, but not E2 alone (data not shown). These results are 444

consistent with previous findings that E1-mediated growth suppression does not require E2 or 445

viral genome replication (12, 13). 446

Inhibition of NFκB activation enhances E1-dependent replication of HPV16 genomes. 447

As induction of NFκB activation by E1 has never been described, we were interested in whether 448

this activation of NFκB affects E1-dependent replication of the viral genome. To this end, we 449

examined effects of IκBα overexpression, which prevents activation of NFκB, on E1-dependent 450

replication. As shown in Fig.1, the tetON E1+E2 cells proved to be a heterogeneous cell 451

population in which roughly 40% of the cells express HA-tagged proteins upon DOX treatment. 452

We therefore tried to obtain a clonal cell population expressing E1 and E2 more homogenously 453

upon induction. A total of 50 clones were isolated by limiting dilution and examined for the copy 454

number of HPV16 genomes and the levels of E1 and E2 expression in the presence or absence of 455

23

DOX. There were variations in the basal copy number of HPV16 genomes and the induction 456

levels of E1 among clones; some clones contained only a few copies of HPV16 genomes while 457

others contained 1,000 copies (data not shown). In most clones, the induction levels of E1 were 458

correlated with the increase in HPV16 copy numbers and levels of E1 and IκBα were inversely 459

correlated (Fig 4A) as expected from the results of the pooled population (Fig.2A). In clones A5 460

and B2, we were able to detect a band at a size corresponding to HA16E2 (Fig.4A, HA long 461

exposure), which could not be detected in the parental pooled population upon DOX treatment 462

(Fig.1F). We chose clones A5 and B2 for further studies as these showed the sharpest increase of 463

the viral genomes after DOX addition. The basal copy number of HPV16 genomes in clones A5 464

and B2 were the average of the clones. Wild type IκBα (IκBαWT), an IκBαS32A/S36A mutant 465

(IκBαMT), which is resistant to phosphorylation and subsequent degradation, or an empty vector 466

(MCS) as a control were transduced to clones A5 and B2 by retroviral gene transfer. 467

Accumulation of exogenous IκBαWT as well as IκBαMT regardless of E1 and E2 expression 468

was confirmed while reduction of endogenous IκBα level upon induction of E1 and E2 469

expression was evident in the control cells (Fig.4B, shown for clone B2). TNFα treatment which 470

activates NFκB via degradation of IκBα to parental HCK1T-HPV16 was also included as a 471

positive control (Fig.4B, lanes 1 and 2). The NFκB target genes, TNF-α, IL6 and IL8, were 472

extremely up-regulated following incubation with DOX in the control cells (Fig 4C). In clone B2, 473

the mRNA levels of TNFα, IL6 and IL8 were increased by 65+/-7.6, 88 +/-24.6 and 289 +/-42 474

fold, respectively, upon DOX treatment. The higher levels of these mRNAs compared to those 475

detected in the parental heterogeneous population (Fig.2C) was most likely due to homogeneity 476

of the cells expressing high levels of E1 (Fig.4A left, HA bottom panel). Consistent with the 477

IκBα protein level, transcriptional up-regulation of the NFκB target genes upon induction of E1 478

24

and E2 expression was strongly inhibited although not completely abrogated in the cells 479

expressing IκBαWT as well as IκBαMT. These data verified that over-expression of IκBαWT as 480

well as IκBαMT indeed prevented NFκB activation induced by E1 expression. Of note, basal 481

levels of the NFκB target genes in the absence of DOX were significantly lower in the cells 482

expressing IκBαWT as well as IκBαMT compared to the control cells (MCS), suggesting that 483

over-expression of IκBα also suppresses constitutive activation of NFκB seen in the presence of 484

HPV16 genomes (Fig.3C). We then analyzed replication of the HPV16 genome and cell 485

proliferation upon E1 and E2 expression in the presence of IκBα over-expression (Fig. 4D and E). 486

Interestingly, the copy number of HPV16 genomes following DOX incubation in the cells 487

expressing IκBαWT as well as IκBαMT was significantly higher than that in the control cells, 488

suggesting that the activation of NFκB suppresses E1-dependent replication of the HPV16 489

genome (Fig.4D). The basal copy number of HPV16 genomes in the cells expressing IκBαWT as 490

well as IκBαMT was slightly higher than that in the control cells. Although the difference was 491

not statistically significant, it is possible that the basal activity of NFκB also influences the 492

control of HPV16 copy numbers in the absence of exogenous E1 as discussed later. We noted 493

that the protein level of HA-E1 in the cells expressing IκBαMT appeared to be higher than that in 494

MCS control cells. The elevated level of HA-E1 might have contributed to augmented 495

replication of the HPV16 genome in cells expressing IκBαMT. In contrast, the inhibitory action 496

of E1 on cell proliferation was not affected by over-expression of IκBαWT or IκBαMT (Fig. 4E), 497

suggesting that activation of NFκB is not involved in E1-induced growth suppression or 498

apoptosis. The levels of viral transcripts containing spliced E1^E4 ORF were approximately 1.5 499

and 1.7 fold higher in the cells expressing IκBαWT and IκBαMT, respectively, than that in the 500

control cells in the absence of DOX, implying that the basal activity of NFκB might suppress 501

25

p97 to some extent. The viral transcripts were increased by 2 fold in the control cells and by 5.6 502

fold in the cells with IκBαWT or IκBαMT upon DOX treatment. We still considered these results 503

that activation of NFκB suppresses p97 and that over-expression of IκBαWT or IκBαMT 504

substantially alleviated the NFκB-mediated suppression (Fig.3D). Overall, these data suggest 505

that activation of NFκB limits E1-dependent replication of the HPV16 genome while it is not 506

involved in suppressive effects of E1 on cell proliferation. We repeated the same experiments 507

using clone A5 and obtained essentially the same results as with clone B2 (data not shown). 508

E1 induces NFκB activation by eliciting DNA damage response (DDR) pathways. 509

NFκB can be activated by a variety of upstream signals including DDR. Upon generation of 510

double stranded breaks (DSBs), ATM has been shown to activate NFκB through phosphorylation 511

dependent degradation of IκBα (43). Because the wild type but not the helicase defective mutant 512

of HPV16 E1 has been shown to activate ATM, we initially hypothesized that E1 induces NFκB 513

activation by eliciting DDR, most likely via activation of ATM. To test this hypothesis, 514

activation of DDR was analyzed in heterogeneous cell populations of tetON E1+E2, tetON 515

E1K483A+E2, tetON E1 or tetON E2 or several clones of the tetON E1+E2 cells by Western 516

blotting. Checkpoint kinase 1 and 2 (CHK1 and CHK2) are known phosphorylation targets of 517

ATR and ATM that mediate signal transduction downstream of ATR and ATM, respectively. 518

Antibodies specific to phosphorylated ATM, pATM(S1981) and CHK2, pCHK2(T68), or CHK1, 519

pCHK1(S345), were used as markers for ATM or ATR activation, respectively. NBS1 was also 520

analyzed as it has been shown to be phosphorylated by ATM (5, 44). Upon DOX incubation, 521

phosphorylation of ATM and CHK1 was increased in the cell populations of tetON E1+E2 and 522

tetON E1 though basal levels of the phosphorylated CHK1 appeared also higher in these cells 523

26

(Fig.5A, left panel). A slower migrating pattern of NBS1 and CHK2 upon DOX incubation was 524

detected, indicating that this was attributable to ATM-mediated phosphorylation of these proteins. 525

In clones of the tetON E1+E2 cells, the phosphorylation of ATM, CHK1 and CHK2 was also 526

increased following incubation with DOX and a slower migrating pattern of NBS1 and CHK2 527

was detected. On the other hand, none of those indications for DDR activation could be detected 528

in the cell populations of tetON E1K483A+E2 and tetON E2, as expected. These results 529

indicated that ATM and ATR pathways are both activated upon induction of E1 and the 530

activation of DDR depends on its helicase activity. We then examined reduction of the IκBα 531

level in the presence of DDR inhibitors such as Wortmannin and KU55933 upon E1 and E2 532

expression. Wortmannin is a broad inhibitor of PI3Ks shown to inhibit ATM, ATR and DNA-533

PKcs while KU55933 is a specific inhibitor of ATM. Clone B2 was pre-incubated with indicated 534

inhibitors at 2 hour prior to addition of DOX and the cell lysates were collected at the indicated 535

time points in the presence or absence of the inhibitors. Activation of DDR and reduction of the 536

IκBα level were analyzed by Western blotting (Fig.5B). A high level of E1 expression was 537

detected at 8 hours following DOX addition. Concomitantly, a slower migrating pattern of NBS1 538

and CHK2 and the phosphorylated form of CHK1 at S345 and CHK2 at T68 were detected and 539

they continued to accumulate up to 24 hours in DMSO treated control cells. Reduction of the 540

IκBα level was also evident as early as 8 hours following DOX addition and continued thereafter 541

(Fig.5B, lanes 1 to 4). In the presence of Wortmannin, a slower migrating pattern of NBS1 was 542

less prominent though present, and appearance of the phosphorylated CHK1 and CHK2 delayed; 543

they were first detected at 16 hours following incubation with DOX as compared to 8 hours in 544

DMSO treated controls. The time dependent accumulation of the phosphorylated CHK1 and 545

CHK2 was reduced although not completely abrogated in the presence of Wortmannin, 546

27

indicating that ATM as well as ATR mediated signal transduction was suppressed albeit not 547

completely. Interestingly, the IκBα level following DOX addition was maintained in the 548

presence of Wortmannin, suggesting that activation of DDR signals results in reduction of the 549

IκBα level (Fig.5B, lanes 5 to 8). In the presence of KU55933, a slower migrating pattern of 550

NBS1 was almost completely absent while the phosphorylation of CHK1 was increased at a level 551

and with kinetics similar to that seen in DMSO treated cells. The phosphorylated form and 552

slower migrating pattern of CHK2 were reduced though not completely abrogated in the 553

presence of KU55933, indicating that ATM but not ATR mediated signals were suppressed as 554

expected (Fig.5B, lanes 9 to 12). The inhibitory effects of KU55933 on reduction of the IκBα 555

level following induction of E1 and E2 expression was much weaker than that of Wortmannin. In 556

the presence of KU55933, reduction of the IκBα level was not observed at 8 hours but became 557

evident at 24 hours following DOX incubation, suggesting that ATM signals partially contribute 558

to reduction of the IκBα level upon E1 and E2 expression. Furthermore, activation of the NFκB 559

target genes was strongly suppressed in the presence of Wortmannin upon induction of E1 and 560

E2 expression, corresponding with the level of IκBα (Fig.5C, left panel). Similar to the 561

overexpression of IκBα, the basal levels of the NFκB target genes in the absence of DOX were 562

significantly lower in the presence of Wortmannin than that in a control. The activation of NFκB 563

target genes was also suppressed in the presence of KU55933. The difference in those mRNA 564

levels between the presence and absence of KU55933 was statistically significant. However, the 565

suppression by KU55933 treatment was not as potent as by Wortmannin treatment, consistent 566

with the extent of the IκBα reduction (Fig.5C, right panel). The levels of the NFκB target genes 567

in the absence of DOX were comparable in the presence and absence of KU55933. The levels of 568

viral early transcripts containing E1^E4 spliced ORF were comparable between DOX treated and 569

28

untreated samples regardless of the presence of the inhibitors. These results suggested that the 570

activation of DDR upon E1 expression results in activation of NFκB, most likely by promoting 571

IκBα degradation. We also examined the effects of Wortmannin and KU55933 on E1-dependent 572

replication of the HPV16 genome (Fig.5D). The viral copy number following DOX incubation 573

was significantly higher in the presence of Wortmannin than that in a DMSO control. On the 574

other hand, they were only slightly higher in the presence of KU55933 following DOX 575

incubation, without statistical significance. The copy number of HPV16 genomes without DOX 576

incubation was comparable among the cells treated with inhibitors and a DMSO control. These 577

results suggested that activation of DDR results in suppression of E1-dependent replication of 578

the viral genome and this suppression is primarily mediated by DDR pathways other than the 579

ATM mediated pathways. Furthermore, when the effects of Wortmannin and KU55933 were 580

investigated, reduction of cell numbers after incubation with DOX was observed regardless of 581

the presence of inhibitors (Fig.5E). Given the fact that Wortmannin as well as KU55933 582

treatment itself modestly inhibited cell proliferation in the absence of DOX, it was not clear 583

whether activation of DDR is involved in inhibitory action of E1 on cell proliferation. Similar 584

results were obtained upon treatment of those inhibitors to parental cell populations of tetON 585

E1+E2 and some of other clones (data not shown). Importantly, the effects of Wortmannin and 586

KU55933 on E1-dependent replication of the viral genome were well correlated with those on 587

reduction of the IκBα level and proved collinear with overexpression of IκBα (Fig.4). 588

Collectively, these results suggest that activation of DDR signals limits E1-dependent replication 589

of HPV16 genome through activation of NFκB. The subtle effects of KU55933 on E1-dependent 590

replication and suppression of cell growth were consistent with previous reports (12, 13). 591

29

We noted that the protein level of E1 was highest at 8 hours following DOX incubation and 592

then gradually decreased, despite the continued presence of DOX (Fig.5B, lanes 1 to 4). In 593

contrast, such decrease in the E1 protein level was completely blocked and accumulation during 594

the observation period was apparent up to 24 hours with Wortmannin treatment (Fig.5B, lanes 5 595

to 8). The decrease in the E1 level was modestly alleviated by KU55933 treatment (Fig.5B, lanes 596

9 to 12). The results were reproducible in several independent experiments. These observations 597

led us to hypothesize that the DDR-dependent activation of NFκB disturbs the protein stability of 598

E1. To eliminate the possibility that Wortmannin somehow enhanced transcription of E1 from a 599

tetracycline inducible promoter, the mRNA level of exogenous HA-E1 was analyzed in the 600

presence or absence of Wortmannin (Fig.5F). The mRNA level of HA-E1 at 24 hours following 601

DOX induction was comparable between the cells treated with Wortmannin and DMSO, 602

indicating that the higher level of E1 protein in the presence of the inhibitor was not due to 603

enhanced transcription. We also considered the possibility that lysis of E1 is partial under our 604

lysis conditions and that treatment of Wortmannin may alter solubility of E1. To address whether 605

our lysis method is responsible for the seemingly increased level of E1 in the presence of 606

Wortmannin, we lysed an insoluble fraction in an SDS sample buffer after extraction of a soluble 607

fraction and the levels of E1 were analyzed in those fractions from equal numbers of cells. In 608

addition, the cells were directly lysed in an SDS sample buffer for comparison (Fig.5G). 609

Minichromosome maintenance protein (MCM) 2, histone H3 and vinculin were also detected to 610

indicate solubility of nuclear, chromatin and cytoplasmic proteins, respectively, under these 611

conditions. Under our lysis conditions, E1 was detected mostly in the soluble fraction (Fig.5G, 612

lanes 1, 3 and 5) and very little E1 remained in the insoluble fraction (Fig.5G, lanes 2, 4 and 6). 613

MCM2 and vinculin were detected mostly in the soluble fraction while histone H3 was detected 614

30

in both. All proteins were well extracted after direct lysis in an SDS sample buffer as expected 615

(Fig.5G, lanes 7 to 9). The level of E1 was higher in the presence of Wortmannin compared to 616

the vehicle treated control regardless of the lysis methods tested, demonstrating that the 617

increased level of E1 was not due to increased solubility (Fig.5G, lanes 3, 5, 8 and 9). 618

ATR, but not DNA-PKcs and p38 mitogen-activated protein kinase (p38MAPK), is 619

involved in E1-dependent activation of NFκB 620

It is reported that p38MAPK activated upon UV exposure induces NFκB activation through 621

casein kinase II (CKII)- and p38MAPK-dependent phosphorylation of IκBα at its C-terminal 622

proline, glutamic acid, serine and threonine-rich (PEST) domain (45, 46). UV induced activation 623

of p38MAPK is thought to be independent of ATM. Because our results suggested that E1 624

induces reduction of the IκBα level by eliciting DDR and this reduction is not primarily mediated 625

by kinase activity of ATM, we questioned whether p38MAPK might be involved. Activation of 626

p38MAPK was investigated using an antibody specific to phosphorylated p38MAPK at T180 627

and Y182, an indicator of p38MAPK activation. Phosphorylation of p38MAPK was increased 628

following incubation with DOX in the pooled cell populations of tetON E1+E2 and tetON E1 629

(Fig.6A) and in clones of tetON E1+E2 cells (Fig.6B, lanes 1 and 2), while it remained 630

unaffected in the cell populations of tetON E1K483A+E2 and tetON E2. These results suggested 631

that E1 induces activation of p38MAPK in a manner dependent on its helicase activity, akin to 632

the activation of DDR. To investigate if the E1 induced activation of p38MAPK is mediated by 633

DDR, the effects of Wortmannin and KU55933 on p38MAPK was examined in clone B2 634

(Fig.6B). In the presence of KU55933, the phosphorylation of p38MAPK was increased to a 635

comparable level as in the presence of the DMSO control upon incubation with DOX, indicating 636

31

that the E1-induced activation of p38MAPK is independent of ATM. Phosphorylation of 637

p38MAPK was increased during 2 hours of pre-incubation with Wortmannin. Although further 638

accumulation of the phosphorylated form was not observed after incubation with DOX, it was 639

not clear whether the E1-induced activation of DDR other than ATM is involved in the 640

activation of p38MAPK. We also examined activation of DNA-PKcs upon E1 and E2 expression 641

as phosphorylation of DNA-PKcs at Serine 2056 (S2056) has been shown to occur in response to 642

DNA damage (47). Phosphorylation of DNA-PKcs was indeed increased upon E1 and E2 643

expression and this was abrogated in the presence of Wortmannin (Fig.6B). The treatment of 644

KU55933 showed little to no effect on the phosphorylation of DNA-PKcs, suggesting that the 645

activation of DNA-PKcs upon E1 and E2 expression is independent of ATM. To explore which 646

one of these molecules mediates the E1 dependent activation of NFκB, the levels of E1 and IκBα 647

at 24 hours after addition of DOX were investigated in the presence of VE-822, Nu7026 and 648

SB203580, specific inhibitors of ATR, DNA-PKcs and p38MAPK, respectively (Fig.6C). In 649

comparison with a vehicle treated control, increased levels of E1 were detected in the presence of 650

Wortmannin and VE-822, among those inhibitors, implying that ATR activity is involved in 651

regulation of the protein stability of E1 (Fig.6C, bottom panel for quantification). On the other 652

hand, apparent inhibition of IκBα reduction was only seen in the presence of Wortmannin. To 653

further analyze the effects of VE-822 on the levels of IκBα and E1, a time course experiment 654

was carried out with HCK1T-HPV16 tetON E1+E2 clone B2 as described in Fig.5. In the 655

presence of VE-822, the phosphorylation of CHK1 was almost completely abrogated, verifying 656

that VE-822 indeed inhibits ATR activity. Importantly, progressive decrease of E1 as well as 657

IκBα levels following DOX addition was prevented, at least till 16 hours, then became evident at 658

24 hours after DOX addition in the presence of VE-822, suggesting that ATR is involved in the 659

32

E1-dependent activation of NFκB and subsequent destabilization of E1. We then examined E1-660

dependent replication of the HPV16 genome and found it was enhanced in the presence of VE-661

822, compared to the control. The basal copy number of HPV16 genomes was also slightly 662

increased in the absence of DOX (Fig.6E). On the other hand, inhibition of cell proliferation was 663

still seen in the presence of VE-822 upon expression of E1 and E2 (Fig.6F). Because the 664

treatment of VE-822 itself modestly suppressed cell proliferation in the absence of DOX, it was 665

not clear whether activation of ATR mediates inhibition of cell proliferation by E1 expression. 666

We found no difference in the levels of E1 and IκBα examined by time course experiments and 667

in E1-dependent replication of the viral genomes in the presence of Nu7026 or SB203580 668

compared to the control (data not shown). The effect of VE-822 in inhibiting reduction of the 669

IκBα level was more potent than that of KU55933 but not Wortmannin, suggesting that ATR and 670

ATM play major and minor roles, respectively, in the E1-dependent activation of NFκB. Since 671

the expression level of E2 could also affect viral genome replication, we analyzed the stability of 672

E2 in the presence of Wortmannin and VE-822 (Fig.6G). To this end, we generated a 673

heterogeneous cell population of HCK1T-HPV16 with tetON inducible expression of HA-E1 674

and 3xFLAG-tagged E2 by lentivirus gene transfer, as HA-tagged E2 was very difficult to detect 675

in our hands. HCK1T-HPV16 tetON HA-E1 and FLAG-E2 induced replication of the HPV16 676

genome as well as original tetON E1+E2 cells did with DOX incubation (data knot shown). A 677

time course experiment revealed that the expression level of FLAG-E2 was overall constant for 678

at least 24 hours and not affected by the presence of Wortmannin or VE-822, indicating that 679

DDR and/or NFκB induced-destabilization is specific to E1. All results could be replicated with 680

clone A5 (data not shown). 681

33

Activation of NFκB mediates DDR induced-destabilization of E1 and promotes 682

polyubiquitination of E1. 683

To address whether the activation of NFκB indeed mediates DDR induced destabilization of 684

E1, a time course experiment was performed with HCK1T-HPV16 tetON E1+E2 clone B2 685

transduced with IκBαWT, IκBαMT or MCS. Following DOX incubation, phosphorylation of 686

CHK1 was increased and a slower migrating pattern of NBS1 appeared, with kinetics and levels 687

comparable among the cells transduced with MCS, IκBαWT and IκBαMT (Fig.7A). These 688

results indicated that over-expression of IκBα does not disrupt the activation of ATM or ATR 689

mediated DDRs. As expected, progressive decrease of the E1 level was evident in the MCS 690

control cells while this was alleviated in the cells expressing IκBαMT, though accumulation of 691

E1 protein was not so evident as that in Wortmannin-treated cells. In the cells expressing 692

IκBαWT, progressive decrease of the E1 level was also observed, but the level remained 693

consistently higher than that in the MCS control cells. The mRNA levels of HA-E1 were 694

comparable between the MCS control and cells expressing IκBαWT, while it was modestly 695

decreased in cells expressing IκBαMT (Fig.7B), indicating that the protein level of E1 in 696

IκBαMT expressing cells was increased independently of transcriptional up-regulation. 697

Furthermore, we examined the effects of a proteasome inhibitor, Epoximicin, on the protein 698

levels of HA-E1 and IκBα (Fig.7C). Because treatment of Epoximicin caused significant damage 699

to cells regardless of DOX incubation, cell lysates of HCK1T-HPV16 tetON E1+E2 clone B2 700

were isolated in the presence or absence of Epoximicin at 12 hours after incubation with vehicle 701

or DOX, the earliest possible time at which destabilization of E1 could be detected. As expected, 702

the protein level of E1 was elevated in the presence of Epoximicin compared to the control 703

(Fig.7C, lanes 2 and 4). Proteasome inhibitors have been shown to inhibit degradation of IκBα 704

34

and subsequent activation of NFκB (48). Although the treatment with Epoximicin somehow 705

reduced the steady state level of IκBα in the absence of DOX, further reduction of IκBα upon E1 706

and E2 expression was abrogated in the presence of Epoximicin. These results could be 707

replicated with clone A5 (data not shown). While the findings were compatible with our 708

hypothesis that activation of DDR-NFκB may induce proteasomal degradation of E1, 709

involvement of proteasomes in destabilization of E1 needed to be further clarified. Therefore, we 710

carried out an ubiquitin conjugation assay using 293FT cells to directly analyze the 711

polyubiquitination of E1. A lentivirus vector expressing HA-E1 under control of the CMV 712

promoter, CSII-CMV-HA16E1, and an expression vector of 6xHis tagged ubiquitin (pCMV-713

6xHis-Ub) were co-transfected into 293FT cells with an expression vector of IκBαMT or an 714

empty vector and cell lysates were collected at 48 hours thereafter. The HA-E1 was 715

immunoprecipitated using magnetic beads conjugated with anti-HA antibodies and subsequently 716

analyzed by Western blotting. The effect of Wortmannin was investigated by incubation from 20 717

hours post-transfection till harvesting. The GFP expression vector was also included in all 718

transfections to monitor the efficiency of the transfections. We found that E1 expression did not 719

conspicuously reduce the IκBα level in 293FT cells (Fig.7D, lanes 7 and 8). To rule out the 720

possibility that co-expression of His-tagged ubiquitin hinders the E1-induced reduction of IκBα, 721

293FT cells were also transfected without pCMV-6xHis-Ub. Regardless of co-expression of His-722

tagged ubiquitin, reduction of the IκBα level was barely detected and effects of Wortmannin on 723

levels of E1 and IκBα were inconspicuous (Fig.7D, lanes 1, 2, 3 and 7, 8, 12). To potentiate 724

NFκB activation in 293FT cells expressing E1, TNFα was added at 20 hours-post transfection. 725

As expected, this TNFα treatment reduced the IκBα level and, to lesser extent, that of E1, 726

compared to the vehicle treated control (Fig.7D, compare lane 2 with lane 4 and lane 8 with lane 727

35

10). In cells co-transfected with an IκBαMT expression vector, the level of E1 appeared lower, 728

probably due to the lower efficiency of the transfection as indicated by GFP expression (Fig.7D, 729

lanes 5, 6, 11 and 12). Nonetheless, protein ladders were detected in the immunoprecpitates at a 730

size above 100 kDa, bigger than the E1 (around 70kDa), indicating polyubiquitination of E1 731

(Fig.7D right). Strikingly, TNFα treatment strongly enhanced polyubiquitination of E1 and this 732

was abolished with the expression of IκBαMT (Fig.7D, lanes 16 and 18). Consistent with the 733

results of Western blotting, effects of Wortmannin on polyubiquitination of E1 were barely 734

detected. These results suggest that activation of NFκB pathway promotes ubiquitin-dependent 735

proteasomal degradation of E1, though the precise mechanisms remained unclear. 736

E1 and E2 expression results in accumulation of cells in S and G2 phases. 737

Several studies have shown that expression of E1 and E2 results in accumulation of cells in S 738

and G2 phases (11-13). To investigate a temporal relationship between the levels of E1 and the 739

cell cycle progression in our system, the cell cycle profile was analyzed at the indicated time 740

points following DOX addition in clones B2 (Fig.8). Accumulation of cells in S and G2/M 741

phases was already evident at 8 hours and increased by 16 hours following induction of E1 and 742

E2 expression. The percentage of the cells in S phase was consistently higher (32% in S and 23% 743

in G2 at 8 hours) than that in G2 phase until 16 hours after DOX addition (46% in S and 26% in 744

G2 phase at 16 hours), suggesting that expression of E1 and E2 delayed S phase progression, 745

consistent with previous reports (12, 13). A proportion of the cells progressed to G2 phase by 24 746

hours. The percentage of the cells in G2 phase (40%) exceeded that of the cells in S phase (33%) 747

at 24 hours and remained consistently higher up to 40 hours with incubation of DOX. Cells 748

containing less than 2N DNA (sub-G1) appeared at 24 hours, coinciding with the detection of 749

36

cleaved forms of PARP-1 and caspase-3 (Fig.1F). These results were replicated with clone A5 750

(data not shown). Taken together with the observation that the level of E1 was highest between 8 751

and 16 hours and progressively reduced by 24 hours (Fig.4, 5 and 6), these results suggest that 752

E1 and E2 expression initially induces cell cycle retardation in S phase, which is attenuated as 753

the level of E1 reduces. 754

NFκB activity regulates copy number of the HPV genome during maintenance replication. 755

As mentioned above, endogenous levels of the NFκB target genes were consistently higher in 756

human keratinocytes stably maintaining episomal HPV genomes than in HCK1T cells (Fig.3C 757

and D). We also noted a slight increase of the HPV16 genome copy number in HCK1T-HPV16 758

tetON E1+E2 cells introduced with IκBαMT compared to MCS in the absence of DOX (Fig.4D). 759

Since our results suggested that NFκB activation limits E1-dependent replication, we postulated 760

that NFκB activity could be involved in maintenance of the HPV genome in proliferating 761

keratinocytes. Therefore, IκBαMT was introduced into parental HCK1T-HPV16 by retrovirus 762

gene transfer as described in Fig.4 and the copy number of HPV16 genomes was measured at 5 763

days after drug selection was completed (Fig.9A, black columns). As expected, the copy number 764

was increased by 30% with statistical significance in the cells expressing IκBαMT compared to 765

the MCS control. Conversely, the copy number of HPV16 genomes was decreased by 766

approximately 50% with incubation of TNFα for 5 days. The copy number of the HPV31 767

genome in CIN612-9E cells was also decreased by TNFα in a dose dependent manner (Fig.9B, 768

black columns). While expression of IκBαMT did not affect cell proliferation, TNFα treatment 769

modestly suppressed cell proliferation in both cell lines (Fig.9A and B, white columns). These 770

37

results imply that constitutive activation of NFκB in the presence of episomal HPV is involved in 771

regulation of maintenance replication of the viral genome. 772

773

38

Discussion 774

Papillomaviruses undergo three phases of genome replication, the initial amplification, 775

maintenance replication and productive amplification (38). While the underlying mechanisms for 776

the differentiation-dependent productive amplification are becoming gradually clear, virtually 777

nothing is known about the molecular mechanisms for transition from the initial amplification to 778

maintenance replication within the basal compartment. Our results showed that NFκB is 779

activated when E1-dependent replication of the HPV16 genome is induced in undifferentiated, 780

basal-like human cervical keratinocytes and inhibition of this NFκB activation enhances E1-781

dependent replication of the HPV16 genome. Thus, our results suggest that E1 and NFκB may 782

form a negative feedback loop to regulate E1-dependent replication of the HPV16 genome 783

(Fig.9C). The stability of E1 was augmented when activation of NFκB was abrogated in the 784

presence of DDR inhibitors or IκBαMT and activation of NFκB by TNFα facilitated 785

polyubiquitination of E1, suggesting that DDR-NFκB signaling may promote degradation of E1. 786

Furthermore, constitutive activation of NFκB was evident in human keratinocytes stably 787

maintaining episomal HPV genomes and inhibition or potentiation of the NFκB activity 788

respectively resulted in increase or decrease of the viral copy numbers (Fig.9A and B). Based on 789

these results, we propose a possible model featuring involvement of an E1-NFκB feedback loop 790

in the establishment and maintenance of persistent HPV infection. This feedback loop could be 791

further ensured by transcriptional inhibition of the viral early promoter, which drives the 792

expression of early genes including E1, by active NFκB. To our knowledge, this is the first 793

evidence implicating NFκB activity in regulation of papillomavirus genome replication. 794

Activation of NFκB by E1 through DDR 795

39

Our results showed that NFκB is activated as a consequence of E1-induced DDR, primarily 796

via the ATR mediated pathway (Fig.6). These results were rather surprising to us since the best 797

studied pathway of NFκB activation upon DNA damage is ATM-mediated induction of IκBα 798

degradation. ATM has been shown to activate the IκB kinase (IKK) complex by directly 799

phosphorylating its regulatory subunit, the NFκB essential modulator (NEMO), and this 800

activation of IKK leads to phosphorylation-dependent degradation of IκBα (49). Interestingly, it 801

was recently shown that degradation of NEMO is enhanced in human keratinocytes containing 802

episomal HPV16 genomes, compared to primary human keratinocytes (50). Therefore, activation 803

of NFκB by the ATM-mediated pathway through NEMO could be inherently limited in HCK1T-804

HPV16 cells. We failed to detect phosphorylation of the IKK complex upon E1 and E2 805

expression and found that an inhibitor of IKKs, IKK inhibitor VII, had no effect on E1-806

dependent replication (T.Nakahara and T.Kiyono, unpublished data). These results are consistent 807

with a modest effect of KU55933 on reduction of the IκBα level, as the ATM mediated 808

activation of NFκB was shown to be dependent of IKKs. The absence of IKK phosphorylation 809

also implies that ATR mediated reduction of IκBα might be independent of IKKs. Similar to our 810

results, it was reported that degradation of IκBα was induced by doxorubicin, a DNA damaging 811

agent, in mouse embryonic fibroblasts (MEFs) devoid of IKKs and this was independent of 812

phosphorylation at S32 and S36 as well as the PEST domain of IκBα. LY294002, a PI3K 813

inhibitor, partially blocked the degradation of IκBα (51). Whereas the level of IκBα was 814

maintained throughout a 24 hours observation period in the presence of Wortmannin, it was 815

decreased after 16 hours following DOX addition even though phosphorylation of CHK1 was 816

more efficiently suppressed throughout in the presence of VE-822 (Fig.6D). The greater effect of 817

Wortmannin in inhibiting E1-induced reduction of IκBα could be due to its pleiotropic effects, 818

40

suppressing both ATM and ATR pathways. Alternatively, it is possible that other PI3Ks are 819

involved. Since no data are available directly implicating ATR in NFκB activation in the 820

literature thus far, further studies are necessary to elucidate the mechanism(s) for ATR mediated 821

activation of NFκB. We also found that E1 induces activation of p38MAPK in a manner 822

dependent on its helicase activity but independent of DDR (Fig.6). Although it is beyond a scope 823

of our current study, the significance of p38MAPK activation in the HPV life cycle warrants 824

future investigation. 825

Inhibition of E1-dependent replication of the viral genome by NFκB. 826

The time course experiments in the present study revealed that the expression level of E1 827

progressively decreased and this was abrogated in the presence of Wortmannin, VE-822 and 828

IκBαMT expression, suggesting that activation of NFκB causes destabilization of E1 (Fig.5, 6 829

and 7). Since the results with a proteasome inhibitor, Epoxicimin, did not provide a direct answer 830

to whether NFκB dependent-reduction of E1 is mediated by an ubiquitin-proteasome pathway, 831

we carried out an ubiquitin conjugation assay in 293FT cells and found polyubiquitination of E1 832

to be greatly enhanced by TNFα treatment (Fig.7D). Thus, it is likely that NFκB promotes 833

ubiquitin-dependent proteasomal degradation of E1. On the other hand, E1 expression scarcely 834

induced reduction of IκBα in 293FT cells. We observed increased phosphorylation of ATM and 835

CHK1 in 293FT cells expressing E1 (data not shown). The lack of conspicuous effect of E1 836

expression on the IκBα level in 293FT cells might be due to the presence of adenovirus E1A, 837

since it has been shown to suppress IκBα degradation (52), or to a cell type difference 838

downstream of DDR activation. It was previously shown that E1 of BPV-1 is targeted by 839

anaphase-promoting complex (APC) for degradation (53, 54). Although the question of whether 840

41

E1 proteins of HPVs, including HPV16, are also targeted by APC has not been resolved, it is 841

possible that the reduction of HPV16 E1 seen in this study was mediated by APC as HPV16 E1 842

contains three putative D-boxes (RxxL) and one KEN box, known recognition motifs for APC. 843

APC is a multicomponent ubiquitin ligase well known to regulate the cell cycle. Several recent 844

studies have indicated that APC is activated upon DNA damage in ATM-dependent as well as 845

ATM-independent manners (55-57). Although it is not clear whether NFκB was involved in the 846

DDR induced APC activation in these studies, it is reasonable to speculate that E1-dependent 847

activation of DDR induces activation of APC, possibly through NFκB. The fact that Wortmannin 848

treatment completely blocked the reduction of E1 while over-expression of IκBαMT blocked 849

reduction of E1 somewhat less efficiently suggests that NFκB-dependent as well as -independent 850

mechanisms for E1 degradation may exist downstream of DDR (Fig.5, 6 and 7). In the BPV1 851

study, polyubiquitination of BPV1 E1 was enhanced in the presence of BPV1 E2 and a plasmid 852

containing the replication origin of BPV-1 in vivo. We observed that a majority of the cells 853

accumulated in S phase by 16 hours and a proportion of these cells progressed to G2 phase by 24 854

hours following DOX treatment, indicating that the degradation of E1 in our experiments most 855

likely occurs during S phase in the presence of E2 and the viral genome (Fig.8). These results are 856

consistent with the finding that polyubiqutination of BPV-1 E1 is facilitated in the S phase 857

extract of 293 cells in vitro. Therefore, it seems likely that APC-mediated degradation of BPV-1 858

E1 in vivo also occurs in S phase and APC is activated by E1-induced DDR. However, our 859

results do not rule out the possibility that other E3 ubiquitin ligases are involved in DDR and/or 860

NFκB-dependent degradation of E1. Whereas several studies have highlighted a significance of 861

ATM activation for productive amplification, contribution of ATR activation to the HPV life 862

cycle has not been indicated. Our results also suggest that ATR may negatively regulate viral 863

42

genome replication through NFκB activation. Previously, King et al. reported that activation of 864

DDR by etoposide had no effect on E1-dependent replication in a transient replication assay 865

using 293T cells (58). Their results are in agreement with our finding of a minor effect of ATM 866

since etoposide primarily induces DSBs by inhibiting topoisomerase II (59). However, they 867

showed elevated phosphorylation of CHK1 by etopside treatment suggesting that ATR was also 868

activated. The limited effect of ATR activation on E1-dependent replication in their study could 869

be because 293T cells were used. The effect of E1 expression on IκBα reduction in 293FT cells 870

was likewise minor in our hands. On the other hand, Edwards et al. recently showed that 871

knockdown of ATR by siRNA and treatment with a CHK1 inhibitor for 2 days resulted in 872

reduction of the viral genome in W12 cells, isolated from a CIN1 biopsy containing HPV16, 873

contrary to our results (60). Since we incubated VE-822 at most for 26 hours, the longer duration 874

and/or differences in approaches to inhibition of the ATR-CHK1 pathway might have affected 875

the results through an NFkB-independent pathway. 876

It was shown recently that DNA damage induces simultaneous activation of NFκB and IRF7 877

and that IFN α and λ, but not ß, were subsequently produced in HeLa cells (61). Several studies 878

have demonstrated that chronic IFNß treatment reduces episomal copies of papillomavirus 879

genome in W12 cells for HPV16, in CIN612 cells for HPV31 and in mouse C127 cells 880

containing BPV-1(62-65). Conversely, it was found that IFN inducible genes were down-881

regulated in cells containing an episomal HPV genome and that loss of viral episomes is 882

associated with endogenous activation of type I IFN-induced antiviral genes (30, 66). The gene 883

product of IFN-induced protein with tetratricopeptide repeats 1 (IFIT1, a.k.a. ISG56) was 884

demonstrated to inhibit E1-dependent replication of viral DNA by direct binding to HPV18 E1 in 885

transient replication assays (67, 68). Our results do not exclude the possibility that E1-dependent 886

43

activation of NFκB results in up-regulation of type I IFNs and these subsequently inhibit the 887

activity of E1 with regard to viral genome replication. This might explain our finding that 888

expression of IκBαMT enhanced E1-dependent replication of the viral genome very well while it 889

attenuated the degradation of E1 less efficiently than did Wortmannin treatment. 890

A possible function of the NFκB-mediated feedback loop in the viral life cycle 891

In our previous study, we demonstrated that E1 of HPV16 is required for initial and productive 892

amplification while it is dispensable for maintenance replication. Interestingly, Hoffmann et al. 893

reported that HPV16 genomes in W12 cells are maintained by replicating in a once-per-S-phase 894

fashion while HPV31 genomes in CIN612-9E cells are maintained by a random-choice 895

mechanism and introduction of E1 expression from an exogenous promoter in W12 cells 896

switched HPV16 genome replication to a random-choice mechanism. The authors concluded that 897

there are two modes of genome replication, at least for HPV16 during the maintenance phase, 898

and choice of modes is associated with the expression level of E1 (69). In agreement with 899

previous studies (11, 12), our results also showed that excess expression of E1 could be 900

detrimental to cell proliferation in basal-like cervical keratinocytes (Fig.1). Thus, regulation of 901

the E1 protein level appeared to be an essential element in the viral life cycle for establishing 902

viral persistence and determining the mode of viral genome replication. Results of our present 903

study suggest that NFκB activity is involved in regulation of the E1 level and that NFκB 904

activation is induced by E1 itself, constituting a negative feedback loop in proliferating 905

keratinocytes. We propose that E1-dependent initial amplification following virus infection 906

induces NFκB activation and this in turn terminates the initial amplification by inducing 907

degradation of E1, mediating a transition to maintenance replication. Although the expression 908

44

level of exogenous E1 in our experiments could be much higher than that during initial 909

amplification, a lower level of E1 might be sufficient to induce NFκB activation. Karim et al. 910

reported that increase in NFκB activity was detected within 24 hours after infection with HPV16 911

virions isolated from raft cultures (50). Although their interpretation was that NFκB is activated 912

as part of an innate immune response, their results did not exclude the possibility that E1-913

dependent initial amplification contributes to activation of NFκB. Indeed, it is plausible that 914

innate immune responses to virus infection potentiate E1-induced activation of NFκB during the 915

initial amplification. In cervical keratinocytes stably maintaining episomal HPV genomes, 916

constitutive activation of NFκB was detected and manipulation of NFκB activity resulted in 917

increase or decrease of the viral copy numbers (Fig.3 and Fig.9). Although we cannot provide 918

direct evidence that the endogenous E1 level was changed by such manipulation, given that an 919

antibody sufficiently sensitive to detect endogenous E1 has yet to be developed, the results imply 920

that endogenous E1, if present, may induce constitutive activation of NFκB in the presence of 921

episomal HPV genomes and the NFκB activity regulates viral copy numbers through limiting or 922

completely abolishing E1. Thus, we speculate that NFκB activity may play a key role in 923

determining the mode of maintenance replication. Since several groups have shown that E1s of 924

various papillomaviruses induce activation of DDR (11-13), we believe that a negative feedback 925

loop between E1 and NFκB might constitute a common mechanism to regulate E1 levels in the 926

basal compartment of the infected tissue. Interestingly, it has been reported that NFκB is up-927

regulated in basal cells of a fraction of CIN1 when compared with normal tissues (22). The 928

reported percentage NFκB positivity in low grade cervical lesions has varied from study to study; 929

some authors have indicated up-regulation of NFκB is detected only in cervical cancer while 930

others indicated that it is already present in the basal compartment in 30% of CIN1 (17, 19). The 931

45

varied patterns of NFκB activation in the basal compartment of CIN1 may reflect the presence or 932

absence of E1 during the stable maintenance phase. It is also possible that expression of other 933

viral proteins such as E6 contributes to constitutive activation of NFκB in the presence of HPV 934

genomes, as E6 of HPV16 has been shown to induce NFκB by at least two mechanisms (28, 70). 935

Recently, Gunasekharan and Laimins reported that a cellular microRNA, miR-145, contains a 936

seed sequence matching E1 and E2 ORFs of HPV31 and that its over-expression resulted in 937

decrease of viral copy numbers in CIN612-9E under undifferentiating as well as differentiating 938

culture conditions. The level of miR-145 was found to be high in a monolayer culture but 939

suppressed upon differentiation, suggested that miR-145 suppresses E1 and/or E2 levels in basal 940

cells and that the suppression is removed upon differentiation. Interestingly, they found two 941

RELA binding sites in the promoter of miR-145. Although experimental data that NFκB 942

upregulates miR-145 has yet been provided, effects of TNFα on viral genomes could also have 943

been mediated by up-regulation of miR-145 in the case of CIN612-9E. Together with our results, 944

these studies clearly underscore an importance of NFκB activity for viral persistence of HPVs. 945

Currently, we are not sure whether NFκB activation and/or its inhibition of E1-dependent 946

replication occur during productive amplification. As NFκB activity and components of NFκB 947

pathways are intricately associated with differentiation processes in keratinocytes, the outcomes 948

as well as the pathways of NFκB activation could be different in differentiating cells. 949

Interestingly, Satsuka et al. reported that TNFα treatment slightly reduces the copy number of 950

HPV18 replicons in monolayer cultures of keratinocytes whereas it enhanced productive 951

amplification in raft cultures (71). On the other hand, it is also conceivable that NFκB activation 952

is prevented by increased levels of E6 and E7, as these may suppress NFκB activity by various 953

mechanisms (24, 30). 954

46

NFκB is a major transcription factor that up-regulates inflammatory cytokines including TNFα, 955

IL6 and IL8 which in turn activate NFκB itself. A number of studies have provided compelling 956

evidence that chronic inflammation increases risk of cancer development, including that of 957

cervical neoplasms. The ability of NFκB to suppress viral genome replication may facilitate loss 958

of episomal HPV and emergence of viral genome integration, potentiating cancer progression 959

when ectopically activated in basal cells. Clearly, uncovering NFκB functions in the viral life 960

cycle is important to elucidate molecular mechanisms underlying viral persistence and 961

pathogenesis of HPV infection. 962

963

47

Acknowledgements 964

We would like to thank Keiko Ishiyama for her technical assistance. This work was supported 965

in part by Grants-in-Aid for Young Scientists (B) (23790517 and 25860349) to T.N and a Grant-966

in-Aid for Cancer Research from the Ministry of Health, Labor and Welfare of Japan to T.Y. and 967

T.K. 968

969

48

970

971

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62. Pett MR, Herdman MT, Palmer RD, Yeo GS, Shivji MK, Stanley MA, Coleman N. 1147 2006. Selection of cervical keratinocytes containing integrated HPV16 associates with 1148 episome loss and an endogenous antiviral response. Proceedings of the National 1149 Academy of Sciences of the United States of America 103:3822-3827. 1150

63. Turek LP, Byrne JC, Lowy DR, Dvoretzky I, Friedman RM, Howley PM. 1982. 1151 Interferon induces morphologic reversion with elimination of extrachromosomal viral 1152 genomes in bovine papillomavirus-transformed mouse cells. Proc. Natl. Acad. Sci. U. S. 1153 A. 79:7914-7918. 1154

64. Chang YE, Laimins LA. 2000. Microarray analysis identifies interferon-inducible genes 1155 and Stat-1 as major transcriptional targets of human papillomavirus type 31. J Virol 1156 74:4174-4182. 1157

65. Chang YE, Pena L, Sen GC, Park JK, Laimins LA. 2002. Long-term effect of 1158 interferon on keratinocytes that maintain human papillomavirus type 31. J Virol 76:8864-1159 8874. 1160

66. Nees M, Geoghegan JM, Hyman T, Frank S, Miller L, Woodworth CD. 2001. 1161 Papillomavirus type 16 oncogenes downregulate expression of interferon-responsive 1162 genes and upregulate proliferation-associated and NF-κB-responsive genes in cervical 1163 keratinocytes. Journal of virology 75:4283-4296. 1164

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1178 1179

1180

53

Figure legends 1181

Figure.1 Conditional induction of E1-dependent replication in immortalized human 1182

cervical karatinocytes containing HPV16 genome (HCK1T-HPV16). (A) A representative 1183

image of Southern blotting for HPV16 genomes in 10 μg of total genomic DNA isolated from 1184

cells with the indicated gene expression at 24 hours after DOX (1μg/ml) addition. The serially 1185

diluted, linearized full length of HPV16 genomes were included as copy number standards (lanes 1186

1 to 3). The DNAs with BamHI digestion which linearizes the HPV16 genome, and Xho I 1187

digestion which does not cut the HPV16 genome, are shown in lanes 4 to 7 and lanes 8 to 11, 1188

respectively. Arrowheads indicate HPV16 DNAs which correspond to Form I, a supercoiled 1189

DNA, and Form II, a relaxed circular DNA. (B) qPCR results for the copy number of HPV16 1190

genomes with the indicated gene expression at 24 hours and (C) at the indicated time points 1191

following DOX incubation. (D) Indirect immunoflorescence analysis (IFA) for HA-E1 and E2. 1192

As a negative control, HCK1T-HPV16 with tetON E1+E2 incubated with a vehicle control is 1193

shown. (E) The number of cells with the indicated gene expression that stayed attached to tissue 1194

culture plates is shown. (F) Western blot analysis for the levels of E1 and the markers of 1195

apoptosis, PARP-1 and caspase-3, in HCK1T-HPV16 cells with the indicated gene expression 1196

following incubation with DOX (lanes 2, 4, 6 and 8) or vehicle as a control (lanes 1, 3, 5 and 7). 1197

Arrowheads indicate cleaved forms of PARP-1 and caspase-3. α-tubulin was detected as a 1198

loading control. The averages of results from at least three independent experiments are shown. 1199

P-values were evaluated by Student’s t-test. 1200

Figure 2. Activation of NFκB upon induction of E1 and/or E2 expression in HCK1T-1201

HPV16 cells. (A) The status of NFκBp65 (RELA) and its inhibitor, IκBα, was analyzed by 1202

54

Western blotting for HCK1T-HPV16 cells with the indicated gene expression at 24 hours after 1203

DOX addition. α-tubulin was included as a loading control. (B) Localization of RELA and HA- 1204

E1 and/or E2 was examined in HCK1T-HPV16 tetON E1+E2 by indirect IFA. A rabbit 1205

polyclonal antibody to RELA and a mouse monoclonal antibody to HA were simultaneously 1206

incubated and secondary antibodies, Alexa 488 conjugated anti-rabbit IgG and Alexa 596 1207

conjugated anti-mouse IgG, respectively, were used for detection. Nucleus was counterstained by 1208

DAPI. (C) Relative mRNA levels of indicated NFκB target genes and viral transcripts containing 1209

E1^E4 spliced ORF were measured by RT-qPCR. The mRNAs of the indicated genes were 1210

normalized to ß-actin mRNA. Fold increase of the indicated mRNAs in DOX treated samples to 1211

vehicle treated samples are shown. The averages of at least three independent experiments are 1212

shown. 1213

Figure 3. Activation of NFκB upon induction of E1 and/or E2 expression in HCK1T in the 1214

absence of an episomal HPV16 genome. (A) The level of IκBα and expression of E1 and E2 1215

were analyzed in parental HCK1T that do not contain an HPV16 genome by Western blotting. 1216

HCK1T-tetON cells were included as a negative control. Cell lysates were prepared at 24 hours 1217

following incubation with DOX (lanes 2, 4, 6 and 8) or a vehicle (lanes 1, 3, 5 and 7). Vinculin 1218

was detected as a loading control. (B) qRT-PCR analysis for mRNA levels of the NFκB target 1219

genes in HCK1T-tetON E1+E2 or E1 alone. The mRNAs of the indicated genes were normalized 1220

to ß-actin mRNA. Fold increase of the indicated mRNAs in DOX treated samples to vehicle 1221

treated samples are shown. The averages of at least three independent experiments are shown. 1222

(C) RT-PCR analysis for mRNA levels of the NFκB target genes in HCK1T, HCK1T-HPV16 1223

and CIN612-9E cells. As a negative control, an RNA isolated from HCK1T not subjected to RT 1224

reaction was used as a template for PCR reactions (lane 1). 1225

55

Figure 4. Effects of over-expression of IκBα on E1-dependent replication of HPV16 and cell 1226

proliferation of subclones of HCK1T-HPV16 cells with tetON E1+E2. (A) Shown are results 1227

of Western blot analysis (left) for E1 and E2 levels and the steady state levels of IκBα in 1228

representative clones and the results of qPCR for HPV16 genomes (right) following incubation 1229

with DOX or vehicle for 24 hours. Images with short and long exposure with the anti-HA 1230

antibody were included to indicate expression of HA-E2. α-tubulin is shown as a loading control 1231

for the top three panels and vinculin for the HA panel comparing clones to the parental 1232

population. (B) Western blot analysis to verify the over-expression of IκBα in clone B2 1233

transduced with retroviruses carrying an empty vector designated as MCS (lanes 3 and 4) or the 1234

vector expressing IκBαS32/36A mutant (IκBαMT) (lanes 5 and 6) or wild type IκBα (IκBαWT) 1235

(lanes 7 and 8). Parental HCK1T-HPV16 treated with TNFα for 15 minutes was included as a 1236

positive control for degradation of IκBα (lanes 1 and 2). Vinculin was detected as a loading 1237

control. (D) RT-qPCR analysis of NFκB target genes and viral early transcripts containing 1238

spliced E1^E4 ORF. Shown are levels of the indicated mRNAs relative to MCS control without 1239

DOX treatment. Two asterisks indicate a p-value < 0.01 for the comparison of the mRNAs in 1240

MCS with those in IκBαWT- or IκBαMT- expressing cells in the absence of DOX. Differences 1241

for all of the mRNA levels in between MCS and IκBαWT- or IκBαMT- expressing cells with 1242

DOX treatment were statistically significant (P<0.01). Highlighting statistical significance was 1243

omitted for DOX treated samples to avoid making graphs too busy. (D) The copy number of 1244

HPV16 genomes was measured by qPCR with indicated gene expression. An asterisk indicates a 1245

p-value <0.05. (E) The number of cells staying attached to tissue culture plates is shown. An 1246

asterisk indicates a p-value < 0.05. The averages of at least three independent experiments are 1247

shown. 1248

56

Figure 5. Effects of inhibitors of DDR on activation of NFκB, E1-dependent replication of 1249

HPV16 genomes and cell proliferation. (A) Western blot analysis for activation of ATM and 1250

ATR mediated DDR pathways. The left panel (lanes 1 to 8) shows the results of original cell 1251

populations of HCK1T-HPV16 with the indicated gene expression. The right panel (lanes 9 to 1252

18) shows the results for representative clones of HCK1T-HPV16 tetON E1+E2 cells. Vinculin 1253

or α-tubulin indicates a loading control in each panel. White arrowheads indicate a slower 1254

migration pattern of NBS1 and CHK2. (B) A time course experiment for DDR activation and the 1255

level of IκBα in the presence of DMSO as a control, 10 μM of Wortmannin or 2.5 μM of 1256

KU55933. (C) Transcriptional activation of NFκB target genes and the viral early transcripts was 1257

examined by RT-qPCR in the presence or absence of the inhibitors incubated with or without 1258

DOX for 24 hours. An asterisk indicates a p-value < 0.05 for the comparison of the mRNAs in 1259

between DMSO treated and Wortmannin treated cells in the absence of DOX. The mRNAs of 1260

NFκB target genes but not the viral early transcripts in Wortmannin or KU55933 treated cells 1261

compared to those in DMSO treated cells in the presence of DOX were significantly reduced 1262

(P<0.05). Highlighting p-values less than 0.05 was omitted for DOX treated samples. (D) The 1263

copy number of HPV16 genomes was measured by qPCR at 24 hours after DOX addition in the 1264

presence of the indicated inhibitors. (E) The number of cells staying attached to tissue culture 1265

plates in the presence or absence of inhibitors was counted. An asterisk indicates a p-value < 1266

0.05. (F) Relative mRNA levels of HA-E1 were analyzed by RT-qPCR in the presence or 1267

absence of Wortmannin following incubation with DOX or vehicle for 24 hours. (G) Western 1268

blot analysis for solubility of E1 in the presence of Wortmannin or vehicle. S and I indicate 1269

soluble (lanes 1, 3 and 5) and insoluble (lanes 2, 4 and 6) fractions, respectively. The averages or 1270

representative images of at least three independent experiments are shown. 1271

57

Figure 6. ATR, but not DNA-PKcs and p38MAPK, is involved in the E1-dependent 1272

reduction of IκBα. (A) Activation of p38MAPK was examined in HCK1T-HPV16 populations 1273

with the indicated gene expression. (B) Effects of Wortmannin and KU55933 on activation of 1274

p38MAPK and DNA-PKcs in clone B2. Cells lysate were prepared at 0 and 24 hours after DOX 1275

addition following pre-incubation with the inhibitors for 2 hours. Vinculin was detected as a 1276

loading control. (C) Western blot analysis for the levels of IκBα and E1 at 24 hours after 1277

incubation with DOX in the presence of indicated inhibitors. A bottom graph shows intensities of 1278

E1 with inhibitors relative to that with a vehicle control. The intensities of E1 were normalized to 1279

that of vinculin. Concentrations of inhibitors used were 10 μM for Wortmannin, 0.5 nM for VE-1280

822 (ATR inhibitor), 20 μM for Nu7026 (DNA-PKcs inhibitor), 2.5 μM for KU55933 and 10μM 1281

for SB203580 (p38MAPK inhibitor). (D) Western blot analysis for a time course experiment in 1282

the presence of Wortmannin (lanes 5 to 8), VE-822 (lanes 9 to 12) or a vehicle control (lanes 1 to 1283

4). (D) The copy number of HPV16 genomes was measured by qPCR and (E) the number of 1284

cells staying attached was examined after incubation with DOX or vehicle in the presence of 1285

DMSO or VE-822 for 24 hours. Astarisks indicate p<0.05. (G) Western blot analysis for the 1286

stability of E2 in the presence of indicated inhibitors as described in Fig.5. The averages or 1287

representative images of at least three independent experiments are shown. 1288

Figure 7. Activation of NFκB mediates destabilization of E1 and promotes 1289

polyubiquitination. (A) Time course experiments for clone B2 transduced with MCS (lanes 1 to 1290

4), IκBαWT (lanes 9 to 12) or IκBαMT (lanes 5 to 8) following incubation with DOX. A white 1291

arrowhead indicates a slower migrating pattern of NBS1. Vinculin was detected as a loading 1292

control. (B) RT-qPCR analysis for HA-E1 in the indicated cells upon DOX incubation. (C) 1293

Western blot analysis for the levels of E1 and IκBα in clone B2 incubated with vehicle (lanes 1 1294

58

and 2) or 10nM of Epoximicin (lanes 3 and 4) for 12 hours. (D) An ubiquitin conjugation assay 1295

was performed as described in the Materials and Methods. The left panel (lanes 8 to 13) shows 1296

results of Western blot analysis for input cell lysates subjected to immunoprecipitation (IP). The 1297

right panel (lanes 14 to 19) shows results of IP-WB to detect His-Ub conjugation to HA-E1. The 1298

cell lysates from 293FT cells transfected without a His-Ub expression vector (lanes 1 to 6) were 1299

also included. Wor and TNF indicate treatment with 10 μM of Wortmannin and 20 ng/ml of 1300

TNFα, respectively, from 20 hours post-transfection. IkB indicates co-transfection with an 1301

expression vector of IκBαMT. 1302

Figure 8. E1 and E2 expression induces accumulation of cells in S and G2 phases. 1303

Clones B2 were collected at indicated time points after a vehicle (above) or DOX (bottom) 1304

addition and cell cycle profiles were analyzed by flow cytometry. Representative profiles are 1305

shown. The graphs indicate the percentages of cells in the cell cycle phases. 1306

Figure 9. The effect of constitutive activation of NFκB in maintenance replication of HPV 1307

genome. (A) The copy number of HPV16 genomes in HCK1T-HPV16 transduced with 1308

retrovirus carrying an empty (MCS) or IκBαMT expressing vector was measured at 5 days after 1309

drug selection was completed. (B) The copy number of HPV16 or HPV31 was measured at 5 1310

days after incubation of TNFα at indicated concentrations (black columns). The cell proliferation 1311

was also compared (White columns). (C) A schematic presentation for a negative feedback loop 1312

between E1 and NFκB. E1 induces activation of NFκB most likely through degradation of IκBα 1313

as a consequence of DDR activation. NFκB activation in turn limits E1 dependent replication 1314

and disrupts the stability of E1 through facilitating proteasomal degradation. 1315

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E1+E2 E1+-DOX(24h)

D

Figure.1 Conditional induction of E1-dependent replication in immortalized human cervical karatinocytes containing HPV16 genome (HCK1T-HPV16). (A) A representative image of Southern blotting for HPV16 genomes in 10 μg of total genomic DNA isolated from cells with the indicated gene expression at 24 hours after DOX (1μg/ml) addition. The serially diluted, linearized full length of HPV16 genomes were included as copy number standards (lanes 1 to 3). The DNAs with BamHI digestion which linearizes the HPV16 genome, and Xho I digestion which does not cut the HPV16 genome, are shown in lanes 4 to 7 and lanes 8 to 11, respectively. Arrowheads indicate HPV16 DNAs which correspond to Form I, a supercoiled DNA, and Form II, a relaxed circular DNA. (B) qPCR results for the copy number of HPV16 genomes with the indicated gene expression at 24 hours and (C) at the indicated time points following DOX incubation. (D) Indirect immunoflorescence analysis (IFA) for HA-E1 and E2. As a negative control, HCK1T-HPV16 with tetON E1+E2 incubated with a vehicle control is shown. (E) The number of cells with the indicated gene expression that stayed attached to tissue culture plates is shown. (F) Western blot analysis for the levels of E1 and the markers of apoptosis, PARP-1 and caspase-3, in HCK1T-HPV16 cells with the indicated gene expression following incubation with DOX (lanes 2, 4, 6 and 8) or vehicle as a control (lanes 1, 3, 5 and 7). Arrowheads indicate cleaved forms of PARP-1 and caspase-3. α-tubulin was detected as a loading control. The averages of results from at least three independent experiments are shown. P-values were evaluated by Student’ s t-test.

HCK1T-HPV16 tetON E1+E2

RELA HA DAPI Merge

Vehicle

DOX(24h)

02468

1012141618

TNFα IL6 IL8 E1^E4spliced

Vehicle+DOX (24h)

Fold

Incr

ease

HCK1T-HPV16 tetON E1+E2

02468

1012141618

TNFα IL6 IL8 E1^E4spliced

Vehicle+DOX(24h)

HCK1T-HPV16 tetON E1K483A+E2

B

C

A HCK1T-HPV16 tetON

HAE

1+E

2

E1K

483A

+E2

- + - +DOX(24h)

IκBα

α−tubulin

p65(RELA)

1 2 3 4

- + - +

E2

E1

5 6 7 8

Figure 2

Figure 2. Activation of NFκB upon induction of E1 and/or E2 expression in HCK1T-HPV16 cells. (A) The status of NFκBp65 (RELA) and its inhibitor, IκBα, was analyzed by Western blotting for HCK1T-HPV16 cells with the indicated gene expression at 24 hours after DOX addition. α-tubulin was included as a loading control. (B) Localization of RELA and HA- E1 and/or E2 was examined in HCK1T-HPV16 tetON E1+E2 by indirect IFA. A rabbit polyclonal antibody to RELA and a mouse monoclonal antibody to HA were simultaneously incubated and secondary antibodies, Alexa 488 conjugated anti-rabbit IgG and Alexa 596 conjugated anti-mouse IgG, respectively, were used for detection. Nucleus was counterstained by DAPI. (C) Relative mRNA levels of indicated NFκB target genes and viral transcripts containing E1^E4 spliced ORF were measured by RT-qPCR. The mRNAs of the indicated genes were normalized to ß-actin mRNA. Fold increase of the indicated mRNAs in DOX treated samples to vehicle treated samples are shown. The averages of at least three independent experiments are shown.

A HCK1T tetON

1 2 3 4 5 6 7 8

- + - + - +

IκBα

Vinculin

HA(E1)

E1+

E2

E2

E1

DOX(24h)

FLAG(E2)

p65(RELA)

- +

Vehicle +DOX (24h)

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TNFα IL6 IL8 TNFα IL6 IL8

E1+E2 E1

B HCK1T tetON

C

HC

K1T

HC

K1T

-HP

V16

TNFαIL6IL8

β-Actin

CIN

612-

9E

no R

T

1 2 3 4

Figure 3

Figure 3. Activation of NFκB upon induction of E1 and/or E2 expression in HCK1T in the absence of an episomal HPV16 genome. (A) The level of IκBα and expression of E1 and E2 were analyzed in parental HCK1T that do not contain an HPV16 genome by Western blotting. HCK1T-tetON cells were included as a negative control. Cell lysates were prepared at 24 hours following incubation with DOX (lanes 2, 4, 6 and 8) or a vehicle (lanes 1, 3, 5 and 7). Vinculin was detected as a loading control. (B) qRT-PCR analysis for mRNA levels of the NFκB target genes in HCK1T-tetON E1+E2 or E1 alone. The mRNAs of the indicated genes were normalized to ß-actin mRNA. Fold increase of the indicated mRNAs in DOX treated samples to vehicle treated samples are shown. The averages of at least three independent experiments are shown. (C) RT-PCR analysis for mRNA levels of the NFκB target genes in HCK1T, HCK1T-HPV16 and CIN612-9E cells. As a negative control, an RNA isolated from HCK1T not subjected to RT reaction was used as a template for PCR reactions (lane 1).

Figure 4

Figure 4. Effects of over-expression of IκBα on E1-dependent replication of HPV16 and cell proliferation of subclones of HCK1T-HPV16 cells with tetON E1+E2. (A) Shown are results of Western blot analysis (left) for E1 and E2 levels and the steady state levels of IκBα in representative clones and the results of qPCR for HPV16 genomes (right) following incubation with DOX or vehicle for 24 hours. Images with short and long exposure with the anti-HA antibody were included to indicate expression of HA-E2. α-tubulin is shown as a loading control for the top three panels and vinculin for the HA panel comparing clones to the parental population. (B) Western blot analysis to verify the over-expression of IκBα in clone B2 transduced with retroviruses carrying an empty vector designated as MCS (lanes 3 and 4) or the vector expressing IκBαS32/36A mutant (IκBαMT) (lanes 5 and 6) or wild type IκBα (IκBαWT) (lanes 7 and 8). Parental HCK1T-HPV16 treated with TNFα for 15 minutes was included as a positive control for degradation of IκBα (lanes 1 and 2). Vinculin was detected as a loading control. (D) RT-qPCR analysis of NFκB target genes and viral early transcripts containing spliced E1^E4 ORF. Shown are levels of the indicated mRNAs relative to MCS control without DOX treatment. Two asterisks indicate a p-value < 0.01 for the comparison of the mRNAs in MCS with those in IκBαWT- or IκBαMT- expressing cells in the absence of DOX. Differences for all of the mRNA levels in between MCS and IκBαWT- or IκBαMT- expressing cells with DOX treatment were statistically significant (P<0.01). Highlighting statistical significance was omitted for DOX treated samples to avoid making graphs too busy. (D) The copy number of HPV16 genomes was measured by qPCR with indicated gene expression. An asterisk indicates a p-value <0.05. (E) The number of cells staying attached to tissue culture plates is shown. An asterisk indicates a p-value < 0.05. The averages of at least three independent experiments are shown.

TNFα IL6

A HCK1T-HPV16 tetON E1+E2 clones

2

4

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8

10

12

14

MCS IκBαMTIκBαWT

HP

V16

gen

ome

copy

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Vehicle +Dox(24h)

*

*

Cell N

umbe

r (x1

05 )

IκBαWT IκBαMTMCS

Vehicle

+DOX (24h)

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1.0

1.5

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120

B

TNF α

+ MC

S

IkBα

WT

IkBα

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Vinculin

HA(E1)

tetON E1+E2 clone B2

--DOX(24h)

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MCS IκBαWT IκBαMT

IL8

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MCS IκBαWT IκBαMT

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MCS IκBαWT IκBαMT

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MCS IκBαWT IκBαMT

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- + - + - + - + - +

A42

B3 A26

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DOX(24h)

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αtubulin

HA(short)

1 2 3 4 6 7 85 109

HA(long)

E1

E2

E1

- +

Par

enta

l po

p

E1HA(short)

Vinculin11 12

** ** ** ** ** **

** **

HCK1T-HPV16

IκBα

Figure 5. Effects of inhibitors of DDR on activation of NFκB, E1-dependent replication of HPV16 genomes and cell proliferation. (A) Western blot analysis for activation of ATM and ATR mediated DDR pathways. The left panel (lanes 1 to 8) shows the results of original cell populations of HCK1T-HPV16 with the indicated gene expression. The right panel (lanes 9 to 18) shows the results for representative clones of HCK1T-HPV16 tetON E1+E2 cells. Vinculin or α-tubulin indicates a loading control in each panel. White arrowheads indicate a slower migration pattern of NBS1 and CHK2. (B) A time course experiment for DDR activation and the level of IκBα in the presence of DMSO as a control, 10 μM of Wortmannin or 2.5 μM of KU55933. (C) Transcriptional activation of NFκB target genes and the viral early transcripts was examined by RT-qPCR in the presence or absence of the inhibitors incubated with or without DOX for 24 hours. An asterisk indicates a p-value < 0.05 for the comparison of the mRNAs in between DMSO treated and Wortmannin treated cells in the absence of DOX. The mRNAs of NFκB target genes but not the viral early transcripts in Wortmannin or KU55933 treated cells compared to those in DMSO treated cells in the presence of DOX were significantly reduced (P<0.05). Highlighting p-values less than 0.05 was omitted for DOX treated samples. (D) The copy number of HPV16 genomes was measured by qPCR at 24 hours after DOX addition in the presence of the indicated inhibitors. (E) The number of cells staying attached to tissue culture plates in the presence or absence of inhibitors was counted. An asterisk indicates a p-value < 0.05. (F) Relative mRNA levels of HA-E1 were analyzed by RT-qPCR in the presence or absence of Wortmannin following incubation with DOX or vehicle for 24 hours. (G) Western blot analysis for solubility of E1 in the presence of Wortmannin or vehicle. S and I indicate soluble (lanes 1, 3 and 5) and insoluble (lanes 2, 4 and 6) fractions, respectively. The averages or representative images of at least three independent experiments are shown.

C

A HCK1T-HPV16 tetON HCK1T-HPV16 tetON E1+E2 clones

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DMSO Wortmannin KU55933

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TNFα IL6 IL8 E1^E4 spliced

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Vinculin

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HA(E1)

HCK1T-HPV16 tetON E1+E2 clone B2

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Figure 5

GHCK1T-HPV16 tetON E1+E2 clone B2

S I S I S IHA(E1)

MCM2

Histone H3

Vinculin

DOX(24h)Wortmannin

- + + + ++ +

2xSDS buffer

-

lysis buffer

1 2 3 4 5 6 7 8 9

Figure 6. ATR, but not DNA-PKcs and p38MAPK, is involved in the E1-dependent reduction of IκBα. (A) Activation of p38MAPK was examined in HCK1T-HPV16 populations with the indicated gene expression. (B) Effects of Wortmannin and KU55933 on activation of p38MAPK and DNA-PKcs in clone B2. Cells lysate were prepared at 0 and 24 hours after DOX addition following pre-incubation with the inhibitors for 2 hours. Vinculin was detected as a loading control. (C) Western blot analysis for the levels of IκBα and E1 at 24 hours after incubation with DOX in the presence of indicated inhibitors. A bottom graph shows intensities of E1 with inhibitors relative to that with a vehicle control. The intensities of E1 were normalized to that of vinculin. Concentrations of inhibitors used were 10 μM for Wortmannin, 0.5 nM for VE-822 (ATR inhibitor), 20 μM for Nu7026 (DNA-PKcs inhibitor), 2.5 μM for KU55933 and 10μM for SB203580 (p38MAPK inhibitor). (D) Western blot analysis for a time course experiment in the presence of Wortmannin (lanes 5 to 8), VE-822 (lanes 9 to 12) or a vehicle control (lanes 1 to 4). (D) The copy number of HPV16 genomes was measured by qPCR and (E) the number of cells staying attached was examined after incubation with DOX or vehicle in the presence of DMSO or VE-822 for 24 hours. Astarisks indicate p<0.05. (G) Western blot analysis for the stability of E2 in the presence of indicated inhibitors as described in Fig.5. The averages or representative images of at least three independent experiments are shown.

HA(E1)

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+DOX(h)

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tman

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pDNA-PKc(S2056)

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DOX(24h)

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HCK1T-HPV16 tetON E1+E2 Clone B2

VinculinFLAG(E2)

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DMSO Wortmannin VE-822

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DOX(h)

HCK1T-HPV16 tetON HA-E1+FLAG-E2

Figure 6

Figure 7

Figure 7. Activation of NFκB mediates destabilization of E1 and promotes polyubiquitination. (A) Time course experiments for clone B2 transduced with MCS (lanes 1 to 4), IκBαWT (lanes 9 to 12) or IκBαMT (lanes 5 to 8) following incubation with DOX. A white arrowhead indicates a slower migrating pattern of NBS1. Vinculin was detected as a loading control. (B) RT-qPCR analysis for HA-E1 in the indicated cells upon DOX incubation. (C) Western blot analysis for the levels of E1 and IκBα in clone B2 incubated with vehicle (lanes 1 and 2) or 10nM of Epoximicin (lanes 3 and 4) for 12 hours. (D) An ubiquitin conjugation assay was performed as described in the Materials and Methods. The left panel (lanes 8 to 13) shows results of Western blot analysis for input cell lysates subjected to immunoprecipitation (IP). The right panel (lanes 14 to 19) shows results of IP-WB to detect His-Ub conjugation to HA-E1. The cell lysates from 293FT cells transfected without a His-Ub expression vector (lanes 1 to 6) were also included. Wor and TNF indicate treatment with 10 μM of Wortmannin and 20 ng/ml of TNFα, respectively, from 20 hours post-transfection. IkB indicates co-transfection with an expression vector of IκBαMT.

0 8 16 24 0 8 16 24 0 8 16 24NBS1

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IκBα

HA(E1)

Vinculin

+DOX(h)MCS IκBαWTIκBαMT

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pNBS1

Vinculin

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A HCK1T-HPV16 tetON E1+E2 clone B2

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Vehicle +DOX

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G1 S G2/M

subG1

Figure 8

Figure 8. E1 and E2 expression induces accumulation of cells in S and G2 phases.Clones B2 were collected at indicated time points after a vehicle (above) or DOX (bottom) addition and cell cycle profiles were analyzed by flow cytometry. Representative profiles are shown. The graphs indicate the percentages of cells in the cell cycle phases.

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Cell proliferationGenome copy

Vehicle TNFα(5ng/ml)

TNFα(10ng/ml)

CIN612-9E

* **

***

*

E1

ATR/ATM activation

replication

IκB degradation

NFκB activation

degradation?

?Wortmannin

E2

?

A

C

Figure 9BHCK1T-HPV16

Figure 9. The effect of constitutive activation of NFκB in maintenance replication of HPV genome. (A) The copy number of HPV16 genomes in HCK1T-HPV16 transduced with retrovirus carrying an empty (MCS) or IκBαMT expressing vector was measured at 5 days after drug selection was completed. (B) The copy number of HPV16 or HPV31 was measured at 5 days after incubation of TNFα at indicated concentrations (black columns). The cell proliferation was also compared (White columns). (C) A schematic presentation for a negative feedback loop between E1 and NFκB. E1 induces activation of NFκB most likely through degradation of IκBα as a consequence of DDR activation. NFκB activation in turn limits E1 dependent replication and disrupts the stability of E1 through facilitating proteasomal degradation.