The contribution of Kaposi’s sarcoma-associated herpesvirus ORF7 and its zinc-finger motif to viral genome cleavage and capsid formation

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of endothelial and B cell malignancies. During KSHV lytic infection, lytic-related proteins are synthesized, viral genomes are replicated as a tandemly repeated form, and subsequently, capsids are assembled. The herpesvirus terminase complex is proposed to package an appropriate genome unit into an immature capsid, by cleavage of terminal repeats (TRs) flanking tandemly linked viral genomes. Although the mechanism of capsid formation in α- and β-herpesviruses are well-studied, in KSHV, it remains largely unknown. It has been proposed that KSHV ORF7 is a terminase subunit, and ORF7 harbors a zinc-finger motif, which is conserved among other herpesviral terminases. However, the biological significance of ORF7 is unknown. We previously reported that KSHV ORF17 is essential for the cleavage of inner scaffold proteins in capsid maturation, and ORF17 knockout (KO) induced capsid formation arrest between the procapsid and B-capsid stages. However, it remains unknown if ORF7-mediated viral DNA cleavage occurs before or after ORF17-mediated scaffold collapse. We analyzed the role of ORF7 during capsid formation using ORF7-KO-, ORF7&17-double-KO (DKO)-, and ORF7-zinc-finger motif mutant-KSHVs. We found that ORF7 acted after ORF17 in the capsid formation process, and ORF7-KO-KSHV produced incomplete capsids harboring non-spherical internal structures, which resembled soccer balls. This soccer ball-like capsid was formed after ORF17-mediated B-capsid formation. Moreover, ORF7-KO- and zinc-finger motif KO-KSHV failed to appropriately cleave the TR on replicated genome and had a defect in virion production. Thus, our data revealed that ORF7 contributes to terminase-mediated viral genome cleavage and capsid formation. IMPORTANCE In herpesviral capsid formation, the viral terminase complex cleaves the TR sites on newly synthesized tandemly repeating genomes and inserts an appropriate genomic unit into an immature capsid. Herpes simplex virus 1 (HSV-1) UL28 is a subunit of the terminase complex that cleaves the replicated viral genome. However, the physiological importance of the UL28 homolog, KSHV ORF7, remains poorly understood. Here, using several ORF7-deficent KSHVs, we found that ORF7 acted after ORF17-mediated scaffold collapse in the capsid maturation process. Moreover, ORF7 and its zinc-finger motif were essential for both cleavage of TR sites on the KSHV genome and virus production. ORF7-deficient KSHVs produced incomplete capsids that resembled a soccer ball. To our knowledge, this is the first report showing ORF7-KO-induced soccer ball-like capsids production and ORF7 function in the KSHV capsid assembly process. Our findings provide insights into the role of ORF7 in KSHV capsid formation.

which resembled soccer balls. This soccer ball-like capsid was formed after 37 ORF17-mediated B-capsid formation. Moreover, ORF7-KO-and zinc-finger motif  failed to appropriately cleave the TR on replicated genome and had a defect in virion 39 production. Thus, our data revealed that ORF7 contributes to terminase-mediated viral 40 genome cleavage and capsid formation. 41

IMPORTANCE 44
In herpesviral capsid formation, the viral terminase complex cleaves the TR sites 45 on newly synthesized tandemly repeating genomes and inserts an appropriate genomic unit 46 into an immature capsid. Herpes simplex virus 1 (HSV-1) UL28 is a subunit of the terminase 47 Introduction from the outer capsid shell, resulting in its angularization (16,17,18). The HSV-1 terminase 94 complex cleaves the tandemly linked viral genomes and binds to the portal on the capsid, 95 resulting in the packaging of an appropriate genomic unit into an immature capsid (9,11,19). 96 Although it is unclear when the terminase-mediated DNA cleavage and DNA packaging into 97 capsids occur, these events are thought to take place between (i) procapsid formation and 98 (iv) C-capsid formation. B-capsids are formed as unsuccessful capsids when the decayed 99 scaffold is not eliminated from the capsid during the aforementioned (i), (ii), and (iii) stages. 100 However, the B-capsid may also be an intermediate capsid in the C-capsid formation 101 process. 102 In HSV-1, the mechanism of capsid formation has been revealed using 103 recombinant viruses. In contrast, KSHV capsid formation has not been well-studied. We 104 previously showed that the KSHV protease ORF17 was essential for both the cleavage of 105 HSV-1 terminase is required for viral DNA cleavage and DNA packaging into the 112 immature capsids (9). The terminase cleaves the viral DNA at the TR sites, which flank the 113 tandem repeats of the viral genome. Thus, a correctly cleaved genome is inserted into an 114 immature capsid. Three HSV-1 proteins, UL15, UL28, and UL33 are components of the 115 HSV-1 terminase complex (21,22). In cells infected with a temperature-sensitive UL28 116 mutant, capsids containing viral genomes were not observed, but capsids containing 117 compact internal scaffolds were detected (23). Moreover, UL28-deficient HSV-1 is capable 118 of replicating viral DNA, but it is unable to cleave replicated viral concatemer DNA and fails 119 to produce infectious progeny virus (24). Epstein-Barr virus (EBV) BALF3 and KSHV ORF7 120 are homologs of HSV-1 UL28. EBV BALF3 is required for viral DNA replication and mature 121 capsid formation (25). The putative zinc-finger motif of UL28 122 (C197-X2-C200-X22-C223-X-H225) is conserved among the UL28 homologs of all 123 herpesviruses, including KSHV ORF7 (25). This motif is essential for HSV-1 terminase 124 function, because recombinant HSV-1 lacking the putative zinc-finger motif of UL28 failed to 125 package viral DNA (21). Thus, the function of terminase and its components has been 126 extensively studied in the context of HSV-1 and EBV. In KSHV, at least three viral proteins 127 (ORF7, ORF29, and ORF67.5) are thought to form the terminase complex because of their 128 protein sequence homology to terminase proteins from other viruses; however, the precise composition of the terminase complex from KSHV remains unknown. 130 We previously generated an ORF7-KO KSHV bacterial artificial chromosome 131 (BAC) to analyze the role of ORF7 in viral replication. We found that ORF7 was dispensable 132 for viral DNA replication but was essential for virus production (26). Furthermore, ORF7 133 interacted with ORF29 and ORF67.5, which are homologs of HSV-1 UL15 and UL33, 134 respectively (26). However, it still remains unknown whether ORF7 is a component of the 135 KSHV terminase complex. Specifically, it is unclear whether ORF7 and its zinc-finger motif 136 contribute to viral DNA cleavage. It remains unknown if ORF7-mediated viral DNA cleavage 137 occurs before or after ORF17-mediated scaffold collapse. Therefore, in order to address 138 these questions, we analyzed the virological properties of ORF7 KO-, ORF7&ORF17 139 and ORF7 zinc-finger motif mutant-KSHVs. We found that ORF7 acted after ORF17 in the 140 capsid formation process, and KSHV ORF7 and its zinc-finger motif were required for viral 141 DNA cleavage and the capsid formation. 142

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ORF7-KO KSHV produces unique incomplete capsids which resemble soccer balls. 145 We previously generated and analyzed two types of ORF7-KO KSHV, 146 frameshift-induced ORF7 (FS-ΔORF7 or ORF7-fsKO) BAC16 and stop codon-induced 147 ORF7 (ST-ΔORF7 or ORF7-stKO) BAC16. It was revealed that ORF7 is essential for the 148 production of infectious virions (26). However, the role of ORF7 in capsid formation remains 149 unclear. Therefore, to evaluate the contribution of ORF7 in KSHV capsid formation, we 150 observed capsids formed in wild-type (WT) BAC16-, ORF7-fsKO BAC16-, and ORF7-stKO 151 BAC16-harboring iSLK cells (WT-iSLK, ORF7-fsKO-iSLK, and ORF7-stKO-iSLK, 152 respectively) by TEM. The cells were treated with Dox and NaB for 72 h in order to induce a 153 lytic infection via RTA expression. The lytic cells were sampled to analyze the capsids 154 formed in the nucleus (Fig. 1). C-capsids were detected in WT-iSLK (Fig. 1A, indicated by 155 the black arrowhead), and a few A-capsids were also found in the nuclei (Fig. 1A, indicated 156 by the white arrowhead). A small number of B-capsids were also observed in different visual The capsids produced in ORF7-KO-iSLK cells contain a common internal structure that 162 appears to be a telstar pattern (coin dot) of the soccer ball. Therefore, we refer to these 163 capsids as 'soccer ball-like capsids' in this manuscript. In contrast to WT-iSLK, C-capsids 164 were not observed in ORF7-KO-iSLK cells ( KSHV. 199 We previously showed that the KSHV protease ORF17 was involved in capsid 200 maturation as a viral protease which digests scaffold proteins, resulting in scaffold shell 201 disruption. Moreover, either complete KO of ORF17 or a mutant deficient in ORF17 protease 202 activity induced capsid formation arrest between the procapsid and B-capsid stages (20). 203 Here, we found that ORF7 possessed KSHV genome TR cleavage activity (Fig. 2B). The 204 herpesvirus terminase complex contributes to viral genome cleavage and packaging of the 205 cleaved genomes into immature capsids. This immature capsid is thought to be assembled 206 after the formation of procapsids or internal scaffold shell-disrupted procapsids (29, 30). 207 However, there are no reports describing which event (i.e., ORF7-mediated genome 208 cleavage or ORF17-mediated internal scaffold disruption) occurs earlier during capsid 209 formation. Thus, in order to determine the order of ORF7-and ORF17-mediated effects on 210 capsid assembly, we constructed ORF7 and ORF17 DKO (designated as ORF7&17-DKO) 211 KSHVs. We then compared viral events including capsid formation among WT, ORF7-KO, 212 ORF17-KO, and ORF7&17-DKO KSHVs. If ORF7 acts earlier than ORF17 in capsid 213 formation, then the ORF7&17-DKO KSHV should have the same phenotype as ORF7-KO 214 should present the same phenotype as ORF17-KO KSHV. Thus, we constructed 216 ORF7&17-DKO-BAC16 from ORF7-fsKO-BAC16 by insertion of the ORF17 KO frame shift 217 mutation (which has been previously described) (20) and confirmed the DKO mutation by 218 Sanger sequencing (Fig. 3A). The ORF7&17-DKO-BAC16 clone was transfected into the 219 iSLK cell line, and stable cells harboring the BAC16 were selected by hygromycin B. Next, 220 ORF17-mediated effects on capsid assembly (Fig. 3). Therefore, ORF7-deficent KSHV may 268 induce capsid formation arrest between the B-capsid and mature C-capsid stages during the lytic phase. Meanwhile, it is known that viral transcription and viral genome replication occur 270 independently of capsid formation. Therefore, it was not surprising that the KO of ORF7, 271 ORF17, and both ORF7&17 did not impact viral transcription and viral genome replication. 272 In order to evaluate the importance of both ORF7 and ORF17 in virus production, 5A). In addition to measuring viral DNA, we also evaluated the virus titers in the culture 284 supernatants. In order to evaluate infectious virion production, the culture medium was 285 added to uninfected HEK293T cells, and the numbers of GFP-positive cells were counted by 286 flow cytometry. We found that the infectivity of the virions produced from ORF7&17-DKO-iSLK cells were recovered by exogenous ORF7-2xS (ORF7-2S) and 288 3xFLAG-ORF17 (3F-ORF17) co-expression (Fig. 5B). These complementation assays that 289 evaluated the effects of exogenous ORF expression on virus production and infectivity 290 indicated that ORF7 and ORF17 play crucial roles in virus production. 291

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The conserved zinc-finger motif of ORF7 is important for infectious KSHV production. 293 The KSHV ORF7 harbors a putative zinc-finger motif 294 (C182-X 2 -C185-X 22 -C208-X 1 -H210), which is highly conserved among other human 295 herpesvirus homologous genes (25). The zinc-fingers, which binds zinc ion, are speculated 296 to be formed by three C and one H (i.e., C182, C185, C208, and H210) within this motif 297 (C182-H210). In order to investigate the importance of the ORF7 zinc-finger motif for 298 infectious virus production, we generated a series of ORF7 zinc-finger mutants. These 6A) were transiently transfected into ORF7-fsKO-iSLK cells, and virus production was analyzed by qPCR and viral infectivity was assessed by flow cytometry. The ORF7 WT-or 306 ORF7 mutant-transfected cells were cultured with medium containing Dox and NaB for lytic 307 induction. The amount of virus production in the culture supernatant was determined by 308 measurement of viral DNA using qPCR. Virus production was efficiently recovered by ORF7 309 WT expression; however, empty vector and all of the zinc-finger motif mutants (mC2, 310 mC1H1, and mZf) did not induce virus production recovery (Fig. 6B). The protein expression 311 of ORF7 WT and the ORF7 mutants were confirmed by Western blotting (the lower panels 312 of Fig. 6B). Next, the amount of infectious virus in the culture supernatant, prepared using 313 the same method as described for Fig. 6B, was determined. The culture medium was added 314 to fresh HEK293T cells, and the number of GFP-positive cells were counted by flow 315 cytometry to evaluate the infectious virion production. Our results agreed with the qPCR 316 data (Fig. 6B). Specifically, the infectivity of the virions produced from ORF7-fsKO-iSLK cells 317 were recovered by ORF7 WT, whereas all of the ORF7 zinc-finger motif mutants did not 318 facilitate the recovery of viral infectivity (Fig. 6C). These results indicated that the zinc-finger 319 motif of ORF7 is essential for the production of infectious KSHV. 320 321 Virological analysis of ORF7 zinc-finger motif mutant KSHV. 322 In order to further explore whether the zinc-finger motif of ORF7 is important for virus production, we constructed ORF7 mC1H1 (C208A, H210A) BAC16. Since the mC1H1 324 mutation requires the narrowest mutation region compared to the other two mutations, the 325 mC1H1 mutation was introduced into WT-BAC16 to generate an ORF7 zinc-finger motif 326 mutant. To construct ORF7 mC1H1-BAC16, the alanine mutations (C208A, H210A) were 327 introduced into WT-BAC16 using the same method ( virus production was significantly suppressed, and the infectivity was abolished compared to WT-iSLK cells (Figs. 7D and E). Thus, our reverse genetic approach established that the 342 zinc-finger motif of ORF7 is essential for virion production during KSHV lytic infection. 343 Finally, we asked whether the ORF7 zinc-finger motif was required for cleavage of 344 the KSHV TR during lytic infection in ORF7 mC1H1-iSLK cells. The contribution of the 345 ORF7 zinc-finger motif to TR cleavage activity in BAC16 was examined by the same 346 Southern blotting method as described for Fig. 2. Uncleaved TRs were detected in all of the 347 tested KSHV-(WT-, ORF7-fsKO-, ORF7-stKO-, and ORF7 mC1H1-BAC16) harboring cells. 348 DNA fragments from cleaved TRs were detected in WT-iSLK cells but were not detected in 349 ORF7-fsKO-iSLK and ORF7-stKO-iSLK cells (Fig. 8). Interestingly, two aberrantly-sized 350 fragments, that differed from the banding pattern observed in WT-iSLK cells, were detected 351 in the ORF7 mC1H1-iSLK cells (Fig. 8). These results suggested that the ORF7 zinc-finger 352 motif regulates cleavage (or cleavage length) at the TR site. Thus, these data demonstrated 353 that the zinc-finger motif of ORF7 is necessary for proper KSHV TR cleavage. In addition, 354 ORF7 may function as a subunit of the KSHV terminase complex that cleaves the TRs on 355 KSHV genomes during lytic infection. 356

Discussion 358
In this study, we showed that KSHV ORF7 is required for viral DNA cleavage and 359 packaging, which suggested that it is important for KSHV terminase function. In addition, 360 TEM analysis using both ORF7 and ORF17-deficient BAC16 confirmed that ORF7 acts after 361 ORF17 in the KSHV capsid formation process. It was also revealed that the putative 362 zinc-finger motif of ORF7 is essential for infectious virus production and contributes to TR cleavage (Fig. 8). Uncleaved TRs were detected in WT-BAC16, ORF7-fsKO-BAC16, 446 ORF7-stKO-BAC16, and ORF7 mC1H1-BAC16-harboring cells. WT-KSHV produced cleaved fragments from the BAC16 TR, whereas ORF7-KO-KSHV failed to produce cleaved 448 fragments. Interestingly, the ORF7 mC1H1-KSHV produced two major aberrantly-sized 449 fragments, which was dissimilar to the cleavage pattern of WT-KSHV. These data indicated 450 that the ORF7 zinc-finger motif may distinguish the appropriate TR cleavage sites for the 451 terminase, rather than regulating the nuclease activity of the terminase. 452 We found that KSHV ORF7 contributed to capsid formation as well as viral DNA 453 cleavage, which is a main function of the viral terminase complex. This study provides the 454 first evidence that ORF7 is necessary for KSHV terminase function. Since the machinery for Plasmids 473 The C-terminal 2xS-tagged and 3xFLAG-tagged ORF7 expression plasmids and 474 the N-terminal 3xFLAG-tagged ORF17 expression plasmid have been previously described 475 (20,26     T G C c t a g a a T G C … T G C a g t C A C 5' … … 3'