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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
3
4
Tadashi Watanabe1, Mayu Nishimura1, Taisuke Izumi2+, Kazushi Kuriyama1, 5
Yuki Iwaisako1, Kouhei Hosokawa1, Akifumi Takaori-Kondo2 and Masahiro 6
Fujimuro1* 7
8
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
17
Running title: KSHV ORF66 is essential for late gene expression 18
19
*: 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: fuji2@mb.kyoto-phu.ac.jp 23
24
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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
48
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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
64
65
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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
119
120
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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
132
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
269
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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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18
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
373
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19
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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20
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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21
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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22
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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23
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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26
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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27
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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28
(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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18. Davis ZH, Hesser C, Park J, Glaunsinger BA. 2015. Interaction Between 707 ORF24 and ORF34 in the Kaposi's Sarcoma-Associated Herpesvirus 708 Late Gene Transcription Factor Complex is Essential For Viral Late Gene 709 Expression. J Virol doi:10.1128/jvi.02157-15. 710
19. Davis Zoe H, Verschueren E, Jang Gwendolyn M, Kleffman K, Johnson 711 Jeffrey R, Park J, Von Dollen J, Maher MC, Johnson T, Newton W, Jäger 712 S, Shales M, Horner J, Hernandez Ryan D, Krogan Nevan J, 713 Glaunsinger Britt A. 2015. Global Mapping of Herpesvirus-Host Protein 714 Complexes Reveals a Transcription Strategy for Late Genes. Mol Cell 715 57:349-360. 716
20. Nishimura M, Watanabe T, Yagi S, Yamanaka T, Fujimuro M. 2017. 717 Kaposi's sarcoma-associated herpesvirus ORF34 is essential for late 718 gene expression and virus production. Sci Rep 7:329. 719
21. Brulois K, Wong LY, Lee HR, Sivadas P, Ensser A, Feng P, Gao SJ, Toth 720 Z, Jung JU. 2015. Association of Kaposi's Sarcoma-Associated 721 Herpesvirus ORF31 with ORF34 and ORF24 Is Critical for Late Gene 722 Expression. J Virol 89:6148-54. 723
22. Castañeda AF, Glaunsinger BA. 2019. The Interaction between ORF18 724 and ORF30 Is Required for Late Gene Expression in Kaposi's Sarcoma-725 Associated Herpesvirus. J Virol 93:e01488-18. 726
23. Gong D, Wu NC, Xie Y, Feng J, Tong L, Brulois KF, Luan H, Du Y, Jung 727
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
33
JU, Wang CY, Kang MK, Park NH, Sun R, Wu TT. 2014. Kaposi's 728 sarcoma-associated herpesvirus ORF18 and ORF30 are essential for 729 late gene expression during lytic replication. J Virol 88:11369-82. 730
24. Tischer BK, Smith GA, Osterrieder N. 2010. En passant mutagenesis: a 731 two step markerless red recombination system. Methods Mol Biol 732 634:421-30. 733
25. Brulois KF, Chang H, Lee AS, Ensser A, Wong LY, Toth Z, Lee SH, Lee 734 HR, Myoung J, Ganem D, Oh TK, Kim JF, Gao SJ, Jung JU. 2012. 735 Construction and manipulation of a new Kaposi's sarcoma-associated 736 herpesvirus bacterial artificial chromosome clone. J Virol 86:9708-20. 737
26. Fakhari FD, Dittmer DP. 2002. Charting Latency Transcripts in Kaposi's 738 Sarcoma-Associated Herpesvirus by Whole-Genome Real-Time 739 Quantitative PCR. J Virol 76:6213-6223. 740
27. Nandakumar D, Glaunsinger B. 2019. An integrative approach identifies 741 direct targets of the late viral transcription complex and an expanded 742 promoter recognition motif in Kaposi's sarcoma-associated herpesvirus. 743 PLoS Pathog 15:e1007774. 744
28. Zhang Y. 2008. I-TASSER server for protein 3D structure prediction. 745 BMC Bioinformatics 9:40. 746
29. Roy A, Kucukural A, Zhang Y. 2010. I-TASSER: a unified platform for 747 automated protein structure and function prediction. Nat Protoc 5:725-38. 748
30. Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y. 2015. The I-TASSER 749 Suite: protein structure and function prediction. Nat Methods 12:7-8. 750
31. Yang J, Roy A, Zhang Y. 2013. Protein-ligand binding site recognition 751 using complementary binding-specific substructure comparison and 752 sequence profile alignment. Bioinformatics 29:2588-95. 753
32. Yang J, Roy A, Zhang Y. 2013. BioLiP: a semi-manually curated 754 database for biologically relevant ligand-protein interactions. Nucleic 755 Acids Res 41:D1096-103. 756
33. Aubry V, Mure F, Mariame B, Deschamps T, Wyrwicz LS, Manet E, 757 Gruffat H. 2014. Epstein-Barr virus late gene transcription depends on 758 the assembly of a virus-specific preinitiation complex. J Virol 88:12825-759 38. 760
34. Dunn W, Chou C, Li H, Hai R, Patterson D, Stolc V, Zhu H, Liu F. 2003. 761 Functional profiling of a human cytomegalovirus genome. Proc Natl Acad 762 Sci U S A 100:14223-14228. 763
35. Zhang W, Li H, Li Y, Zeng Z, Li S, Zhang X, Zou Y, Zhou T. 2010. 764 Effective inhibition of HCMV UL49 gene expression and viral replication 765 by oligonucleotide external guide sequences and RNase P. Virol J 7:100. 766
36. Murakami K, Tsai K-L, Kalisman N, Bushnell DA, Asturias FJ, Kornberg 767 RD. 2015. Structure of an RNA polymerase II preinitiation complex. 768 Proceedings of the National Academy of Sciences 112:13543-13548. 769
37. Chapa TJ, Du Y, Sun R, Yu D, French AR. 2017. Proteomic and 770 phylogenetic coevolution analyses of pM79 and pM92 identify 771 interactions with RNA polymerase II and delineate the murine 772 cytomegalovirus late transcription complex. The Journal of general 773 virology 98:242-250. 774
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
34
38. Sato Y, Watanabe T, Suzuki C, Abe Y, Masud H, Inagaki T, Yoshida M, 775 Suzuki T, Goshima F, Adachi J, Tomonaga T, Murata T, Kimura H. 2019. 776 S-Like-Phase Cyclin-Dependent Kinases Stabilize the Epstein-Barr Virus 777 BDLF4 Protein To Temporally Control Late Gene Transcription. J Virol 93. 778
39. Bryant DM, Datta A, Rodriguez-Fraticelli AE, Peranen J, Martin-Belmonte 779 F, Mostov KE. 2010. A molecular network for de novo generation of the 780 apical surface and lumen. Nat Cell Biol 12:1035-45. 781
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
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
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.
*
*
*
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
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
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
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
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
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
iSLK-WT iSLK-Δ66iSLK-WT/ Control / 3xFLAG-ORF66
iSLK-WT iSLK-Δ66iSLK-WT/ Control / 3xFLAG-ORF66
iSLK-WT iSLK-Δ66iSLK-WT/ Control / 3xFLAG-ORF66
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
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
(ORF34)Blot: Anti-Myc
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
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
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
(ORF34)Blot: Anti-Myc
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
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
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
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
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
(ORF34)Blot: Anti-Myc
(ORF66)Blot: Anti-S
(ORF34)Blot: Anti-Myc
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
(ORF34)Blot: Anti-Myc
(ORF66)Blot: Anti-S
(ORF34)Blot: Anti-Myc
: S
WCE
(kDa)
55
55
45
55
Pull-down
(ORF34)Blot: Anti-Myc
(ORF66)Blot: Anti-S
(ORF34)Blot: Anti-Myc
: S
WCE
(kDa)
55
55
45
55
Pull-down
(ORF34)Blot: Anti-Myc
(ORF66)Blot: Anti-S
(ORF34)Blot: Anti-Myc
: 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
(ORF34)Blot: Anti-Myc
Short exposure
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
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
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
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.
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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