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Kaposi’s Sarcoma-Associated Herpesvirus Inhibitor of cGAS (KicGAS), Encoded by ORF52, Is an Abundant Tegument Protein and Is Required for Production of Infectious Progeny

Wenwei Li, Denis Avey, Bishi Fu, Jian-jun Wu, Siming Ma, Xia Liu, Fanxiu Zhu Department of Biological Science, Florida State University, Tallahassee, Florida, USA

ABSTRACT Although Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF52 (also known as KSHV inhibitor of cGAS [KicGAS]) has been detected in purified virions, the roles of this protein during KSHV replication have not been characterized. Using specific mono- clonal antibodies, we revealed that ORF52 displays true late kinetics and confirmed its cytoplasmic localization in both transfected and KSHV-infected cells. We demonstrated that ORF52 comigrates with other known virion proteins follow- ing sucrose gradient centrifugation. We also determined that ORF52 resides inside the and remains partially asso- ciated with when extracellular virions are treated with various detergents and/or salts. There results indicate that ORF52 is a tegument protein abundantly present in extracellular virions. To characterize the roles of ORF52 in the KSHV life cycle, we engineered a recombinant KSHV ORF52-null mutant and found that loss of ORF52 results in reduced virion production and a further defect in infectivity. Upon analysis of the virion composition of ORF52-null viral particles, we observed a decrease in the incorporation of ORF45, as well as other tegument proteins, suggesting that ORF52 is important for the packaging of other virion proteins. In summary, our results indicate that, in addition to its immune evasion function, KSHV ORF52 is re- quired for the optimal production of infectious virions, likely due to its roles in virion assembly as a tegument protein.

IMPORTANCE The tegument proteins of herpesviruses, including Kaposi’s sarcoma-associated herpesvirus (KSHV), play key roles in the viral life cycle. Each of the three subfamilies of herpesviruses (alpha, beta, and gamma) encode unique tegument proteins with special- ized functions. We recently found that one such gammaherpesvirus-specific protein, ORF52, has an important role in immune evasion during KSHV primary , through inhibition of the host cytosolic DNA sensing pathway. In this report, we fur- ther characterize ORF52 as a tegument protein with vital roles during KSHV lytic replication. We found that ORF52 is important for the production of infectious viral particles, likely through its role in virus assembly, a critical process for KSHV replication and pathogenesis. More comprehensive investigation of the functions of tegument proteins and their roles in viral replication may reveal novel targets for therapeutic interventions against KSHV-associated diseases.

aposi’s sarcoma-associated herpesvirus (KSHV), also known 40% of the virion mass (8). While capsid proteins are conserved Kas herpesvirus 8 (HHV-8), is the etiologic agent of among all herpesviruses, several tegument proteins are unique to Kaposi’s sarcoma (KS) (1) and, also, two lymphoproliferative dis- each subfamily. Regarding the functions of virion proteins, those orders, primary effusion lymphoma (PEL) (2) and multicentric of capsid and envelope proteins are generally better characterized Castleman disease (MCD) (3). KSHV belongs to the than those of tegument proteins. Most of our knowledge pertain- genus in the subfamily and is related to rhe- ing to tegument proteins is derived from studies on alpha- and sus rhadinovirus (RRV), herpesvirus saimiri (HVS), and murine betaherpesviruses. Studies of the tegument of gammaherpesvi- gammaherpesvirus 68 (MHV-68). The closest relative of KSHV ruses, including KSHV and EBV, are lagging because they do not among the known human herpesviruses is Epstein-Barr virus replicate as robustly as alpha- and betaherpesvirus in cultured (EBV), which belongs to the same subfamily (4, 5). cells. Like all herpesviruses, KSHV has two alternative life cycles: latent and lytic. During latency, only a few viral latent genes are expressed. During the lytic replication cycle, the full complement Received 16 October 2015 Accepted 8 March 2016 of viral genes are expressed in a temporal cascade, beginning with Accepted manuscript posted online 23 March 2016 immediate early (IE) genes, followed by early (E) genes, and then Citation Li W, Avey D, Fu B, Wu J-J, Ma S, Liu X, Zhu F. 2016. Kaposi’s sarcoma- late (L) genes, whose expression depends on viral DNA replica- associated herpesvirus inhibitor of cGAS (KicGAS), encoded by ORF52, is an tion. Successful completion of this lytic replication culminates in abundant tegument protein and is required for production of infectious progeny viruses. J Virol 90:5329–5342. doi:10.1128/JVI.02675-15. the release of progeny virions (6, 7). Editor: J. U. Jung A typical herpesvirus virion consists of a linear double- Address correspondence to Fanxiu Zhu, [email protected]. stranded viral DNA core enclosed within an icosahedral capsid, an Supplemental material for this article may be found at http://dx.doi.org/10.1128 outer envelope with viral , and a tegument layer lo- /JVI.02675-15. cated between the capsid and envelope. Among these, the tegu- Copyright © 2016, American Society for Microbiology. All Rights Reserved. ment is the most complex in composition and accounts for about

June 2016 Volume 90 Number 11 Journal of jvi.asm.org 5329 Li et al.

Our laboratory has long been interested in tegument proteins diluted primary antibodies for2hatroom temperature or overnight at of KSHV, especially those that are specific to gammaherpesvi- 4°C. Anti-rabbit and anti-mouse IgG conjugated to horseradish peroxi- ruses. Our previous work on a gammaherpesvirus-specific tegu- dase (Pierce) were used as the secondary antibodies. SuperSignal chemi- ment protein, ORF45, revealed its crucial functions in many facets luminescence reagents (Pierce) were used for detection. of the KSHV lytic life cycle, including evasion of the host antiviral Immunofluorescence staining. Cells were cultured on coverslips in innate immune responses by suppression of IRF7 (9–11), modu- 12-well plates, fixed with 2% formaldehyde in phosphate-buffered saline (PBS) for 10 min, permeabilized with 0.2% Triton X-100 in PBS (PBST) lation of cellular kinase signaling (12–15), and transport of freshly for 20 min on ice, blocked with 3% bovine serum albumin in PBST for 30 assembled viral particles along microtubules (16). min, and then incubated with primary antibody for 1 h. After three washes KSHV ORF52 is predicted to encode a protein of 131 amino with PBST, the cells were incubated with Alexa Fluor-conjugated second- acids (aa) that is conserved among gammaherpesviruses. ORF52 ary antibodies (Invitrogen, Carlsbad, CA) for 1 h. After another three itself, as well as its homologues in MHV-68, RRV, and EBV washes, the cells were counterstained with DAPI (4=,6-diamidino-2-phe- (BLRF2), have all been detected in virions by mass spectrometry nylindole; Sigma, St. Louis, MO) and then mounted in antifade reagent (17–21). ORF52 of MHV-68 has been characterized as a tegument (Invitrogen, Carlsbad, CA) and visualized with a fluorescence micro- protein with a key role in the tegumentation and secondary envel- scope. The plasmids carrying endomembrane gene markers were pro- opment of virions in the (22–24). ORF52 of RRV has vided by David Meckes from Florida State University. The plasmids ex- recently been shown to be a tegument protein required for the pressing TGN (catalog number 55145), ␣-tubulin (catalog number maturation and vesicle-mediated egress of viral particles (25). 49149), and microtubule-associated protein 4 (MAP4; catalog number Phosphorylation of EBV BLRF2 by /-rich protein 55076) were purchased from Addgene. Virus stock preparation and treatment. As previously described (15, kinase 2 (SRPK2) has been shown to be important for viral repli- 19), six or more T150 flasks with cells were induced for 5 days, and then cation (26). the medium was collected and centrifuged to remove debris. Virions We originally became interested in ORF52 not only because it were pelleted at 100,000 ϫ g for1hona25%sucrose cushion with a is a gammaherpesvirus-specific virion protein but also because it Beckman SW28 rotor. The virus pellets were dissolved overnight in 1/100 is one of only a few KSHV proteins which contain a conserved volume of TNE (10 mM Tris-Cl, pH 8.0, 150 mM NaCl, 1 mM EDTA) phosphorylation motif (RXRXXS/T) of the ORF45-activated cel- buffer and stored at Ϫ80°C. lular kinase p90 ribosomal S6 kinase (RSK) and, thus, represents a For trypsin treatment, as previously described (19), purified virions potential viral substrate of RSK (12–15). Moreover, we recently were treated with trypsin (4 ␮g/ml; Promega, Madison, WI) in 100 ␮lof found that KSHV ORF52 plays a role in innate immune evasion by buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM CaCl2) at 37°C for directly inhibiting enzymatic activity of the host cytosolic DNA 1 h. Trypsin digestions were terminated by adding phenylmethylsulfonyl sensor, cGAS (27, 28). Because ORF52 is the first reported viral fluoride (PMSF) to a concentration of 0.5 mM and 1/100 volume of pro- inhibitor of cGAS and its functions were previously uncharacter- tease inhibitors (Sigma). In parallel, Triton X-100 was added to a final concentration of 1% to remove the viral envelope and expose the tegu- ized, we named it KicGAS (KSHV inhibitor of cGAS). Although mented capsid to the protease. Samples were then analyzed by Western we have shown that ORF52/KicGAS inhibits the innate immune blotting. response during KSHV primary infection, the exact roles of this For detergent treatment, purified virions were treated with different protein during the KSHV life cycle remain unknown. Here, we concentrations of detergents in TNE buffer for 30 min at 37°C. For salt report the characterization of KSHV ORF52 and its roles during treatment, virions were treated with 1% Triton X-100 containing different KSHV lytic replication, made possible through the generation and concentrations of NaCl in TNE buffer for 30 min at 37°C. The reaction use of an ORF52-null bacterial artificial chromosome 16 (BAC16) mixture was separated into two fractions, supernatant and pelleted tegu- mutant. Our results demonstrate that ORF52 is an abundant teg- ment-nucleocapsid, by centrifugation at 100,000 ϫ g for 1 h. Equal ument protein that is required for the production of infectious amounts of protein from the supernatant and pellet were analyzed by virions. Western blotting. Genetic manipulation of KSHV BAC16 . Mutagenesis of MATERIALS AND METHODS BAC16 was performed as previously described (15) by using a recom- bineering system as described by Tischer et al. (31, 32). In brief, the Kan/ Cell culture and . HeLa cells were cultured under 5% CO2 at 37°C in Dulbecco’s modified Eagle’s medium (DMEM) supplemented I-SceI cassettes were amplified from plasmid pEPKan-S by PCR with with 10% fetal bovine serum (FBS) and antibiotics. iSLK cells were cul- primers KS52-STOP-5= (5=-ACATCTACGCGTACCTGACATGGCCGC tured in DMEM containing 10% FBS, 450 ␮g/ml G418, and 1 ␮g/ml GCCCAGGGGCAGAAAGCTTTGATTAATTGACCCAAAAAGGACCT puromycin. Transient were performed in 12-well plates with TACGATAGGATGACGACGATAAGTAGGG-3=) and KS52-STOP-3= FuGENE 6 transfection reagent (Promega, Madison, WI) or in 100-mm (5=-TCTTTGCGGTTAGGTCTTCCATCGTAAGGTCCTTTTTGGGTC dishes with calcium phosphate methods. AATTAATCAAAGCTTTCTGCCCCTGGGCGCGGCCAGCCAGTGTT Antibodies and Western blot analysis. Monoclonal antibodies ACAACCAATTAACC-3=) for mutant BAC16-Stop52; primers KS52- against ORF52 were generated by the FSU hybridoma facility. The de- S123A-5= (5=-ACCGCCTCCTGGTGCCAATAACAGGCGACGAAGAG tailed procedures for the production of antibodies were described in our GAGCCGCGACAACACGGGCGGGGGTTGAAGGATGACGACGATA previous study (29). Antibodies against ORF26, ORF65, ORF33, ORF38, AGTAGGG-3=) and KS52-S123A-3= (5=-GCTGGTCCGCGGTTCAGTC and ORF45 used in this study were described previously (15, 29, 30). ATCAACCCCCGCCCGTGTTGTCGCGGCTCCTCTTCGTCGCCTGT Antibody against RTA was offered by Ke Lan at Institut Pasteur of Shang- GCCAGTGTTACAACCAATTAACC-3=) were used for mutant BAC16- hai. Anti-PF8 antibody was provided by Robert Ricciardi at the University 52S123A. The purified PCR fragment was electroporated into BAC16- of Pennsylvania. Anti-K8.1 antibody was obtained from Bala Chandran at containing GS1783 cells that had been induced at 42°C for 15 min. The Rosalind Franklin University of Medicine and Science. A monoclonal recombinant clones were selected at 32°C on LB plates containing 34 antibody against ␤-actin was purchased from Sigma. For Western blot ␮g/ml chloramphenicol and 50 ␮g/ml kanamycin and then characterized analysis, proteins were resolved by SDS-PAGE and transferred to nitro- by restriction fragment length polymorphism (RFLP). Positive clones cellulose membranes. The membranes were blocked in 5% dry milk in 1ϫ were induced again at 42°C and plated on LB plates containing 1% L-ar- phosphate-buffered saline with 0.2% Tween 20 and then incubated with abinose for secondary recombination. Then, replicas of the clones were

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FIG 1 KSHV ORF52 is expressed with late gene kinetics. (A, B) Anti-ORF52 mouse monoclonal antibodies (4H4 and 8C8) specifically recognize ORF52 protein in both transiently transfected cells (A) and induced BCBL-1/iSLK.219 cells (B). (C) The two anti-ORF52 monoclonal antibodies were used for epitope mapping by Western blot analysis, using purified glutathione S-transferase (GST)-ORF52 proteins encompassing a series of 10-aa internal deletion mutants. (D) iSLK.BAC16 cells were induced in the presence or absence of PAA for the indicated times (days postinduction [dpi]). Viral protein levels were assessed by Western blotting with antibodies to the indicated proteins. IE, immediate early gene; E, early gene; L, late gene.

picked from L-arabinose plates onto plates with 34 ␮g/ml chlorampheni- . The reaction was stopped by the addition of 10 mM EDTA fol- col alone or plates with 34 ␮g/ml chloramphenicol plus 50 ␮g/ml kana- lowed by heating at 70°C for 15 min with 0.4 mg of proteinase K (Qiagen, mycin. The kanamycin-sensitive clones were considered second-recom- Valencia, CA) in 0.5ϫ buffer AL (Qiagen, Valencia, CA). Then, the mix- binant clones and confirmed by RFLP and sequencing. ture was extracted with phenol-chloroform. The DNA was precipitated by To make a revertant mutant, we replaced the mutant ORF52 with a ethanol with glycogen as a carrier, and the DNA pellet was dissolved in 40 wild-type ORF52 sequence by a homologous recombination strategy sim- ␮l of Tris-EDTA buffer. Two microliters of such DNA was used in a ilar to that described above. For constructing the BAC16-Rev52 revertant real-time quantitative PCR with SYBR dyes. Viral DNA copy numbers mutant, the Kan/I-SceI cassettes were amplified from plasmid pEPKan-S were calculated with external standards of known concentrations of by PCR with the following primers: KS52-STOP-R-5= (5=-ACATCTACG BAC16 DNA. The primers ORF73-LCN (5=-CGCGAATACCGCTATGT CGTACCTGACATGGCCGCGCCCAGGGGCAGACCCAAAAAGGAC ACTCA-3=) and ORF73-LCC (5=-GGAACGCGCCTCATACGA-3=) were CTTACGATAGGATGACGACGATAAGTAGGG-3=) and KS52-STOP- previously described by Krishnan et al. (34). R-3= (5=-TCTTTGCGGTTAGGTCTTCCATCGTAAGGTCCTTTTTG Viral infection and FACS. Infection was carried out as previously GGTCTGCCCCTGGGCGCGGCCAGCCAGTGTTACAACCAATTA described (15, 33). Briefly, HEK293 cells were plated into 24-well plates ACC-3=). the day before infection and then incubated with concentrated virus with Reconstitution of recombinant KSHVs. Briefly, iSLK cells seeded in a Polybrene (4 ␮g/ml) and spun at 800 ϫ g for1hatroom temperature. The 24-well plate were transfected with 0.5 ␮g of BAC DNAs by using Effect- plates were then incubated at 37°C for another 2 h, and the inocula were ene (Qiagen). One day after transfection, cells were subcultured into a then removed and replaced with fresh medium with 5% FBS. The next T150 flask with fresh medium containing 450 ␮g/ml G418 and 1 ␮g/ml day, the medium was replaced with fresh medium containing 1% FBS. puromycin. The next day, hygromycin was added to a final concentration After 48 h of infection, the cells were examined by fluorescence-activated of 500 ␮g/ml for selection. After about 12 days of selection, hygromycin- cell sorting (FACS) for the expression of green fluorescent protein (GFP). resistant colonies were trypsinized, pooled, and subcultured at a 1:9 dilu- Briefly, the cells were trypsinized, washed with PBS, and then fixed in 2% tion every 3 days. To induce viral lytic replication, BAC-containing iSLK paraformaldehyde in PBS for 10 min at room temperature. The cells were cells were seeded into 6-well plates or T150 flasks and, 1 day later, the then washed and resuspended in PBS, followed by analysis with a BD medium was replaced with fresh medium containing 2 ␮g/ml doxycycline FACSCanto analyzer. and 1 mM butyrate. Preparation and quantification of viral genomic DNA. The prepara- RESULTS tion and quantification of viral genomic DNA were performed as previ- ously described (33). Briefly, the medium from induced BAC-iSLK cells KSHV ORF52 can be phosphorylated by p90RSK. We recently was collected, centrifuged, and passed through a 0.45-␮m filter to clear identified several putative substrates of ORF45-activated RSK cell debris. Treatment of 100 ␮l of the cleared supernatant with 10 units of (35). Of these, ORF52 was one of the few viral proteins for which Turbo DNase (Ambion, Austin, TX) at 37°C for 1 h degraded extravirion phosphorylation was detected at the consensus RSK phosphory-

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FIG 2 ORF52 exhibits cytoplasmic localization. (A) HeLa cells transfected with an ORF52-expressing vector were fixed 24 h after transfection and stained with mouse monoclonal antibodies to ORF52 (4H4 and 8C8) and DAPI. (B) Cytoplasmic localization of ORF52 was confirmed in induced iSLK.BAC16 (top) and BCBL-1 (bottom) cells using antibody 4H4. (C) SLK.iBAC-GFP52 cells were cultured in the presence (induced) or absence (uninduced) of doxycycline for 72 h. Cells were then fixed and stained with monoclonal anti–␣-tubulin antibody and DAPI. lation motif RXRXXS*/T* (asterisks indicate phosphorylated res- phosphonoacetic acid (PAA), which inhibits viral DNA replica- idues) (36). We used bioinformatic tools to scan the of tion. As expected, we observed that the expression of early genes gammaherpesviruses and identified dozens of open reading was minimally affected, while ORF52 expression was abolished frames (ORFs) bearing this motif. However, only the (Fig. 1D, lanes 6 to10), confirming that ORF52 is a true late gene. RXRXXS*/T* motif in ORF52 is positionally conserved (see Fig. KSHV ORF52 is a cytoplasmic protein. Although the ORF52s S1A in the supplemental material). We found that ORF52 is ef- of RRV and MHV-68 were found to be mostly cytoplasmic (23– ficiently phosphorylated by RSK at the predicted Ser123 resi- 25), EBV BRLF2 was observed mostly in the nucleus (26). We and due and that this phosphorylation is increased by ORF45 (see others have shown that GFP-tagged or myc-tagged KSHV ORF52 Fig. S1B and C). We have further confirmed ORF45-ORF52 exhibits mostly cytoplasmic localization (27, 39). Here, we used interaction (see Fig. S1D), which is consistent with previous our monoclonal antibodies to examine the subcellular localiza- observations (37, 38). tion of ORF52, and both antibodies detected specific signals of KSHV ORF52 is expressed as a true late gene. Although the transfected ORF52 exclusively in the cytoplasm (Fig. 2A). Impor- ORF52s of MHV-68 and RRV have been shown to be required for tantly, when induced iSLK/BAC16 or BCBL-1 cells were stained virion morphogenesis (23, 25), the roles of KSHV ORF52 have not by these antibodies, ORF52 was also observed predominantly in been characterized. In order to investigate the characteristics and the cytoplasm (Fig. 2B). These results indicate that ORF52 is a functions of ORF52, we first generated two monoclonal antibod- cytoplasmic protein. ies against it, 4H4 and 8C8. Both antibodies detected specific sig- In addition to its clear cytoplasmic localization, we also ob- nals of the expected size of ORF52 (131 aa) in transfected cells served some punctate and bundled signals, which are similar to (Fig. 1A). The antibodies also detected signals in tetradecanoyl what had been previously observed in MHV-68 ORF52-trans- phorbol acetate (TPA)-induced BCBL-1 cells and doxycycline- fected cells (24) and RRV-infected cells (25). The punctate struc- induced iSLK cells but not in uninduced cells (Fig. 1B), further tures are reminiscent of the endomembrane system. To further confirming the specificities of these antibodies and also suggesting characterize these punctate structures, we cotransfected KSHV that ORF52 is expressed only during the lytic phase. Using a series ORF52 with markers for different components of the endomem- of ORF52 internal deletion mutants, we revealed the distinct brane system. We observed modest signal overlap of KSHV epitopes of these two antibodies (Fig. 1C). ORF52 with the trans-Golgi network (TGN), and, to a lesser ex- We next investigated the kinetics of ORF52 expression. As tent, with the endoplasmic reticulum (ER) but detected no over- shown by the results in Fig. 1D, ORF52 was expressed only after lap with endosomal markers (see Fig. S2 in the supplemental ma- cells were induced to undergo lytic replication. The expression terial). The bundled and filamentous structure of ORF52 is kinetics are similar to the kinetics of the late gene (L) and capsid suggestive of microtubules. Indeed, we observed that transfected triplex dimer protein, ORF26, and slower than the kinetics of RTA ORF52 colocalized with ␣-tubulin, as well as MAP4 (see Fig. S2B (immediate early [IE]) and PF8/ORF59 (early [E]), suggesting and C). To further confirm this colocalization in KSHV-infected that ORF52 is a late gene. To provide further evidence that ORF52 cells, we modified iBAC (a BAC16 mutant that has been engi- is indeed a true late gene, we induced cells in the presence of neered to express RTA from a doxycycline-inducible promoter

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lope, we treated virions with trypsin in the presence and absence of Triton X-100, a detergent that dissolves membranes. As expected, the envelope K8.1 was degraded even in the absence of Triton X-100, the tegument proteins ORF45 (19, 40), ORF33, and ORF38 (30) were degraded only in the presence of the deter- gent, and the capsid protein ORF26 remained resistant to diges- tion. Similarly to tegument proteins, ORF52 was degraded by trypsin only when the viral envelope was disrupted by Triton X-100, suggesting that ORF52 is located inside the viral envelope (Fig. 4A). To investigate the association between ORF52 and capsid, we treated virions with increasing concentrations of detergent and then separated the capsid from the dissociated proteins by centrif- ugation through a 25% sucrose cushion. Both the supernatant (containing dissociated proteins) and pellet (containing capsid and associated proteins) were analyzed by Western blotting. As expected, envelope protein K8.1 became soluble and was detected in the supernatant in the presence of detergent. In contrast, capsid protein ORF26 was always detected in the pellet, as was ORF33 (Fig. 4B), a panherpesvirus conserved tegument protein that ap- parently associates directly with capsid (30, 41). With the deter- gent treatment, ORF52 and ORF45 became dissociated from cap- sids. However, ORF52 protein was more easily stripped off from the virion into the supernatant than ORF45 (Fig. 4B). At a higher concentration of detergent, about half of the ORF52 was dissoci- ated from the capsid, while the majority of the ORF45 was still associated with the capsid. Similar trends were obtained upon treatment of purified virions with increasing concentrations of the FIG 3 ORF52 is a virion protein. (A) Cell lysates of uninduced and induced anionic detergent (SDS) (Fig. 4C). As the iSLK.BAC16 cells (lanes 1 and 2) and gradient-purified KSHV virions (lane 3) concentration of SDS was increased, most of the ORF52 was de- were resolved by SDS-PAGE and analyzed by Western blotting with antibodies tached from the capsid, while more than half of the ORF45 re- to the indicated proteins. (B) KSHV virions were fractionated on a 20-to-60% sucrose gradient. Fractions and input were analyzed by Western blotting with mained associated with the capsid. At the highest concentration of antibodies to the indicated proteins. (C) Viral DNA of each fraction in shown SDS, more than 98% of the ORF52 was stripped off into the su- in panel B was extracted and analyzed by quantitative PCR (qPCR) (red), and pernatant, while about 50% of the ORF45 stayed in the pellet. the percentage of sucrose of each fraction was calculated from the refractive Under the same conditions, small portions of capsid protein index (green). The two values were plotted for each fraction collected. ORF26 and inner tegument protein ORF33 became detectable in the supernatant, indicating that the capsid had disintegrated. We also treated virions with increasing concentrations of [35]) by fusing GFP to the C terminus of ORF52. Stable cells NaCl. Again, the tegument proteins ORF52 and ORF45 were de- harboring this designed KSHV BAC DNA (SLK.iBAC-GFP52) tected in both fractions. At higher NaCl concentrations, more were then cultured in the presence or absence of doxycycline. The ORF52 protein was detected in the supernatant than in the pellet ORF52-GFP signals were only detected in the induced cells and (Fig. 4D). In contrast, the majority of ORF45 protein remained often displayed punctate and bundled patterns in the perinuclear associated with the viral pellet even at the highest NaCl concen- region. The IFA results confirmed significant signal overlap be- tration (Fig. 4D). All of these experiments suggested that ORF52 is tween ORF52 and ␣-tubulin (Fig. 2C). a tegument protein and is more loosely associated with the viral KSHV ORF52 is an abundant tegument protein. Although capsid than ORF45. ORF52 was identified in purified KSHV virions by mass spectrom- KSHV ORF52 is required for production of infectious viri- etry (19), its localization in virions was not investigated. We con- ons. In an attempt to characterize the roles of ORF52 in the firmed that ORF52 is present in purified KSHV virions, along with KSHV life cycle, we generated an ORF52-null mutant of the other known virion proteins, such as capsid protein ORF26 and infectious bacterial artificial chromosomal clone of KSHV, tegument protein ORF45 (Fig. 3A). When concentrated KSHV BAC16, using a seamless recombineering technique (Fig. 5A) virions were analyzed by sucrose gradient centrifugation, the (31, 32, 42). We terminated ORF52 translation prematurely by ORF52 protein was found to cofractionate well with ORF45 and introducing a triple stop codon into its coding region near the the majority of ORF52 protein comigrated with the capsid protein N terminus (after the 7th codon) (Fig. 5B, red letters). To fa- ORF26 (Fig. 3B, fractions 9 to 16). The peak fractions for ORF26 cilitate analysis of the recombinants, a HindIII site was also protein content (Fig. 3B, fractions 11 to 15) also contained large introduced (Fig. 5B, underlined letters). To ensure that any proportions of ORF52 and ORF45 signals, as well as viral genomic phenotypic changes are indeed caused by the designed muta- DNA (Fig. 3B and C), indicating that these fractions likely repre- tion rather than unintentional secondary mutations, we fur- sent intact viral particles. ther generated a revertant, BAC16-Rev52, in which the ORF52 To determinate whether ORF52 resides within the viral enve- coding sequence was restored to the wild type (Fig. 5B). To

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FIG 4 ORF52 is a tegument protein. (A) Purified virions were left untreated (lane 1) or treated with trypsin either in the absence (lane 2) or in the presence (lane 3) of 1% Triton X-100 for1hat37°C. The proteolysis reactions were terminated by the addition of 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and 1ϫ protease inhibitor. The samples were analyzed by Western blotting with antibodies to the indicated proteins. (B) Purified virions were treated with different concentrations (from 2% to 0%) of Triton X-100 in TNE buffer for 30 min and then centrifuged at 100,000 ϫ g for 1 h. The supernatant (S) and pellet (P) were dissolved in SDS-PAGE loading buffer and analyzed by Western blotting with antibodies against virion proteins as indicated. (C) Purified virions were left untreated (no detergent) or treated with different concentrations (from 1% to 0%) of SDS in TNE buffer containing 1% Triton X-100 and 0.5% deoxycholate (DOC) for 30 min and then centrifuged and analyzed as described for panel B. (D) Purified virions were treated with different concentrations (from1Mto150 mM) of NaCl in TNE buffer with 1% Triton X-100 for 30 min or left untreated (input) and then centrifuged and analyzed as in the experiments whose results are shown in panels B and C. Teg, tegument protein; Cap, capsid protein; Env, envelope protein. The percentages of proteins stripped off into the supernatant were plotted for indicated virion proteins based on the results of the Western blotting.

assess the extent to which the ORF45/RSK-dependent phos- polymorphism (RFLP) and further verified by sequencing of the phorylation of ORF52 at Ser123 plays a role in lytic reactiva- affected region. Due to the insertion of a HindIII site into the tion, we mutated this residue to alanine to generate BAC16- ORF52 locus in the BAC16-Stop52 clone, the ϳ5.5-kb HindIII 52S123A (Fig. 5B). fragment was split into two bands of sizes ϳ4.1 kb and ϳ1.4 kb All BAC mutants were analyzed by restriction fragment length (Fig. 5C, red arrows). As expected, digestion of the BAC DNAs

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FIG 5 Construction of ORF52 mutants. (A) Schematic diagram of KSHV BAC16 DNA with expected restriction fragment sizes shown for HindIII (left) and KpnI (right). (B) Schematic diagram of genome structure surrounding ORF52. KpnI/HindIII restriction sites are indicated, and the nucleotide sequences of the designed mutations are shown. Reed letters shown the triple stop codon, and underlined lettered show the HindIII site. (C) Restriction digestion of purified KSHV BAC DNAs with KpnI or HindIII. Red arrows indicate the expected changes in the HindIII digestion pattern of BAC16-Stop52. No nonspecific or spurious rearrangements were observed in any of the mutant BACs. (D) iSLK cells containing either BAC16-Stop52 DNA or its revertant mutant (Rev52) were analyzed by IFA for ORF52 expression (red) and BAC16 DNA (green).

with KpnI revealed no discernible differences between the wild determined viral genome copies by real-time PCR as previously type and mutants (Fig. 5C). described (33, 43). As shown by the results in Fig. 6A, BAC16- The BAC DNAs were transfected into iSLK cells, and stable cell Stop52 produced significantly fewer progeny viruses at each time lines were established after selection with hygromycin (15). Im- point than BAC16-WT and BAC16-Rev52. At 5 days postinduc- munofluorescence assays confirmed the loss of ORF52 expression tion, the defect was about 30-fold. In contrast, the S123A muta- in doxycycline-induced iSLK.BAC16-Stop52 cells (Fig. 5D). To tion seemed to have little effect on progeny virion production. compare the viral growth curves of the mutants and wild-type When the viruses were analyzed by Western blotting, the defect KSHV, we collected extracellular viruses from the medium and in genome copy number was mirrored by the loss of capsid pro-

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FIG 6 ORF52 is required for production of infectious progeny viruses. (A) Stable iSLK cells carrying BAC16 WT or a phosphorylation-deficient (S123A), ORF52-null (Stop52), or revertant (Rev52) viral genome were induced to undergo lytic reactivation. Extracellular virions were collected from the medium on the indicated days postinduction (dpi), and viral genome copies in the medium were determined by qPCR as described in Materials and Methods. (B) Extracellular virions were collected and concentrated (ϳ100-fold) as described in Materials and Methods. Equal volumes of concentrated medium from different cell lines were analyzed by Western blotting with antibodies to the indicated proteins. (C) Purified BAC16-Stop52 and Rev52 viruses were normalized by viral genome copy number (2 ϫ 106 per lane) and then analyzed by Western blotting with anti-ORF26 and anti-ORF52 antibodies. (D) HEK293 cells were infected with twofold serial dilutions of the indicated viruses (normalized by viral genome copy number, starting from 100 viral genome copies per cell) and then analyzed by flow cytometry at 48 h postinfection. Values are the average results of two biological duplicates. teins ORF26 and ORF65, and it appeared that the Stop52 virions we assessed were not significantly affected by the Stop52 mutation were further deficient in the packaging of other tegument pro- (Fig. 7A). We next determined whether ectopic expression of teins, including ORF45 and ORF33 (Fig. 6B). These results con- ORF52 could rescue the deficiency of BAC16-Stop52 in progeny firmed that the loss of ORF52 reduced the production of progeny virion protein in iSLK cells. We transduced iSLK/BAC16-WT with viruses and also suggested that it may impair the tegumentation empty lentiviral vector and iSLK/BAC-Stop52 with either empty process. To compare the infectivities of the extracellular virions, or ORF52-expressing lentiviral vector. The expression of ORF52 we first normalized the viruses by genomic copies of virus. The in iSLK/BAC-Stop52 was partially restored (Fig. 7B, bottom), as comparable amount of viruses was confirmed by the detection of was the yield of extracellular viruses (Fig. 7B, top), further con- similar levels of capsid protein ORF26 in each sample (Fig. 6C). firming that the deficient virion production of BAC16-Stop52 was Twofold serial dilutions of each virus were used to infect HEK293 indeed due to the loss of ORF52. cells, and the outcome of infection was quantified by FACS anal- Because BAC16-Stop52 viruses infect cells less efficiently ysis of the GFP signal at 48 h postinfection. As shown by the results than the wild-type or BAC16-Rev52 viruses (Fig. 6D), we next in Fig. 6D, the infection rate of BAC16-Rev52, as assessed by the sought to determine whether ectopic expression of ORF52 in percentage of GFP-positive cells, was comparable to that of HEK293 cells could rescue the defect in infectivity of BAC16- BAC16-WT, but the infection rate of BAC16-Stop52 virus was Stop52 virions. However, the expression of ORF52 in HEK293 dramatically lower (Fig. 6D). These results suggested that, in ad- cells had no apparent effect on the infection rate of BAC16- dition to reducing viral particle production, the loss of ORF52 Stop52 viruses, suggesting that trans-supplied ORF52 in the compromises the infectivity of progeny virions. infected cells was not sufficient to compensate for the loss of KSHV ORF52 plays a role in virion assembly. To investigate ORF52 in BAC16-Stop52 virions (Fig. 7C). Based on these data the potential mechanism(s) responsible for the observed defect in and those presented in Fig. 6B, we reasoned that BAC16- the production of infectious virions, we first analyzed the levels of Stop52 viruses may have additional defects in virion protein various viral proteins over a time course of lytic reactivation. With components. the exception of the loss of ORF52, the levels of the viral proteins To assess virion composition, we analyzed the virions of

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FIG 7 Complementation of ORF52-null mutant. (A) Stable iSLK cells carrying ORF52-null (Stop52) or the revertant (Rev52) were induced for the indicated times (days postinduction [dpi]). Cell lysates were analyzed by Western blotting with antibodies to the indicated proteins. (B) iSLK.BAC16-Stop52 cells stably transduced by ORF52 or empty control lentiviral expression vector were induced for 5 days, and then viral DNA was extracted from the supernatant and genome copy number was analyzed by qPCR. Values shown are relative to the results for iSLK.BAC16 WT transduced with empty vector. Expression of ORF52 was confirmed by Western blotting (bottom). (C) WT or Stop52 virions were normalized by viral genome copy number and then used to infect HEK293 cells stably transduced by ORF52 or empty control lentiviral expression vector. Cells were analyzed by flow cytometry at 48 h postinfection. Expression of ORF52 was confirmed by Western blotting (right).

BAC16-Rev52 and BAC16-Stop52 following fractionation on a DISCUSSION 20-to-60% sucrose gradient. As shown by the results in Fig. 8A, We initially became interested in ORF52 because it is a gamma- BAC16-Rev52 virion were enriched in fractions 11 to 13, herpesvirus-specific virion protein and, more importantly, be- with the peak containing a concentration of ϳ34% sucrose (Fig. cause it contains a positionally conserved RSK phosphorylation 8A). In contrast, BAC16-Stop52 virions exhibited a distinct shift motif (RXRXXS*/T*) at serine 123. Although we confirmed that in the peak of capsid signal to lighter fractions, fractions 8 to 10, KSHV ORF52 could be phosphorylated by RSK (see Fig. S1B in containing ϳ28% sucrose. This shift in capsid distribution is in- the supplemental material), this phosphorylation was only slightly dicative of a lower buoyant density of Stop52 virions, suggesting increased by coexpression of ORF45 (see Fig. S1C) and was not altered virion composition. To determine which viral proteins apparently affected by inhibition of RSK (data not shown), sug- might be missing in BAC16-Stop52 virions, we normalized the gesting that RSK may not be the sole kinase capable of phospho- number of virions, as well as cell lysate, by ORF26 protein level rylating this site. Recently, Duarte et al. reported that the homo- and analyzed them by Western blotting for envelope protein K8.1 logue of ORF52 in EBV, BRLF2, is phosphorylated by the serine/ and several tegument proteins, including ORF45, ORF33, and arginine-rich protein-specific kinase SRPK2 on near this ORF38. As shown by the results in Fig. 8B, BAC16-Stop52 extra- conserved motif (RaRS*RS* in BRLF2 and RpRS*KS* in MHV-68 cellular virions contained no detectable ORF45 and had signifi- ORF52) and that this phosphorylation is functionally important cantly reduced levels of ORF33, and ORF38 compared to BAC16- for viral replication (26). However, in our work, mutation of ser- Rev52 virions, despite the comparable levels of ORF26 and K8.1 ine 123 of KSHV ORF52 had little effect on the production of between Stop52 and Rev52 virions (Fig. 8B, compare lanes 3 and virions. Further analyses of the ORF45/RSK-mediated phosphor- 6). To assess the integrity of the envelope, we examined the sensi- ylation of ORF52 and other viral substrates are necessary to un- tivity of tegument proteins to trypsin digestion as in the experi- cover the potential regulatory roles of these modifications. ment whose results are shown in Fig. 4A. As shown in Fig. 8C, Mass spectrometric analyses of the purified virions of several despite its lower level in the BAC16-Stop52 virions, ORF38 was gammaherpesviruses, including KSHV, RRV, MHV-68, and EBV, resistant to trypsin digestion in the absence of detergent, suggest- have identified ORF52 as an abundant component of their virions ing that envelopment of virions was not affected by the loss of (17–21). However, there was no experimental evidence demon- ORF52. Altogether, these results suggest that ORF52 is required strating that KSHV ORF52 resides in the tegument layer of viri- for the incorporation of certain tegument proteins, especially ons. Using custom-generated specific monoclonal antibodies, we ORF45, into virions. showed here that KSHV ORF52 is expressed late during the lytic

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FIG 8 ORF52 is required for virion assembly. (A) Crude virion pellets of BAC16-Rev52 (blue) or BAC16-Stop52 (red) were layered over a 20-to-60% sucrose gradient and then ultracentrifuged at 100,000 ϫ g for 1 h. Fractions were collected, separated by SDS-PAGE, and immunoblotted for capsid protein ORF26. The distribution ratio of ORF26 (left axis) and percentage of sucrose (right axis; green) was plotted for each fraction. (B) Equal amounts of uninduced and induced iSLK.BAC16-Rev52 (lanes 1 and 2) or BAC16-Stop52 (lanes 4 and 5) cell lysates or virions (lanes 3 and 6) were resolved by SDS-PAGE and analyzed by Western blotting with antibodies to the indicated proteins. (C) Equal DNA copy numbers of purified virions of BAC16-Stop52 and BAC16-Rev52 were left untreated (lanes 1 and 4) or treated with trypsin in the presence (lanes 3 and 6) or absence (lanes 2 and 5) of Triton X-100. The samples were analyzed by Western blotting with antibodies to the indicated proteins. cycle (Fig. 1D) and localizes to the cytoplasm (Fig. 2). Impor- virions (Fig. 4A). Collectively, these results confirmed that KSHV tantly, we further revealed that ORF52 is readily detected in gra- ORF52 is indeed an abundant tegument protein in KSHV extra- dient-purified virions and cofractionates with other virion com- cellular virions. ponents (Fig. 3). Furthermore, ORF52 resides inside the viral We confirmed that KSHV ORF52 is mostly a cytoplasmic pro- envelope, because it is protected from trypsin digestion of intact tein, consistent with previous studies (27, 39). Interestingly, like

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KSHV ORF52, its homologues in both RRV and MHV-68 were teracts with pUL25 and pUL43 (60, 61), and the loss of pp65 found to be mostly cytoplasmic (23–25), but the homologue in impairs the incorporation of other tegument proteins into virions EBV (BRLF2) was observed mostly in the nucleus (26). We often (62). Interactions among gammaherpesvirus-specific tegument noticed punctuated and bundled structures of KSHV ORF52 in proteins may also exist (38, 48, 52, 63, 64). However, because both transfected and KSHV-infected cells. Noticeable portions of robust lytic replication systems of EBV and KSHV are lacking, the structures seemed to overlap the TGN and microtubules. The studies of gammaherpesvirus assembly are lagging behind those of association of KSHV ORF52 with the TGN suggests its potential alpha- and betaherpesviruses. Studies on alpha- and betaherpes- involvement in tegumentation and/or final envelopment, which virus-specific tegument proteins will shed light on the mecha- has been shown for both RRV and MHV68 ORF52 homologues nisms of virion assembly of gammaherpesviruses. (23–25). Intriguingly, we also observed a potential association of Although ORF52 is considered to be unique to gammaherpes- ORF52 with microtubules. Associations with microtubules or as- viruses, Hew et al. revealed low but discernible structural conser- sociated proteins have been observed for a number of herpesviral vation between the core domain of VP22 (conserved C terminus) tegument proteins—for example, KSHV ORF45 and HSV-1 VP22 and MHV-68 ORF52 (65). More interestingly, the two appear to (see below) (16, 44, 45). It will be interesting to determine the be positionally conserved in the genome; both reside in the same biological significance of the association of ORF52 with microtu- gene block, which is conserved in all herpesviruses, and next to the bules. gene encoding the conserved glycoprotein N (gN; encoded by To reveal the roles of ORF52 in KSHV lytic replication, we ORF53 in KSHV and by UL49.5 in HSV-1) (66). VP22, encoded generated ORF52-null BAC16-Stop52 and its revertant and ana- by UL49, is an abundant alphaherpesvirus-specific tegument pro- lyzed their phenotypes in iSLK cells. We found that BAC16- tein of HSV-1 which is known to be heavily phosphorylated (67, Stop52 produced about 30-fold less progeny virions than the wild- 68) and may partially colocalize with the TGN (69). VP22 was also type or revertant (Fig. 6A and B). Furthermore, this defect was found to colocalize with and induce the stabilization of microtu- largely rescued by ectopic expression of ORF52 (Fig. 7B). These bules (44, 45). This is reminiscent of what we have observed for observations clearly suggest that ORF52 is required for the pro- KSHV ORF52 (Fig. 2C). Furthermore, it has been reported that duction of progeny viruses. When the progeny virions were used loss of VP22 causes reduced incorporation of other viral proteins to infect HEK293, reduced infectivity was also noticed (Fig. 6D). into virions, such as ICP0 (70, 71). VP22 also exists in a virion The reduced infectivity of BAC16-Stop52 is consistent with its role protein interaction network which is important for viral assembly. in inhibiting cGAS DNA sensing during primary infection. How- Besides binding to the tegument protein ICP0, VP22 interacts ever, HEK293 cells do not express cGAS, suggesting that the with its major binding partner VP16, which links to other alpha- observed phenotype must have an additional explanation. herpesvirus-specific tegument proteins (46, 53). Furthermore, Upon further analysis, we noticed a difference between the VP22 is also incorporated into the gE-VP22-gM complex, which composition of Stop52 virions and that of the revertant virions may associate with the UL11-UL16 complex via its interaction (Fig. 8A). In particular, the levels of several tegument proteins, with glycoproteins (72). If ORF52 and VP22 indeed share func- most notably ORF45, were reduced in ORF52-null virions, sug- tional and structural similarities, a logical assumption is that gesting that ORF52 plays a critical role in the assembly of prog- ORF52 is similarly involved in virion assembly through its intri- eny virions (Fig. 8B). cate interactions with other virion proteins. On the other hand, The assembly of herpesvirus virions is a complex process that VP22 may also inhibit cGAS. Consistent with this notion, VP22, depends on intricate interactions among virion proteins. Among like ORF52, is positively charged and has been shown to bind to those tegument proteins, the interaction between ORF64 (homo- DNA (73). logue to pUL36 of HSV-1 and pUL48 of human In the present report, we demonstrate that packaging of KSHV [HCMV]) and ORF63 (homologue to pUL37 of HSV-1 and ORF45 is completely abrogated in ORF52-null virions, which is pUL47 of HCMV) and the interaction between ORF38 (homo- consistent with what has been observed for ORF52-null MHV-68 logue to pUL11 of HSV-1 and pUL99 of HCMV) and ORF33 virus and the knockdown mutants of RRV (23, 25). However, our (homologue to pUL16 of HSV-1 and pUL94 of HCMV) are well results indicate that KSHV ORF52 is more loosely associated with characterized and conserved in all three subfamilies the capsid than is ORF45, in contrast to the result for MHV-68 (38, 46–52). However, the best-characterized functions and the ORF52. Moreover, based on electron microscopy, viral particles most abundant tegument proteins in any herpesvirus are often not derived from MHV-68 ORF52-null and RRV ORF52 knockdown in the conserved core set. Each subfamily of herpesviruses has mutants accumulate in the cytoplasm and lack an envelope, sug- unique tegument proteins, and interactions among these subfam- gesting a possible role of ORF52 in secondary envelopment (23, ily-specific tegument proteins also play important roles in viral 25). As an abundant tegument protein, ORF52 may serve an es- assembly. sential role in linking the capsid to the viral envelope, similar to Several studies have suggested that the alphaherpesvirus-spe- the function of alphaherpesvirus VP16/pUL48 (57–59). However, cific tegument protein VP16 (encoded by pUL48) interacts with KSHV ORF52-null viruses appear to possess an intact membrane several other tegument proteins, including pUL49 (46, 53), pUL41 (Fig. 8C), which is reminiscent of the phenotype observed in (54), pUL36 (46), and pUL46 (46, 55), thereby linking the capsid HSV-1 VP22-null mutants (70, 74). We speculate that KSHV and inner tegument proteins with the outer tegument and mem- ORF52 reinforces the interaction network between virion pro- brane/glycoproteins during viral assembly (56–59). It is worth teins, much as HSV-1 VP22 and HCMV pp65 do (62, 72). The noting that among these interacting partners, all except pUL36 are shared and unique structural features and functions of ORF52 unique to alphaherpesviruses. Similar interactions between tegu- homologues in KSHV, MHV-68, RRV, and EBV allude to the ment proteins of betaherpesviruses have also been reported. The common, yet distinct strategies employed by these viruses to most abundant tegument protein of HCMV, pp65 (pUL83), in- maintain persistent infection of their human host. Future studies

June 2016 Volume 90 Number 11 Journal of Virology jvi.asm.org 5339 Li et al. are necessary to understand the mechanisms by which ORF52 and role in viral lytic replication. J Virol 82:1838–1850. http://dx.doi.org/10 other tegument proteins contribute to key processes throughout .1128/JVI.02119-07. the viral life cycle, including virion assembly and primary infec- 13. Kuang E, Fu B, Liang Q, Myoung J, Zhu F. 2011. Phosphorylation of eukaryotic translation initiation factor 4B (EIF4B) by tion. 45/p90 ribosomal S6 kinase (ORF45/RSK) signaling axis facilitates protein translation during Kaposi sarcoma-associated herpesvirus (KSHV) lytic ACKNOWLEDGMENTS replication. J Biol Chem 286:41171–41182. http://dx.doi.org/10.1074/jbc .M111.280982. We are grateful to Klaus Osterrieder for providing plasmid pEPKan-S, 14. Kuang E, Wu F, Zhu F. 2009. Mechanism of sustained activation of Gregory Smith for providing E. coli strain GS1783, Rolf Renne for provid- ribosomal S6 kinase (RSK) and ERK by Kaposi sarcoma-associated her- ing E. coli strain GS1783 carrying BAC16, and Jinjong Myoung and Don pesvirus ORF45. J Biol Chem 284:13958–13968. http://dx.doi.org/10 Ganem for providing the iSLK cells. We thank Ke Lan for the anti-RTA .1074/jbc.M900025200. monoclonal antibody and Robert Ricciardi for the anti-PF8 polyclonal 15. Fu B, Kuang E, Li W, Avey D, Li X, Turpin Z, Valdes A, Brulois K, antibody. We thank David Meckes for providing endomembrane gene Myoung J, Zhu F. 2015. Activation of p90 ribosomal S6 kinases by ORF45 markers. We thank Timothy Migraw at the Florida State University Col- of Kaposi’s sarcoma-associated herpesvirus is critical for optimal produc- lege of Medicine for assistance with confocal imaging. We thank Ruth tion of infectious viruses. J Virol 89:195–207. http://dx.doi.org/10.1128 Didier at the Florida State University Facility for assis- /JVI.01937-14. 16. Sathish N, Zhu FX, Yuan Y. 2009. Kaposi’s sarcoma-associated herpes- tance with flow cytometry. We thank members of the Zhu laboratory for virus ORF45 interacts with kinesin-2 transporting viral capsid-tegument critical readings of the manuscript and for helpful discussions. complexes along microtubules. PLoS Pathog 5:e1000332. http://dx.doi .org/10.1371/journal.ppat.1000332. FUNDING INFORMATION 17. O’Connor CM, Kedes DH. 2006. Mass spectrometric analyses of purified This work, including the efforts of Fanxiu Zhu, was funded by HHS | rhesus monkey rhadinovirus reveal 33 virion-associated proteins. J Virol National Institutes of Health (NIH) (R01DE016680). This work, includ- 80:1574–1583. http://dx.doi.org/10.1128/JVI.80.3.1574-1583.2006. ing the efforts of Denis R. Avey, was funded by HHS | National Institutes 18. 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