associated herpesvirus orf66 is essential for late gene ... · 8/7/2019 · 4 66. introduction. 67...

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Kaposi’s sarcoma-associated herpesvirus ORF66 is essential for late gene 1

expression and virus production via interaction with ORF34 2

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Tadashi Watanabe1, Mayu Nishimura1, Taisuke Izumi2+, Kazushi Kuriyama1, 5

Yuki Iwaisako1, Kouhei Hosokawa1, Akifumi Takaori-Kondo2 and Masahiro 6

Fujimuro1* 7

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1: Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-9

Shichono-cho 1, Yamashina-ku, Kyoto, Japan. 10

2: Department of Hematology and Oncology, Graduate School of Medicine, 11

Kyoto University, 54 Shogoin Kawasaki-cho, Kyoto, Japan 12

+: Present Address 13

Henry M. Jackson Foundation for the Advancement of Military Medicine Inc., in 14

Support of Military HIV Research Program, Walter Reed Army Institute of 15

Research, Silver Spring, MD, United States 16

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Running title: KSHV ORF66 is essential for late gene expression 18

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*: Address correspondence to Masahiro Fujimuro, Ph.D. 20

Department of Cell Biology, Kyoto Pharmaceutical University, Misasagi-21

Shichono-cho 1, Yamashina-ku, Kyoto 607-8412, Japan; Tel: +81-75-595-4717 22

E-mail: [email protected] 23

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not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (which wasthis version posted August 7, 2019. . https://doi.org/10.1101/728147doi: bioRxiv preprint

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ABSTRACT 25

Kaposi’s sarcoma-associated herpesvirus (KSHV) is closely 26

associated with B-cell and endothelial cell malignancies. After the initial 27

infection, KSHV retains its viral genome in the nucleus of the host cell and 28

establishes a lifelong latency. During lytic infection, KSHV encoded lytic-related 29

proteins are expressed in a sequential manner and are classified as immediate 30

early, early, and late gene transcripts. The transcriptional initiation of KSHV late 31

genes is thought to require the complex formation of the virus specific pre-32

initiation complex (vPIC), which may consist of at least 6 transcription factors 33

(ORF18, 24, 30, 31, 34, and 66). However, the functional role of ORF66 in vPIC 34

during KSHV replication remains largely unclear. Here, we generated ORF66-35

deficient KSHV using a BAC system to evaluate its role during viral replication. 36

While ORF66-deficient KSHV demonstrated mainly attenuated late gene 37

expression and decreased viral production, viral DNA replication was 38

unaffected. CHIP analysis showed that ORF66 bound to the promoters of late 39

gene (K8.1), but did not to those of latent gene (ORF72), immediate early gene 40

(ORF16) and early gene (ORF46/47). Furthermore, we found that three highly 41

conserved C-X-X-C sequences and a conserved leucine-repeat in the C-42

terminal region of ORF66 were essential for interaction with ORF34 and viral 43

production. The interaction between ORF66 and ORF34 occurred in a zinc-44

dependent manner. Our data support a model, in which ORF66 serves as a 45

critical vPIC component to promote late viral gene expression and viral 46

production. 47

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IMPORTANCE 49

KSHV ORF66, a late gene product, and vPIC are thought to contribute 50

significantly to late gene expression during the lytic replication. However, the 51

physiological importance of ORF66 in terms of viral replication and vPIC 52

formation remains poorly understood. Therfore, we generated a ORF66-53

deficient BAC clone and evaluated its viral replication. Results showed that 54

ORF66 played a critical role in virus production and the transcription of L genes. 55

To our knowledge, this is the first report showing ORF66 function in virus 56

replication using ORF66-deficient KSHV. We also clarified that ORF66 57

interacted with the transcription start site of K8.1 gene, a late gene. 58

Furthermore, we identified the ORF34-binding motifs in the ORF66 C- terminus: 59

three C-X-X-C sequences and a leucine-repeat sequence, which are highly 60

conserved among - and -herpesviruses. Our study provides insights into the 61

regulatory mechanisms of not only the late gene expression of KSHV but also 62

those of other herpesviruses. 63

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Introduction 66

KSHV, (also known as human herpesvirus 8 or HHV-8), causes 67

Kaposi’s sarcoma, primary effusion lymphoma (PEL), and multicentric 68

Castleman’s disease (1-3). Since the first discovery of KSHV DNA fragments in 69

a Kaposi's sarcoma lesion of an AIDS patient in 1994 (4), over a quarter of a 70

century has passed, and several aspects of KSHV pathogenesis, life-cycle and 71

viral protein function have been elucidated. Compared with other viruses, 72

herpesvirus has a large number of viral genes in its genome. KSHV encodes 73

not only viral proteins but also miRNAs and LncRNAs. These viral molecules 74

are thought to be essential for KSHV replication and its pathogenesis. One 75

characteristic of the KSHV life-cycle is the establishment of a lifelong latency in 76

the infected individual leading to KSHV-associated malignancy in patients with 77

severe immunosuppression by drugs after organ transplantation or AIDS (1-5). 78

Development of KSHV-associated neoplasm occurs due to infected cells which 79

express few latent KSHV genes (including LANA, v-FLIP, Kaposin, and 80

miRNAs) (6). These genes modulate cell-proliferation and apoptosis pathways 81

(7-11). Another characteristic of the KSHV life-cycle is active virus production, 82

known as lytic infection. The genes related to lytic infection have been 83

categorized into into three groups, immediate early (IE), early (E), and late (L) 84

(12, 13). Sequential and temporal expression of KSHV genes are key to induce 85

efficient viral production during lytic infection. The IE-gene product RTA/ORF50 86

is a transcription factor for triggering transcriptional activation of another IE 87

genes and E genes, and RTA/ORF50 initiates the shift from latency to lytic 88

infection (13, 14). The transcribed products of E genes start viral genome DNA 89


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replication. Finally, the transcriptional products of L genes, contribute to viral 90

particle formation by its encoding viral structure proteins (15). 91

The viral pre-initiation complex (vPIC) has recently been proposed to 92

regulate L gene expression(16). vPIC is composed of several viral proteins 93

conserved among - and -herpesviruses (17), and has functional homology 94

with the host pre-initiation complex, which consists of TATA-binding protein 95

(TBP) and general transcriptional factors (GTFs). Whereas the host pre-96

initiation complex accumulates on the TATA-box of transcriptional start site 97

(TSS) and initiates cellular RNA polymerase II (RNA pol II)-mediated 98

transcription, vPIC accumulates on the “TATT”-box of viral gene TSS and 99

initiates RNA pol II-mediated transcription (17). 100

The function of vPIC machinery and its components has been 101

extensively studied in the context of EBV, MHV-68, CMV and KSHV(16). In 102

KSHV, at least 6 viral proteins contribute to vPIC formation. Viral TBP homolog 103

KSHV ORF24 directly binds to the “TATT”-box on the promoter sequences of 104

the KSHV genome and is essential for the recruitment of host RNA pol II (18, 105

19). We and other groups revealed that KSHV ORF34 acts as a hub for 106

interaction between ORF24 and other vPIC components such as ORF18, 107

ORF30, ORF31, and ORF66 (18, 20, 21). Split luciferase assays and co-108

immunoprecipitation experiments revealed that ORF34 directly/or indirectly 109

interacts with ORF 18, 30, 31 and 66 (18, 20-22). ORF24 binds to the promoter 110

of the L genes with RNA pol II, and ORF34 serves as a bridge between ORF24 111

and a complex of ORF18, 30, 31 and 66 (18, 20, 21). Furthermore, ORF18 (23), 112

ORF30 (21), ORF31 (23) and ORF34 (20) are essential for virus replication and 113


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gene expression. Although ORF66 appears to be a vPIC component (18, 20, 114

22), the importance and its function during KSHV replication remains unknown. 115

Therefore, we established ORF66-deficient KSHV, and evaluated its 116

physiological role during viral replication. Here, we show that ORF66 is 117

essential for virus production and L gene expression via interaction with ORF34. 118

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Results 121

Construction of ORF66-deficient KSHV BAC. 122

We constructed ORF66-deficient recombinant KSHV BAC (ORF66-BAC16) to 123

study the impact of ORF66 during KSHV replication. Three stop codons (3-stop 124

element) were inserted into the ORF66 coding region of KSHV BAC16 using a 125

two-step markerless red recombination system (24, 25) (Fig. 1a). Because 126

ORF66 overlaps with ORF67 (Fig. 1a), the 3-stop codons were inserted within 127

the ORF66 gene to avoid interference with the coding frame of ORF67. The 128

insertion and deletion of a kanamycin resistance gene (KanR) were analyzed by 129

EcoRV-digestion (Fig. 1b). Mutations and the insertion of the 3-stop codons into 130

ORF66-BAC16 were confirmed by Sanger sequencing (Fig. 1c). 131

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ORF66 deficiency abrogates virus production and late gene expression. 133

To efficiently induce recombinant KSHV, tetracycline-inducible (Tet-on) 134

RTA/ORF50-expressing SLK cells (iSLK) and Vero cells (iVero) were used as 135

virus producer cells (20). Recombinant KSHV BAC clone, wild type (WT)-136

BAC16 or ORF66-BAC16, was transfected into iVero or iSLK cells, and then 137

selected with hygromycin to generate recombinant KSHV-inducible stable cell 138

lines, iVero-WT, iVero-ORF66, iSLK-WT, and iSLK-ORF66. To evaluate 139

whether ORF66 is critical for KSHV replication, virus production and virus 140

genome replication in iSLK-ORF66 and iVero-ORF66 cells were analyzed. 141

iSLK-WT, iSLK-ORF66, iVero-WT, and iVero-ORF66 cells were treated with 142

Dox and NaB, and culture supernatants were harvested. The amount of WT-143


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KSHV and ORF66-KSHV in iSLK or iVero culture supernatants were 144

measured by real-time PCR (Fig. 2a and 2e). As a result, the production of 145

ORF66-KSHV in iSLK and iVero cells were about 1000-fold and 100-fold lower 146

than WT-KSHV. In contrast, there was no significant difference in cell-147

associated KSHV DNA levels between WT-KSHV and ORF66-KSHV 148

producing cells (Fig. 2b and 2f). Next, to clarify that the reduction of virus 149

production in iSLK-ORF66 and iVero-ORF66 cells is caused by ORF66-150

deficiency, we tested whether exogenous ORF66 expression could rescue virus 151

production of iSLK- and iVero-ORF66 cells. The iSLK-ORF66 and iVero-152

ORF66 cells stably transfected with empty plasmid or ORF66 expression 153

plasmid were cloned by neomycin (G418) or blastcidin S. The protein 154

expression levels of exogenous 3xFLAG-tagged ORF66 were confirmed by 155

Western blotting (Fig.2c and 2g). Virus production from iSLK-ORF66 and 156

iVero-ORF66 cell lines was partially but significantly recovered when 3xFLAG-157

ORF66 was exogenously expressed (Fig.2d and 2h). These results indicate that 158

ORF66 is crucial for KSHV replication and may function in steps following viral 159

DNA replication. 160

To evaluate the contribution of ORF66 on KSHV gene expression, we 161

performed an RT-qPCR array on viral gene. Total RNA was extracted from 162

iSLK-WT and iSLK-ORF66, stimulated with NaB and Dox for 72 hours. RNA 163

was subjected to RT-qPCR array as previously reported (26). Our data showed 164

a broad reduction of KSHV mRNA in iSLK-ORF66 compared to iSLK-WT 165

(Fig.3), where 7 out of the top 10 down-regulated genes were late genes (K8.1, 166


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ORF17, ORF26, ORF27, K9, ORF75, ORF25). In particular, K8.1, ORF26, 167

ORF27, and ORF25 have been previously reported by Nandakumar et al (27) 168

as direct vPIC targets. In contrast to L genes, Latent and IE genes were mildly 169

down-regulated. 170

If ORF66 functions as a vPIC component, we hypothesized that it 171

would be recruited to the “TATT”-box, which is known to be located 172

approximately 30 bp upstream of the transcription start site (TSS) of L genes 173

(27). To demonstrate this, we evaluated whether ORF66 interacts with L gene 174

promoters by ChIP assay. iSLK-ORF66 and iVero-ORF66 cells stably 175

expressing 3xFLAG-ORF66 were treated with or without NaB and Dox to induce 176

lytic infection, and then cells were subjected to ChIP assay using anti-FLAG 177

antibody (Fig. 4). As a result, immunoprecipitated ORF66 was found to be 178

bound to the promoters of K8.1 (L gene) but not to the promoters of latent gene 179

(ORF72), IE gene (ORF16) and E gene (ORF46/47). These results indicated 180

that a protein complex bearing ORF66 may interact with L gene promoters via 181

an interaction between ORF24 and ORF66. 182

183 The interaction of ORF66-ORF34 and its function in viral production.184

ORF66 was reported to interact with other vPIC components such as 185

ORF31, ORF18 and ORF34 by us and others (18, 20, 22). We also found that 186

ORF34 operated as the vPIC hub, by bridging ORF24 and vPIC components 187

including ORF66. Furthermore, these interactions are thought to be important 188

for functions of vPIC in gene expression. To gain further insight into the 189

interaction between ORF66 and ORF34, we identified the responsible region 190


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within ORF66 for binding with ORF34. We made ORF66 truncated mutants that 191

each had approximately 60 amino acid deletions spanning from the N-terminus 192

to C-terminus of ORF66 (Fig. 5a). The interaction between ORF66 mutants and 193

ORF34 was assayed by pull-down experiments (Fig. 5b). 293T cells were co-194

transfected with 2xS-ORF66 mutants and 6xMyc-ORF34 plasmids, and 2xS-195

ORF66 were precipitated from cell extracts by S-protein-immobilized beads. As 196

a result, ORF66 1-4 truncated mutants interacted with ORF34. However, no 197

interaction with ORF66 5-7 truncated mutants was observed, meaning that 198

the C-terminal region (241a.a.- 429a.a.) of ORF66 is critical for ORF34 binding. 199

Furthermore, we performed a trans-complementation assay using these 200

truncation ORF66 mutants to assess how the loss of ORF34-ORF66 interaction 201

affects virus production. ORF66 mutant plasmids were transfected into iSLK-202

ORF66 and iVero-ORF66, and recovery of virus production was measured. 203

Wild type ORF66 expression significantly increased viral production in iSLK-204

ORF66 and iVero-ORF66 cells, while recovery of virus production was not 205

detected by expressing any of the ORF66 truncated mutants (Fig. 5c and 5d). 206

These results indicate that not only the ORF66 C-terminal domain (i.e., ORF34-207

binding region) but also the entire structure of ORF66 are indispensable for 208

virus production. 209

An amino-acid sequence alignment of the ORF66 C-terminal domain 210

of KSHV and other herpes virus homologs are depicted in Fig. 6a. Conserved 211

amino acids are indicated with a gray background. To identify the key residues 212

in the ORF66 C-termial region for interaction with ORF34, we constructed block 213

alanine-scanning mutants (CR1mut to CR9mut), where several neighboring 214


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conserved amino-acids were substituted to alanine (Fig. 6a), except for proline 215

to avoid disruption the whole protein structure. These mutants were subjected 216

to pull-down assays to investigate the association of ORF34. Results showed 217

that ORF66 CR2, 3, 4 and 6 mutants bind to ORF34, whereas ORF66 CR1, 5, 218

7, 8 and 9mut did not bind (Fig. 6b). Therefore, we focused on specific amino 219

acid sequences of mutants losing the ability to bind to ORF34. In particular, 220

CR1, 5, and 9mut have mutations in the C-X-X-C consensus sequence. On the 221

other hand, CR7 contains a single cysteine residue, and CR8 contains three 222

leucine residues. 223

To obtain farther insight into ORF34 binding via CR1 to CR9 of ORF66 224

in viral production, ORF66 alanine substitution mutants (CR1 to CR9) were 225

subjected to trans-complementation assay using iSLK-ORF66 and iVero-226

ORF66 cells (Fig. 7a and 7b). Compared with ORF66 wild type expression, 227

virus production in both iSLK-ORF66 and iVero-ORF66 cells could not be 228

recovered by the expression of CR1, 5, 7, 8 and 9mut, which failed to interact 229

with ORF34 (Fig. 6b). On the other hand, expression of CR2, 3, 4 and 6mut that 230

maintained interaction with ORF34 showed varying levels of recovery, indicated 231

by red columns (Fig. 7a-b). CR3 and 6mut showed almost the identical levels of 232

recovery compared with ORF66 wild-type in iVero-ORF66 cells and partial 233

recovery in iSLK-ORF66 cells. Recovery by the CR4mut was significant, 234

however, lower than those of CR3 and 6mut. CR2mut had no effect. These data 235

revealed that the binding between ORF66 and ORF34 via the conserved 236

amino-acids of CR1, 5, 7, 8 and 9 regions in ORF66 is necessary, but not 237

sufficient for virus replication. 238


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239

The C-X-X-C sequences and Zinc binding of ORF66 are important for 240

association with ORF34. 241

Based on our results of ORF66 alanine scanning mutants, we found 242

that CR1, CR5 and CR9 regions of ORF66 contain a C-X-X-C sequence which 243

is critical for ORF34 binding (Fig. 6a). Therefore, we generated expression 244

plasmids with single amino acid mutants ORF66 C295A, C298A, C341A, 245

C344A, C424A and C427A in which a cysteine in C-X-X-C was substituted with 246

an alanine. Plasmids of ORF66 G299A, S342A, G345A and G345A mutants in 247

which the conserved glycine or serine around the C-X-X-C sequence was 248

substituted with alanine were also generated. The plasmids of 6xMyc-ORF34 249

and ORF66 alanine mutants were co-transfected into cells, and cell extracts 250

were subjected to affinity purification using S-protein immobilized beads, 251

followed by Western blotting. As a result, C295A, C298A, C341A, C344A, 252

C424A and C427A ORF66 mutants failed to interact with ORF34 (Fig 8a, b and 253

d). Furthermore, two leucine residues at 412 and 413 in CR8 of ORF66 were 254

also important for the association with ORF34 (Fig 8c). These results suggest 255

that a conserved leucine-repeat (412L and 413L) in CR8 and three conserved 256

C-X-X-C sequences in CR1, 5 and 9 are needed for binding to ORF34, leading 257

to the appropriate formation of vPIC. Because the C-X-X-C motif in proteins is 258

often related to the binding to bivalent-cation, we speculate that C-X-X-C 259

sequence contributes to form a higher order structure such as a zinc-finger 260

domain. Next, to elucidate whether capturing zinc ion within ORF66 is 261

necessary for binding to ORF34, we performed pull-down assays using zinc 262


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chelator TPEN and 2xS-ORF66-conjugated S-protein beads. The ORF66-263

conjugated beads were mixed with the cell extracts including overexpressed 264

Myc tagged ORF34 in the presence of TPEN and were pull-down. The 265

precipitates were subjected to western blotting with anti-Myc. As a result, TPEN 266

decreased the interaction of ORF66 with ORF34, indicating that ORF66 may be 267

a zinc-binding protein (Fig. 8e). 268

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Discussion 270

The C-terminus of ORF66 is highly conserved among herpesvirus 271

homologs compared with the N-terminus (Fig.6a). We prepared alanine-272

scanning mutants of ORF66 for amino acid residues that are conserved among 273

MHV, BHV, EBV, HCMV, and HHV-6. The conserved amino acid regions of 274

CR1, 5, 7, 8 and 9 in the ORF66 C-terminal domain is necessary for the binding 275

between ORF66 and ORF34, and virus production. In addition, three conserved 276

C-X-X-C sequences in ORF66 CR1, 5 and 9 were needed for binding to ORF34 277

(Fig. 8). Our results indicate that ORF66 leads to appropriate vPIC formation by 278

the interaction of ORF34 via C-X-X-C sequences in the C-terminal domain of 279

ORF66. We speculate that the conserved C-X-X-C sequences could form a 280

higher order structure such as a zinc-finger domain. Therefore, we approached 281

the protein structure and functional prediction of ORF66 by a server-based 282

helical protein structure simulation, I-TASSER (Iterative Threading ASSEmbly 283

Refinement) (28-30) and generated a full-length homology model of ORF66 284

(Fig. 9). According to a meta-server approach to protein-ligand binding site 285

prediction (COACH) (31, 32), four cysteine residues, C295, C298, C341, and 286

C344 are predicted to associate with a zinc ion. Based on the location of each 287

cysteine residue in the homology model, a pair of cysteines, C295/C298 in 288

CR1/CR2 or C341/C344 in CR5, might chelate a single molecule of zinc, which 289

correlates with our experimental observation using a zinc chelator, TPEN (Fig. 290

8). The zinc chelator TPEN inhibited association between ORF66 and ORF34. 291

This result indicates that ORF66 binds zinc, which is important for its interaction 292

with ORF34. We also performed homology searching of ORF66 by SWISS-293


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model. Interestingly, 319-348 amino acids (including CR5 region) of ORF66 294

have low homology with the TFIIB zinc-ribbon domain of hyperthermophilic 295

archaea Pyrococcus furiosus. As these zinc-associated cysteine residues were 296

mostly located inside the protein structure, except for C341 (Fig. 9; middle 297

panel), it is implies that CR1, CR2, and CR5 residues are involved in 298

maintaining the functional structure formation of ORF66 via zinc interaction. 299

Two leucine residues (L412 and L413) in LLQL of CR8 are essential 300

for binding between ORF66 and ORF34 (Figure 6b and 8c), suggesting that the 301

hydrophobicity of a leucine-rich sequence is also indispensable for ORF66 to be 302

functional. In fact, L412 and L413 residues in CR8 are predicted to be fully 303

exposed on the protein surface, which represented a high degree of 304

hydrophobicity by our structural model (Fig. 9). Two cytosine residues (C424 305

and C427) in CR9 were not predicted to be a zinc-binding domain by our 306

protein-ligand binding prediction, however these residues in ORF66 were also 307

responsible for ORF34 interaction (Figure 6). These residues are exposed on 308

the protein surface (Figure 9; middle panel), and located at the hydrophobic 309

surface including LLQL motif (Figure 9; right panel). Thus, the hydrophobic 310

region surrounding two leucine residues in CR8 and cysteine residues in CR9 is 311

expected to make up the binding surface and be directly involved in the 312

hydrophobic interaction with ORF34. 313

We evaluated the viral replication in KSHV ORF66-producing iVero 314

and iSLK cells, which were stably integrated with an ORF66-dificient KSHV 315

BAC clone. ORF66 plays a critical role in virus production and the transcription 316

of L genes. KSHV ORF24 binds to ORF34, RNA pol II and the TATT-box of the 317


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transcriptional start site (TSS) of the L gene (18, 20, 21). Because direct or 318

indirect interaction of ORF34 with ORF 18, 30, 31 and 66 were indicated by co-319

immunoprecipitation and split luciferase experiments (18, 20, 21), ORF34 has 320

been thought to function as a hub for interactions between ORF24 and other 321

vPIC components. Therefore, these reports, in addition to our viral replication 322

kinetics and viral gene expression data imply that ORF66 engages in L gene 323

expression as a vPIC component. These results are in line with other 324

herpesvirus homologs of KSHV ORF66. For instance, EBV BFRF2 is essential 325

for virus production and contributes to vPIC formation (33). HCMV UL49 is also 326

essential for replication in human foreskin fibroblasts (34, 35). 327

To gain a better understanding of ORF66 within the vPIC complex, we 328

attempted to search for ORF34-binding regions within ORF66. Pull-down 329

assays using truncated ORF66 mutants showed that the C-terminal region 330

(from 241 a.a. to 429 a.a/the C-terminus end) of ORF66 was responsible for 331

binding to ORF34 (Fig. 5b). And the truncated ORF66 mutants were subjected 332

to a trans-complementation assay. However, all truncated ORF66 mutants did 333

not rescue virus productions in both KSHV-ORF66 iVero and iSLK cell lines 334

(Fig. 5c and 5d). Therefore, we speculate that the entire structure of ORF66 is 335

necessary for virus production through vPIC formation. Considering the 336

complex structure of human PIC components (TBP and GTFs) and RNAPII 337

(36), vPIC is might be a crowded complex that consists of not only vPIC factors 338

but also host proteins such as RNAPII, RNAPII binding proteins, and other 339

unknown host. Lacking large regions of ORF66 may also influence the overal 340

structure of the protein and affect interaction with its binding partners. Another 341


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possibility is that the N-terminal and center regions of ORF66 are related to 342

interaction with other vPIC components or unknown host factors. 343

To evaluate the physiological function of conserved amino acids, block 344

alanine-scanning mutants of ORF66 were subjected to a trans-complementation 345

assay. The ORF66 mutants (CR1, 5, 7, 8 and 9mut) failed to associate with 346

ORF34 (Fig. 6) and could not rescue virus production in KSHV-ORF66 cell 347

lines (Fig. 7). In contrast, some ORF66 mutants (CR3 and 6mut), which 348

associate with ORF34, showed full or partial rescue activities, while CR2mut did 349

not. Presumably, the conserved sequence (CLNxG) in the CR2 region is related 350

to binding to other vPIC associated factors. ORF66 rescued activity in iSLK/ 351

66 and iVero/ 66 cell lines, however, recovery rates were lower in iSLK/ 66. 352

The differences in the recovery rates of both cell lines may be due to KSHV 353

production potential and/ or characteristics of each cell line. Efficiency of KSHV 354

production in iSLK is 100-fold higher than iVero. Furthermore, iVero is derived 355

from non-human primates, African green monkey. Slight differences of mutated 356

sites structure and/or species differences of host factors may influence the vPIC 357

formation and accumulation of other host factors, resulting in differences in in 358

the recovery rates. Altogether, association between ORF66 and ORF34 is 359

necessary but not sufficient for virus production. 360

Our results show that the importance and molecular machinery of 361

ORF66 in viral replication and L gene expression, as a vPIC component. 362

Herpesvirus vPICs consist of viral factors as well as RNAPII and several host 363

factors that are engaged in a complex. In MCMV, RNA helicase and cellular 364

factors relating to splicing and translation interact with vPIC (37). Regulation of 365


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vPIC occurs through post-transcriptional modification of vPIC components, such 366

as phosphorylation, which is known to contribute to physiological functions of 367

vPIC in EBV (38). As the dynamics of vPIC in viral replication, vPIC target 368

promoters on KSHV genome has more complexity than estimated. It 369

inextricably linked to genome DNA replication (27). Our efforts to unveil vPIC 370

machinery help to shed light on why - and -herpesviruses have incorporated 371

vPIC machinery into its genome for survival. 372

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Material and Methods 374

Plasmids. 375

pCI-neo-3xFLAG-ORF66, pCI-neo-3xFLAG-ORF66, pCI-neo-6xMyc-ORF34 376

expression plasmids were previously described (20). Truncation and alanine 377

mutant ORF66 coding fragments were obtained by PCR or overlap-extension 378

PCR from ORF66 expression plasmid using primer sets noted in Table 1, and 379

were cloned into pCI-neo-2xS, and pCI-neo-3xFLAG vectors respectively. For 380

pCI-blast plasmid construction, Blastcidin resistant gene (blaR) coding 381

fragments were obtained by PCR from pLKO.1-blast (Addgene plasmid # 382

26655, a kind gift from Dr. Keith Mostov. (39)) using primer sets as noted in 383

Table 1, and were replaced NeoR in pCI-neo mammalian expression vector 384

(Promega, WI, USA). pCI-neo-3xFLAG and pCI-neo-3xFLAG-ORF66 were 385

digested with NheI and NotI sites, and the protein coding fragments were 386

inserted into MCS of pCI-blast to construct of pCI-blast-3xFLAG and pCI-blast-387

3xFLAG-ORF66. 388

389

Mutagenesis of KSHV BAC16. 390

KSHV BAC16 was a kind gift from Jae U. Jung and mutagenesis of KSHV 391

BAC16 was performed according to previous publications (24, 25). The primers 392

for mutagenesis sequences are noted in Table 1. Insertion and deletion of 393

kanamycin resistance cassettes (KanR) in each mutant were analyzed by 394

digestion of EcoRV and agarose-gel electrophoresis. Mutated sites of each 395

BAC clone were confirmed by Sanger sequencing. 396

397


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Establishment of doxycycline-inducible recombinant KSHV-expressing 398

cells and stably ORF66-expressing cells. 399

For maintenance, iSLK cells were cultured in growth medium containing 1 400

μg/mL of puromycin (InvivoGen, CA, USA) and 0.25 mg/mL of G418 (Nacalai 401

Tesque, Kyoto, Japan). iVero cells (20) were cultured in a growth medium 402

containing 2.5 μg/mL of puromycin. KSHV BAC16 wild-type (WT-BAC16) and 403

mutant (ORF66-BAC16) were transfected to iSLK and iVero cells. iSLK and 404

iVero cells were transfected by a calcium phosphate and a lipofection method, 405

respectivly. Transfected cells were selected under 1000 μg/mL of hygromycin B 406

(Wako, Osaka, Japan) and 2.5 μg/mL of puromycin to establish doxycycline-407

inducible recombinant KSHV producing cell lines (iSLK-WT, iSLK-ORF66, 408

iVero-WT, iVero-ORF66). 409

To establish stable ORF66-expressing cells, pCI-blast-3xFLAG-ORF66 410

and empty vector (pCI-blast-3xFLAG) were transfected into iSLK-WT or iSLK-411

ORF66 cells, and transfected cells were selected in 10 μg/mL of Blastcidin S 412

(InvivoGen) and maintained in 7.5 μg/mL of Blastcidin S. Thus, stable cell lines, 413

iSLK-WT/pCI-blast-3xFLAG, iSLK-ORF66/pCI-blast-3xFLAG and iSLK-414

ORF66/pCI-blast-3xFLAG-ORF66, were established. To establish iVero-WT/ 415

pCI-neo-3xFLAG, iVero-ORF66/ pCI-neo-3xFLAG, iVero-ORF66/ pCI-neo-416

3xFLAG-ORF66 stable cell lines, iSLK harboring each KSHV BAC clone was 417

selected and maintained in 1.5 mg/mL of G418. 418

419

420


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Measurement of virus production and viral DNA replication. 421

For quantification of virus production, KSHV virions in culture supernatant were 422

quantified as previously described (20, 40, 41). Briefly, iSLK and iVero cells 423

(iSLK-WT, iSLK-ORF66, iVero-WT, or iVero-ORF66) were treated with 424

Sodium Butyrate (NaB) and doxycycline (iSLK; NaB 0.75 mM/ Dox 4 μg/mL, 425

iVero; NaB 1.5 mM/ Dox 8 μg/mL) for 72 hours to induce to lytic replication and 426

production of recombinant KSHV, and culture supernatants were harvested. 427

Culture supernatants (220 μL) were treated with DNase I (NEB, MA, USA) to 428

obtain only enveloped and encapsidated viral genomes. Viral DNA was purified 429

and extracted from 200 μL of DNase I-treated culture supernatant using the 430

QIAamp DNA blood mini kit (QIAGEN, CA, USA). To quantify viral DNA copies, 431

SYBR green real-time PCR was performed using KSHV-encoded ORF11 432

specific primers. 433

For measurement of KSHV genome replication, each KSHV producing 434

cell line was treated with doxycycline and NaB for 48 hours to induce lytic 435

replication, and harvested. Total cellular DNA containing the KSHV genome 436

were purified and extracted from washed cells using the QIAamp DNA blood 437

mini kit (QIAGEN). Cellular KSHV genome copies were determined by SYBR 438

green real-time PCR and normalized to total DNA. 439

440

Recovery of exogenous gene in BAC harboring cells. 441

The iSLK cells were transfected with pCI-neo-3xFLAG as a control plasmid, and 442

3xFLAG-tagged ORF66 full length, truncated mutant, alanine mutant plasmids 443

using Screenfect A plus (Wako, Tokyo, JAPAN) according to the manufacturer’s 444


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instructions and simultaneously stimulated with NaB 0.75 mM / Dox 4 μg/mL 445

containing medium. The iVero cells were transfected using PEI-MAX MW40000 446

(Polysciences, Inc., Warrington, PA, USA)(42). After 1 day, transfected iVero 447

cells were stimulated with NaB 0.5 mM / Dox 8 μg/mL containing medium. After 448

three days of stimulation, viral supernatant was harvested and KSHV genome 449

was evaluated by real-time PCR. 450

451

RT real-time PCR (RT-qPCR) array. 452

mRNA was extracted from iSLK cells treated with Dox and NaB using FastGene 453

RNA Premium Kit (Nippon Genetics Co. Ltd., Tokyo, Japan). cDNA was 454

synthesized by ReverTra Ace RT-qPCR kit (TOYOBO, Osaka, Japan) and 455

subjected to SYBR green real-time PCR. Gene expression was analyzed by 456

qPCR using specific primers designed by Fakhari and Dittmer (26). Relative 457

KSHV mRNA expression levels were determined by GAPDH expression and 458

Ct methods. 459

460

Chromatin Immuno-precipitation (ChIP) assay. 461

ChIP assay was performed as described previously (43) with slight 462

modifications. Briefly, iSLK-WT/ Control and iSLK-ORF66/ 3xFLAG-ORF66 463

cells were treated with or without 4 μg/mL of Dox and 0.75 mM NaB for 72 464

hours. Formaldehyde-fixed cells were lysed by farnham lysis buffer (5 mM 465

PIPES pH 8.0 / 85 mM KCl / 0.5% NP-40) and the nuclear pellet was collected. 466

The pellet was lysed in SDS lysis buffer and sonicated. The supernatant 467

containing DNA was diluted by CHIP dilution buffer and then subjected to 468


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immunoprecipitation with anti-FLAG (DDDDK-tag) monoclonal antibody (FLA-1; 469

MBL, Nagoya, Japan) or mouse control IgG (Santa-Cruz). Immunoprecipitates 470

containing chromatin and viral DNA were subjected to SYBR green real-time 471

PCR for measuring the amount of promoter DNA of each gene. The amount of 472

immunoprecipitated viral DNA was normalized to 1% of input DNA. The 473

sequences of qPCR primer sets for each transcription start site of ORFs are 474

noted in Table 1. 475

476

Pull-down assay, Western blot, and antibodies. 477

Western blots were performed as described previously (20). For pull-down 478

assays, transfected 293T cells (RCB2202; RIKEN Bio Resource Center, 479

Tsukuba, Japan) were lysed by HNTG buffer (20 mM HEPES (pH 7.9), 0.18 M 480

NaCl, 0.1% NP-40, 0.1 mM EDTA, 10% Glycerol) with protease inhibitors and 481

sonicated. The cell extracts were subjected to affinity purification using S-482

protein immobilized beads (Novagen, MA, USA), and purified proteins 483

(containing 2xS-tagged ORF66 or mutants) were subjected to western blotting. 484

For Zinc chelator TPEN (N,N,N',N'-Tetrakis (2-pyridylmethyl) 485

ethylenediamine; TCI, Tokyo, Japan) treatment, 3xFLAG-taggged ORF66 486

overexpressed in 293T cells, was purified with S-protein immobilized beads in 487

the presence of each dose of TPEN or vehicle (ethanol) for 2 hours. The beads 488

were mixed with cell lysate of 6xMyc-ORF66 overexpressed in 293T in the 489

presence of each dose of TPEN or vehicle (ethanol) for 2 hours. The beads 490

were washed 4 times and subjected to western blotting. 491

Anti-Myc (9E10; Santa-Cruz, CA, USA), Anti-S-tag pAb (MBL, Nagoya, 492


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24

Japan), Anti-FLAG (DDDDK-tag) (FLA-1; MBL), Anti-Actin (AC-15; Santa-Cruz) 493

were used as the primary antibodies. HRP linked anti-mouse IgG antibody (GE 494

Healthcare UK Ltd., Buckinghamshire, UK) or HRP linked anti-rabbit IgG 495

antibody (GE healthcare UK Ltd.) was used as the secondary antibody. Anti-496

FLAG-HRP (M2; Sigma-aldrich, MO, USA) was also used. 497

498

Homology modeling 499

The template structure for ORF66 was initially identified by collecting high-500

scoring structural templates from Local Meta-Threading Sever to generate a 3D 501

structural model. The protein-ligand predictions were then derived by threading 502

the 3D models through a protein function database, BioLiP. All procedures were 503

automatically processed on I-TASSER server program provided by the Zhang 504

lab (URL: https://zhanglab.ccmb.med.umich.edu/I-TASSER/) (28-32). The 505

visualization of the homology model was performed by molecular visualization 506

open-source software, PyMOL. The hydrophobicity in each amino acid was 507

determined by the Eisenberg hydrophobicity scale 508

(https://web.expasy.org/protscale/pscale/Hphob.Eisenberg.html) (44) and 509

visualized by running a color_h.py script on PyMOL. 510

511

Statistics. 512

The two-tailed student’s t-test was used to indicate the differences between the 513

groups. P values are shown in each figure. 514

515

Acknowledgements 516


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25

The BAC16, KSHV BAC clone, was a kind gift from Dr. Kevin Brulois and Dr. 517

Jae U Jung (U.S.C., US). We thank Dr. Gregory A. Smith (Northwestern Univ., 518

US) for the E. coli strain GS1783, and Dr. Nikolaus Osterrieder (Cornell Univ., 519

US) for the plasmid pEP-KanS. We thank Dr. Peter Gee for scientific advice and 520

critical proofreading of the manuscript. This work was supported in-part by a 521

Grant-in-Aid for Scientific Research (C) (18K06642), Young Scientists (B) 522

(16K18925) and Young Scientists (18K14910) from the Ministry of Education, 523

Culture, Sports, Science and Technology of Japan. 524

525

526


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Figure legends 527

Fig.1. Construction of recombinantORF66 KSHV BAC 528

(a) Schematic illustration of the KSHV genome including the ORF66 coding 529

region. Using a two-step Red recombination system, three stop codons were 530

inserted into the ORF66 coding region of KSHV BAC16 (nt113417 - nt113416; 531

Accession number: GQ994935) to construct ORF66-deficient BAC clone 532

(ORF66-BAC16). (b) Agarose gel electrophoresis of the recombinant KSHV 533

BACmids, digested with EcoRV. The asterisks (*) indicate insertion and deletion 534

of a kanamycin-resistance cassette in each BAC clone. (c) DNA sequencing 535

results of ORF66 mutagenesis sites in ORF66-BAC16. 536

537

Fig.2. ORF66 is essential for virus production but not DNA replication of 538

KSHV. 539

Virus production in (a) iSLK-WT, iSLK-ORF66 and (e) iVero-WT, iVero-540

ORF66. Each cell line was cultured for 72 h with medium containing NaB and 541

Dox. KSHV DNA was purified from capsidated KSHV virions in culture 542

supernatants, and KSHV genome copies were determined by real-time PCR. 543

Virus production in (b) iSLK-WT, iSLK-ORF66 and (f) iVero-WT, iVero-544

ORF66. Each cell line was cultured for 48 h with medium containing of NaB 545

and Dox. Cellular DNA containing KSHV genomic DNA was purified from each 546

cell line. KSHV genome copies were determined by real-time PCR and 547

normalized by the total DNA amount. Establishment of exogenous ORF66 548

expressing (c) iSLK-ORF66 and (h) iVero-ORF66 stable cell lines. Western 549


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blot shows exogenous FLAG-tagged ORF66. Rescue of virus production in (d) 550

iSLK-ORF66 and (h) iVero-ORF66 cells by exogenous ORF66 expression. 551

Each stable cell line was cultured with NaB and Dox-containing medium for 3 552

days, and culture supernatant containing virus was harvested and quantified. 553

(a-b, d-f, h) Three or four independent samples were evaluated by real-time 554

PCR. The error bars indicate standard deviations. 555

556

Fig.3. ORF66 is required for the late gene expression 557

iSLK-WT and iSLK-ORF66 cells were cultured for 72h in media with NaB and 558

Dox to induce a lytic state. Total RNA was extracted from cells and subjected to 559

RT-qPCR. The mRNA expression level of each viral gene was normalized by 560

GAPDH expression, and columns indicated fold changes of iSLK-ORF66 561

transcripts compared with iSLK-WT transcripts. Classification of KSHV genes 562

was performed according to Arias C. et al. (45). White, light gray, dark gray, 563

black, and light blue columns indicate Latent, Immeidiate early, Early, Late, and 564

Not classified genes, respectively. Expression levels were assessed using three 565

independent samples, and error bars indicate ± standard deviations. 566

567

Fig.4. ORF66 associates with the transcriptional start site of Late genes 568

iSLK-WT/ Control and iSLK-ORF66/ 3xFLAG-ORF66 cells were treated with 569

(or without) Dox and NaB for 72 hours and subjected to ChIP-qPCR. 3xFLAG-570

ORF66 protein was immunoprecipitated by anti-FLAG or control IgG antibody, 571

and precipitates including chromatin and viral DNA were subjected to SYBR 572

green real-time PCR for measuring the amount of promoter DNA of ORF72 573


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(Latent gene), ORF16 (IE gene), ORF46/47 (E gene) or K8.1 (L gene). The 574

levels of immunoprecipitated viral promoter were normalized to total input DNA. 575

576

Fig.5. ORF66 physically interacts with ORF34 via its C-terminus regions 577

and the entire structure of ORF66 is indispensable for virus production. 578

(a) Schematic representation of tagged ORF34 deletion mutants used in the 579

mapping experiments. The truncated amino acids are numbered next to its 580

corresponding mutant. (b) 293T cells were co-transfected with expression 581

plasmids of 2xS-ORF66 truncated mutant and 6xMyc-ORF34. Transfected cells 582

were lysed, and cell lysates were subjected to pull-down assays using S-583

protein-immobilized beads that capture the 2xS-ORF66. Obtained precipitates 584

including 2xS-ORF66 truncated mutants were probed with indicated antibodies 585

to detect interactions. 586

The (c) iSLK-ORF66 or (d) iVero-ORF66 cells were transfected with control, 587

ORF66 or ORF66-truncated mutant plasmid. Transfected cells were stimulated 588

for 3 days. Progeny KSHV was purified from harvested culture supernatant, and 589

the KSHV genome was quantified by real-time PCR. Viral productivity was 590

assessed using four independent samples, and error bars indicate standard 591

deviations. Transfected virus producing cells were lysed and subjected to 592

Western blotting to confirm ORF66 mutant expression. 593

594

Fig.6. Several conserved residues of ORF66 are essential for physical 595

association with ORF34. 596

(a) Amino acid sequence alignment of C-terminus of ORF66 (241 a.a.- 429 597


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29

a.a.). Herpesvirus homolog amino acid sequences were translated from 598

nucleotide sequences found in the NCBI database (KSHV ORF66 (JSC-1-599

BAC16; Accession number GQ994935), MHV68 ORF66 (strain WUMS; 600

NC_001826), BHV4 ORF65 (strain V; JN133502), EBV BFRF2 (strain B95-8; 601

V01555), CMV UL49 (strain Towne ; FJ616285), HHV6 U33 (strain japan-a1; 602

KY239023)). Raw data of alignment were obtained by using Clustal Omega 603

(EMBL-EBI; https://www.ebi.ac.uk/Tools/msa/clustalo/). Completely conserved 604

amino acids between homologs were indicated by a gray background. Based 605

on this information, several conserved amino acids were split into blocks of 606

alanine scanning ORF66 mutants (CR1mut; ORF66 C295A/C298A/G299A, 607

CR2mut; ORF66 C301A/L302A/N303A/G305A, CR3mut; ORF66 F314A/F320A, 608

CR4mut; ORF66 R323A/D324A/E327A/K328A, CR5mut; ORF66 609

C341A/S342A/C344A/G345A, CR6mut; ORF66 V371A/N375A, CR7mut; 610

ORF66 C393A, CR8mut; ORF66 L412A/L413A/L415A, CR9mut; ORF66 611

C424A/C427A). 612

(b) Block alanine scanning mutants were co-transfected with expression 613

plasmids of 6xMyc-ORF34. Cell lysates were subjected to pull-down assays 614

using S-protein immobilized beads. 615

616

Fig.7. Association between ORF66 and ORF34 is necessary but not 617

sufficient for virus production 618

The (a) iSLK-ORF66 or (b) iVero-ORF66 cells were transfected with control, 619

ORF66 or ORF66 block alanine scanning mutant plasmid. Progeny KSHV was 620

purified and the KSHV genome was quantified. Viral productivities were 621


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assessed using three independent samples, and error bars indicate standard 622

deviations. The colour of each bar indicates the following: Black, control; Gray, 623

ORF66 WT; Blue, ORF66 alanine scanning mutants not binding to ORF34; 624

Red, ORF66 alanine scanning mutants binding to ORF34. Transfected virus 625

producing cells were lysed and subjected to Western blotting analysis for the 626

confirmation of ORF66 mutant expression. 627

628

Fig.8. Identification of individual amino acids of ORF66 responsible for 629

binding to ORF34. 630

ORF66 wild-type, block alanine scanning mutants and single alanine scanning 631

mutants were co-transfected with expression plasmids of 6xMyc-ORF34. Cell 632

lysates were subjected to pull-down assays using S-protein immobilized beads. 633

Blotting showed the association between ORF34 and (a) CR1 mutants 634

(CR1mut; ORF66 C295A/C298A/G299A, ORF66 C295A, ORF66 C298A, 635

ORF66 G299A, (b) CR5 mutants (CR5mut; ORF66 636

C341A/S342A/C344A/G345A, ORF66 C341A, ORF66 S342A, ORF66 C344A, 637

ORF66 G345A), (c) CR8 mutants (CR8mut; ORF66 L412A/L413A/L415A, 638

ORF66 L412A, ORF66 L413A, ORF66 L415A) and (d) CR9 mutants (CR9mut; 639

ORF66 C424A/C427A, ORF66 C424A, ORF66 C427A). (e) Zinc ion chelation 640

influences ORF66 binding abilities to ORF34. Pull-down assay using S-tagged 641

ORF66 binding beads in the presence of Zinc chelator TPEN. 642

643

Fig.9. The protein structure and functional prediction of ORF66. 644


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31

The structural models of ORF66 simulated by I-TASSER, the server completed 645

protein structural and functional prediction, are shown. The cartoon model 646

indicates the position of responsible residues for interaction with ORF34 (left 647

panel). The surface model represents the exposed residues on the protein 648

surface (middle panel). The hydrophobicity of each residue is shown by a red 649

color gradient followed by the Eisenberg hydrophobicity scale (right panel). The 650

predicted Zinc ion-binding cysteins (C295 and C298, C341 and C344) are 651

highlighted with brown. C344 in CR5 is highlighted in magenta. The LLQL in 652

CR8 and C341 in CR5 are shown in cyan and green, respectively. 653

654

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40. Wakao K, Watanabe T, Takadama T, Ui S, Shigemi Z, Kagawa H, Higashi 782 C, Ohga R, Taira T, Fujimuro M. 2014. Sangivamycin induces apoptosis 783 by suppressing Erk signaling in primary effusion lymphoma cells. 784 Biochem Biophys Res Commun 444:135-40. 785

41. Watanabe T, Nakamura S, Ono T, Ui S, Yagi S, Kagawa H, Watanabe H, 786 Ohe T, Mashino T, Fujimuro M. 2014. Pyrrolidinium fullerene induces 787 apoptosis by activation of procaspase-9 via suppression of Akt in primary 788 effusion lymphoma. Biochem Biophys Res Commun 451:93-100. 789

42. Katoh Y, Nozaki S, Hartanto D, Miyano R, Nakayama K. 2015. 790 Architectures of multisubunit complexes revealed by a visible 791 immunoprecipitation assay using fluorescent fusion proteins. J Cell Sci 792 128:2351-62. 793

43. Agata Y, Katakai T, Ye SK, Sugai M, Gonda H, Honjo T, Ikuta K, Shimizu 794 A. 2001. Histone acetylation determines the developmentally regulated 795 accessibility for T cell receptor gamma gene recombination. J Exp Med 796 193:873-80. 797

44. Eisenberg D, Schwarz E, Komaromy M, Wall R. 1984. Analysis of 798 membrane and surface protein sequences with the hydrophobic moment 799 plot. J Mol Biol 179:125-42. 800

45. Arias C, Weisburd B, Stern-Ginossar N, Mercier A, Madrid AS, Bellare P, 801 Holdorf M, Weissman JS, Ganem D. 2014. KSHV 2.0: a comprehensive 802 annotation of the Kaposi's sarcoma-associated herpesvirus genome 803 using next-generation sequencing reveals novel genomic and functional 804 features. PLoS Pathog 10:e1003847. 805

806


https://doi.org/10.1101/728147

1 a.a. 433 a.a.

nt 113436

ORF66

TAGTTAGATAGT

GAAACATG CCCTCCTGnt 113409

stop stop stop

54 a.a.

..... .....

ORF65ORF66ORF67

Two-stepRed recombination

Two-stepRed recombination

KanR

..... .....

(a)

ORF66/ KanR

ΔORF66-BAC16

3-stop element

3-stop element

WT-BAC16

GAAACATGTAGTTAGATAGTCCCTCCTG

OR

F66/ K

an

RΔO

RF66-B

AC

16

WT-B

AC

16

Mark

er

25K

10K

8K

6K

5K

4K

3K

2K

(bp)

2.5K

1.5K

1K

EcoRV

MN S

GA AAC ATG TAG TTA GAT AGT CCC TCC TG

GA AAC ATG CCC TCC TG

P

..... .....113409113436

..... .....

WT-BAC16

ΔORF66-BAC16

3-stop element5’ 3’

(b)

(c)

Fig.1.

*

*

*


https://doi.org/10.1101/728147

Fig.2.

NaB/Dox – + +–

(b)(a) (d)(c)

-WTiSLK

-Δ66iSLK

(×1

05 c

op

ies

/ to

tal

DN

A(n

G))

Ce

llu

lar

KS

HV

ge

no

me

(f)(e)

-WTiVero

-Δ66iVero

-WTiVero

-Δ66iVero

(×1

04 c

op

ies

/ to

tal

DN

A(n

G))

Ce

llu

lar

KS

HV

ge

no

me

(g) (h)

-WT

Vir

us

pro

du

cti

on

P=5.08 x 10-7

iSLK-Δ66

iSLK

P=3.12 x 10-4

1010

109

108

107

106

105

(co

pie

s/m

L)

Vir

us

pro

du

cti

on

(co

pie

s/m

L)

104

108

107

106

105

P=0.129

N.S.

0

1

2

3

4

5

6

2

0

4

6

8

10

12

14

16 P=0.203

N.S.

NaB/Dox – + +–

iSLK

-WT/ C

ontrol

iSLK

-Δ66/ C

ontrol

iSLK

-Δ66/

3xF

LA

G-O

RF66

iVero

-WT/ C

ontrol

iVero

-Δ66/ C

ontrol

iVero

-Δ66/

anti-FLAG

anti-Actin

3xF

LA

G-O

RF66

45

45

(kDa)

Vir

us

pro

du

cti

on

(%

)

10-2

103

102

10

10-1

iVero

-WT/ C

ontrol

iVero

-Δ66/ C

ontrol

iVero

-Δ66/

3xF

LA

G-O

RF66

P=0.0182

iSLK

-WT/ C

ontrol

iSLK

-Δ66/ C

ontrol

iSLK

-Δ66/

3xF

LA

G-O

RF66

Vir

us

pro

du

cti

on

(%

)

10-2

103

102

10

10-1

10-3

P=1.03 x 10-7

anti-FLAG

anti-Actin

45

(kDa)

45


https://doi.org/10.1101/728147

Fig.3.

0.001

0.01

0.1

1 K

8.1

O

RF

17

OR

F26

OR

F27

OR

F75

OR

F45

K9/ vIR

F1

OR

F39

OR

F25

OR

F46

OR

F33

OR

F37

OR

F38

OR

F52

OR

F28

OR

F4

OR

F55

OR

F8

OR

F42

OR

F9

K10

OR

F32

vIR

F3

OR

F47

OR

F65

OR

F31

OR

F30

OR

F22_2

OR

F19

K11

OR

F34

OR

F22_1

OR

F53

OR

F63

K1

OR

F48

OR

F64

OR

F18

OR

F21

OR

F36

OR

F10

OR

F74

OR

F56

OR

F43

OR

F49

K4

OR

F29

OR

F11

OR

F62

OR

F16

OR

F61

OR

F60

OR

F7

K14

OR

F41

OR

F58

OR

F59

OR

F40

OR

F35

K-b

ZIP

O

RF

67

K7/ P

AN

O

RF

66

OR

F24

K12

OR

F54

OR

F73

OR

F23

OR

F72

OR

F57

OR

F6

OR

F70

OR

F2

OR

F69

K3

K6_1

OR

F71

K6_2

OR

F44

OR

F68

K5_2

K5_1

OR

F50

K2

Fo

ld in

du

cti

on

(Δ

66 / W

T p

rod

ucin

g iS

LK

cells)

Latent

Immediate Early

Early

Late

Not classified


https://doi.org/10.1101/728147

Fig.4.

ORF72, Latent, TATA

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

ORF16, IE, TATA

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

ORF46/47, E, TATA

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

K8.1, L, TATT

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

% I

np

ut

% I

np

ut

% I

np

ut

% I

np

ut

P=5.47 x 10-6

P=0.502

N.S.P=0.183

N.S.

P=0.297

N.S.

Control IgG

Anti-FLAG

Control IgG

Anti-FLAG

Control IgG

Anti-FLAG Control IgG

Anti-FLAG

iSLK-WT iSLK-Δ66iSLK-WT/ Control / 3xFLAG-ORF66





https://doi.org/10.1101/728147

Fig.5.

Tag Full length ORF661 429a.a.

66 WT

(Δ2-60)

(Δ61-120)

(Δ121-180)

(Δ181-240)

(Δ241-300)

(Δ301-360)

(Δ361-429)

66 Δ1

66 Δ2

66 Δ3

66 Δ4

66 Δ5

66 Δ6

66 Δ7

61

60 121

120 181

180 241

240 301

300 361

360

(b)(a)

(d)(c)

66 Δ

4

66 Δ

5

66 W

T

66 Δ

3

66 Δ

2

66 Δ

1

66 Δ

6

66 Δ

7

Control

(ORF66WT or mut)Blot: Anti-FLAG

45

Blot: Anti-Actin (kDa)

1

10

100

Vir

us

pro

du

cti

on

(% o

f c

op

ies

/mL

)

1

10

100

Vir

us

pro

du

cti

on

(% o

f c

op

ies

/mL

)iSLK/Δ66 iVero/Δ66

66 Δ

4

66 Δ

5

66 W

T

66 Δ

3

66 Δ

2

66 Δ

1

66 Δ

6

66 Δ

7

Control

(ORF34)Blot: Anti-Myc

(ORF66)Blot: Anti-S


WCE

6xMyc-ORF34

(kDa)

55

66 Δ

466 Δ

5

66 W

T

66 Δ

3

66 Δ

2

66 Δ

1

45

35

55

Pull-

: S

– –

66 Δ

666 Δ

7

down

2xS-ORF66

(ORF66WT or mut)Blot: Anti-FLAG

Blot: Anti-Actin

45

45

(kDa)

45


https://doi.org/10.1101/728147

Fig.6.

1 429

66 WT

Δ5 Δ6 Δ7241

KSHV 241 L P A C E - - - - - - - - - - - - - - - - - - - - Q E G P - - G L V R N L G R R L L - - - - - - - - A Y N V L S P C V S 270

MHV68 224 V P V S S - - - - - - - - - - - - - - - - - - - - S S K Q - - - S V Y D V A I R F I - - - - - - - - G S R I L S P I V R 252

BHV4 244 V P I S G - - - - - - - - - - - - - - - - - - - - S D - - - - S L T H D I H V K I I - - - - - - - - A Y N V L C C Y I S 271

EBV 310 L P I P A V S E G G R K T G G G V G E E L - - V G A G G P - - C L S R D V F V A I V - - - - - - - - S R N V L S C L L N 357

CMV 290 V P L R A L G L H D E T R G G G S T A A A A A V G H A G A G Q Q A R H V E P T K I V L F A L S A A L R G G L I G S V I D 349

HHV6 236 I P V E H N N L V P M V P S - - - - - - - - - - - - - - - - - K P E R G D F P K I L T F A L A T S L K D G L A T S V I S 278

KSHV 271 I P V I C S R V A R - - - - - - - A A L A K R A R C A R A V V C M E C G H C L N F G R G K F - - - H T V N F P P T N V F 320

253 L P I L S R A L A D - - - - - - - - - L A L T G H G H Q I T V C N E C G H C L N L G R D K F - - - L A V N F S P T S M F 300

272 L P I L S R G V - L - - - - - - - T S I G E K G T V Q K F V T C L D C G H C L N F G R G K F - - - K T I N F L P T N I F 320

358 V P A A G P R A Y K C F R S H A S R P V S G P D Y P P L A V F C M D C G Y C L N F G K Q T G V G G R L N S F R P T L Q F 417

350 L P L W C L C R L K C E R H L D - - - - - - - A R S L V A V V C R Q C G H C L N L G K E K L H - - C Q Q N F P L N S M F 400

279 L P V M C Y C K T K C S R F I L - - - - - - - E E S Y I C V I C A K C G H C L N S G K E K L C - - S P Q G F S L S S M F 329

MHV68BHV4EBVCMVHHV6

KSHV 321 F S R D R K E K Q F T I C A T T G R I Y C S Y C G S E H M R V Y P L C D I T G R G T L - - - - A R V V I R A V L A N N A 376

301 Y C R D Q K E K Q F N I C A T T G R I Y C S Y C G A T E F T V Y D M V G R Y A T - - - - - - - G E P F I R A V S S A N S 353

321 Y C R D Q K E K Q A V I C A T T G R I Y C S Y C G S S H I T V L P M M G S D K K I - - - - - - - - S Y L R A V I S N N A 372

418 Y P R D Q K E K H V L T C H A S G R V Y C S N C G S A A V G C Q R L A E P P S A R S G - - - - W R P R I R A V L P H N A 473

401 Y Y R D R Q E K S V I F N T H A E L V H C S L C G S Q R V V R Q R V Y E L V S E T L F G Q R C V R V G W K A V L G L N A 460

330 Y F R D K Q E K N L I Y S M H T D V M Y C S L C G S Q Q L V F E R I Y E M S E H C V L G M K V K T V S W K A V I G T N S 389

MHV68BHV4EBVCMVHHV6

KSHV 377 A L A I R D L D Q T V S F V V P C L G T P D C E - - A A L L K H R D V R G L L Q L T S Q L L E - F C C G K C S S - - - - 429

354 L S I L D N S E Q E C D I L I P C F G K S R - S - - C S I K L R A T F R E L L Y L T A S V D N - F I C Q K C S N K G D E 409

373 A S A I K S I D Q E V H V V V P C L G Q - N C G - - A C I I K R L T I N D L L Y L T A N P N N - L T C F K C T R - - - - 424

474 A Y E L D R G S R L L D A I I P C L G P D R T C M R P V V L R G V T V R Q L L Y L T L R T E A R A V C S I C Q Q R Q A P 591

461 A C A V Y D H R L A F D V I L P C A A R - - T C D S T V V V R G V T V P R L L R L T S H G H G - L L C A R C Q T G E Y R 564

390 A C T I L N D N V K F D V I V P C S C R - - S C Y S T V H L Y N V T V K K L L R L V S H G S D - F Q C Q H C Q H - S F R 470

KSHVMHV68BHV4EBVCMVHHV6

(a)

(b)

WCE

6xMyc-ORF34

(kDa)

55

66 C

R4m

ut

66 C

R5m

ut

66 W

T

66 C

R3m

ut

66 C

R2m

ut

66 C

R1m

ut

55

45

55

Pull-

: S

– –

66 C

R6m

ut

66 C

R7m

ut

down

2xS-ORF66

66 C

R8m

ut

66 C

R9m

ut

45(ORF34)

Blot: Anti-Myc

(ORF66)Blot: Anti-S


CR5mut: ORF66 C341A/S342A/C344A/G345A

CR1mut: ORF66 C295A/C298A/G299A

CR2mut: ORF66 C301A/L302A/N303A/G305ACR3mut: ORF66 F314A/F320A

CR4mut: ORF66 R323A/D324A/E327A/K328A CR6mut: ORF66 V371A/N375A

CR7mut: ORF66 C393A CR8mut: ORF66 L412A/L413A/L415A

CR9mut: ORF66 C424A/C427A

CR1 CR2 CR3

CR4 CR5 CR6

CR7 CR8 CR9


https://doi.org/10.1101/728147

Fig.7

(b)(a)

66 C

R4m

ut

66 C

R5m

ut

66 W

T

66 C

R3m

ut

66 C

R2m

ut

66 C

R1m

ut

66 C

R6m

ut

66 C

R7m

ut

Control

(ORF66WT or mut)Blot: Anti-FLAG45

Blot: Anti-Actin (kDa)

45

1

10

100

Vir

us p

rod

ucti

on

(% o

f co

pie

s/m

L)

1

10

100

Vir

us p

rod

ucti

on

(% o

f co

pie

s/m

L)

66 C

R8m

ut

66 C

R9m

ut

iVero/Δ66iSLK/Δ66

(ORF66WT or mut)Blot: Anti-FLAG45

Blot: Anti-Actin(kDa)

45

5555

66 C

R4m

ut

66 C

R5m

ut

66 W

T

66 C

R3m

ut

66 C

R2m

ut

66 C

R1m

ut

66 C

R6m

ut

66 C

R7m

ut

Control

66 C

R8m

ut

66 C

R9m

ut


https://doi.org/10.1101/728147

Fig.8

(a) (b)

(c) (d)

(e)

6xMyc-ORF34

66 C

344A

66 G

345A

66 W

T

66 S

342A

66 C

341A

66 C

R5m

ut

– –2xS-ORF66

WCE

6xMyc-ORF34

(kDa)

55

66 G

299A

66 W

T

66 C

298A

66 C

295A

66 C

R1m

ut

55

45

55

Pull-

– –

down

2xS-ORF66


(ORF66)Blot: Anti-S


66 L

415A

66 W

T

66 L

413A

66 L

412A

66 C

R8m

ut

– –2xS-ORF66

6xMyc-ORF34

66 W

T

66 C

427A

66 C

424A

66 C

R9m

ut

– –2xS-ORF66

6xMyc-ORF34

TPEN(mM)

: S

WCE

(kDa)

55

55

45

55

Pull-down


(ORF66)Blot: Anti-S


: S

WCE

(kDa)

55

55

45

55

Pull-down


(ORF66)Blot: Anti-S


: S

WCE

(kDa)

55

55

45

55

Pull-down


(ORF66)Blot: Anti-S


: S

45

45

45

4545

Agarose

Lysate

55

45(ORF66)

Blot: Anti-S

– – 5.0 2.5 1.25–

34 34 34 34 34–

– – 66 66 66 66

55


Short exposure


https://doi.org/10.1101/728147

C295 (CR2)

C341 (CR5)

C344 (CR5)C393 (CR7)

C424/C427 (CR9)

LLQL (CR8)

C298 (CR1)

C341 (CR5)

C393 (CR7)

C424/C427 (CR9)

LLQL (CR8)

C341 (CR5)

C393 (CR7)

C424/C427 (CR9)

LLQL (CR8)

Fig. 9.

Cartoon model Surface model Hydrophobicity model


https://doi.org/10.1101/728147

Table 1: Primers for construction of expression plasmids

Primer name Primer sequences (5' -> 3')

[BAC mutagenesis] *a

S_dORF66_3stop_EP ttttgtcatattcggggagcggggtttccagggaaacatgTAGTTAGATAGTccctcctgggccaggcacctTAGGGATAACAGGGTAATCGATTT

As_dORF66_3stop_EP ccagcgacgggcggtccaacaggtgcctggcccaggagggACTATCTAACTAcatgtttccctggaaaccccGCCAGTGTTACAACCAATTAACC

[Cloning pCI-blast plasmid] *b

S_StuI-BlaR-BstBI gcctaggcctaggcttttgcaaaaagcttgattcttctgacacaacagtctcgaacttaaggctagagccaccatggccaagcctttgtctc

As_StuI-BlaR-BstBI ggggttcgaaccccagagtcccgcttagccctcccacacataac

[Cloning expression plasmid] *b

S_XbaI_ORF66 cattctagaATGGCCCTGGATCAGCGCTGGGATC

As_ORF66_NotI gagcggccgcTCAGGAGGAACACTTCCC

- ORF66 truncated mutants *c

S_XbaI_d1(2-60)_ORF66 catctagaATGctgttggaccgcccgtcgctg

S_d2(61-120)_ORF66 GGCCAGGCACtgggcaaaatacctgtcgc

As_d2(61-120)_ORF66 attttgcccaGTGCCTGGCCCAGGAGGG

S_d3(121-180)_ORF66 CACGTAcccacccgtcgcggtgtgg

As_d3(121-180)_ORF66 gacgggtgggTACGTGGCGCGGTATCG

S_d4(181-240)_ORF66 CCCTCGctgcccgcctgcgagcag

As_d4(181-240)_ORF66 ggcgggcagCGAGGGCACCTCCAG

S_d5(241-300)_ORF66 CCACGTAGTGtgtcttaactttggcaggggcaag

As_d5(241-300)_ORF66 agttaagacaCACTACGTGGCGGGACTTAATAAGGCTC

S_d6(301-360)_ORF66 GTGTGGACACgggaccctagcacgcgtc

As_d6(301-360)_ORF66 ctagggtcccGTGTCCACACTCCATGCAC

As_d7(361-429)_ORF66_NotI aagcggccgctcaGCGTCCGGTAATATCGC

- ORF66 block Alanin-scanning mutants *c

S_CR1mut_ORF66 GCAatggagGCTGCAcactgtcttaactttggcag

As_CR1mut_ORF66 TGCAGCctccatTGCcacaaccgccc

S_CR2mut_ORF66 GCTGCTGCAtttGCCaggggcaagtttcatac

As_CR2mut_ORF66 GGCaaaTGCAGCAGCgtgtccacactccatg

S_CR3mut_ORF66 GCTcatactgtcaatGCTcctcccaccaacgtgtttttc

As_CR3mut_ORF66 AGCattgacagtatgAGCcttgcccctgccaaagttaag

S_CR4mut_ORF66 GCTGCAaggaaaGCAGCTcagttcaccatctgtgc

As_CR4mut_ORF66 AGCTGCtttcctTGCAGCgctgaaaaacacgttg

S_CR5mut_ORF66 GCTGCTtacGCTGCAagcgaacatatgagggtgtatc

As_CR5mut_ORF66 TGCAGCgtaAGCAGCgtagatcctccccgtg

S_CR6mut_ORF66 GCTctagctaacGCAgcggcccttgccattc

As_CR6mut_ORF66 TGCgttagctagAGCagccctgattacgacgcgtg

S_CR7mut_ORF66 cctGCCcttgggacgcccgactg

As_CR7mut_ORF66 gtcccaagGGCaggcactacaaaactgacagtttg

S_CR8mut_ORF66 GCAGCTcagGCAacctcacagctgctgg

As_CR8mut_ORF66 TGCctgAGCTGCtccgcgcacgtcac

As_CR9mut_ORF66_NotI tagcggccgctcaggaggaAGCcttcccTGCacagaac

- ORF66 single Alanin-scanning mutants *c

S_CR1_C295A_ORF66 GCAatggagtgtggacactgtcttaactttggcag

As_CR1_C295A_ORF66 tccacactccatTGCcacaaccgccc

S_CR1_C298A_ORF66 tgcatggagGCTggacactgtcttaactttggcag

As_CR1_C298A_ORF66 tccAGCctccatgcacacaaccgccc

S_CR1_G299A_ORF66 tgcatggagtgtGCAcactgtcttaactttggcag

As_CR1_G299A_ORF66 TGCacactccatgcacacaaccgccc

S_CR5_C341A_ORF66 GCTtcttactgtggcagcgaacatatgagggtgtatc

As_CR5_C341A_ORF66 gccacagtaagaAGCgtagatcctccccgtg

S_CR5_S342A_ORF66 tgtGCTtactgtggcagcgaacatatgagggtgtatc

As_CR5_S342A_ORF66 gccacagtaAGCacagtagatcctccccgtg

S_CR5_C344A_ORF66 tgttcttacGCTggcagcgaacatatgagggtgtatc

As_CR5_C344A_ORF66 gccAGCgtaagaacagtagatcctccccgtg

S_CR5_G345A_ORF66 tgttcttactgtGCAagcgaacatatgagggtgtatc

As_CR5_G345A_ORF66 TGCacagtaagaacagtagatcctccccgtg

S_CR8_L412A_ORF66 GCActtcagctcacctcacagctgctgg

As_CR8_L412A_ORF66 gagctgaagTGCtccgcgcacgtcac

S_CR8_L413A_ORF66 ctgGCTcagctcacctcacagctgctgg

As_CR8_L413A_ORF66 gagctgAGCcagtccgcgcacgtcac

As2_CR8_L415A_ORF66_NotI tagcggccgctcaggaggaacacttcccgcaacagaactccagcagctgtgaggtTGCctgaagcagtccgcgcacg

As_CR9_C424A_ORF66_NotI tagcggccgctcaggaggaacacttcccTGCacagaac

As_CR9_C427A_ORF66_NotI tagcggccgctcaggaggaAGCcttcccgcaacagaac

[CHIP qPCR]

CHIP-qPCR_ORF72-F GGCGGGCCATTTGTACTTTC

CHIP-qPCR_ORF72-R ATCTCAGGCCTTCCAGTTTG

CHIP-qPCR_ORF16-F GACGGCAAGGTTTTTATCCC

CHIP-qPCR_ORF16-R CGCAAGTCAAGACACAAGTC

CHIP-qPCR_ORF46/47-F AGCCCCCTTCCGTAATATCTG

CHIP-qPCR_ORF46/47-R TTTTCCGCGGAAGTATGTCG

CHIP-qPCR_K8.1-F ACTCCCACCATGTTGAAGCTTG

CHIP-qPCR_K8.1-R GGGATTTCTGTGCGAATCTGTG

*a : Lowercase indicates homology sequence to KSHV BAC16, underlined uppercase indicates mutagenesis site, and uppercase indicates pEP-KanS sequence.

*b : Underlined lowercase indicates restriction enzyme site.

*c : Underlined lowercase indicates restriction enzyme site, and underlined uppercase indicates mutagenesis site.


https://doi.org/10.1101/728147

associated herpesvirus orf66 is essential for late gene ... · 8/7/2019 · 4 66. introduction. 67...

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