ORF33 and ORF38 of Kaposi's Sarcoma-Associated Herpesvirus Interact and Are Required for Optimal Production of Infectious Progeny Viruses

Abstract

We recently showed that the interaction between Kaposi's sarcoma-associated herpesvirus (KSHV) tegument proteins ORF33 and ORF45 is crucial for progeny virion production, but the exact functions of KSHV ORF33 during lytic replication were unknown (J. Gillen, W. Li, Q. Liang, D. Avey, J. Wu, F. Wu, J. Myoung, and F. Zhu, J Virol 89:4918 -4931, 2015, http://dx.doi.org/10 .1128/JVI.02925-14). Therefore, here we investigated the relationship between ORF33 and ORF38, whose counterparts in both alpha-and betaherpesviruses interact with each other. Using specific monoclonal antibodies, we found that both proteins are expressed during the late lytic cycle with similar kinetics and that both are present in mature virions as components of the tegument. Furthermore, we confirmed that ORF33 interacts with ORF38. Interestingly, we observed that ORF33 tightly associates with the capsid, whereas ORF38 associates with the envelope. We generated ORF33-null, ORF38-null, and double-null mutants and found that these mutants apparently have identical phenotypes: the mutations caused no apparent effect on viral gene expression but reduced the yield of progeny virion by about 10-fold. The progeny virions also lack certain virion component proteins, including ORF45. During viral lytic replication, the virions associate with cytoplasmic vesicles. We also observed that ORF38 associates with the membranes of vesicles and colocalizes with the Golgi membrane or early endosome membrane. Further analyses of ORF33/ORF38 mutants revealed the reduced production of virion-containing vesicles, suggesting that ORF33 and ORF38 are involved in the transport of newly assembled viral particles into cytoplasmic vesicles, a process important for viral maturation and egress. IMPORTANCEHerpesvirus assembly is an essential step in virus propagation that leads to the generation of progeny virions. It is a complicated process that depends on the delicate regulation of interactions among virion proteins. We previously revealed an essential role of ORF45-ORF33 binding for virus assembly. Here, we report that ORF33 and its binding partner, ORF38, are required for infectious virus production due to their important role in the tegumentation process. Moreover, we found that both ORF33 and ORF38 are involved in the transportation of virions through vesicles during maturation and egress. Our results provide new insights into the important roles of ORF33 and ORF38 during viral assembly, a process critical for virus propagation that is intimately linked to KSHV pathobiology.

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 Viruses

“…In contrast, capsid protein ORF26 was always detected in the pellet, as was ORF33 (Fig. 4B), a panherpesvirus conserved tegument protein that apparently associates directly with capsid (30,41). With the detergent treatment, ORF52 and ORF45 became dissociated from capsids.…”

“…The detailed procedures for the production of antibodies were described in our previous study (29). Antibodies against ORF26, ORF65, ORF33, ORF38, and ORF45 used in this study were described previously (15,29,30). Antibody against RTA was offered by Ke Lan at Institut Pasteur of Shanghai.…”

Epstein‐Barr virus tegument protein BGLF2 inhibits NF‐κB activity by preventing p65 Ser536 phosphorylation

“…Recently, BGLF2 has been shown to activate p38 and JNK signaling pathways and enhance AP-1-dependent transcription (34,35). Several orthologs of BGLF2, such as Kaposi's sarcomaassociated herpesvirus ORF33 (66,67), varicella-zoster virus ORF44 (68), and cytomegalovirus (CMV) unique long 94 (UL94) (69), are essential for viral replication. However, herpes simplex virus unique long 16 (UL16) is not crucial for viral proliferation, and deletion of UL16 impairs virus growth.…”

Interaction Between BGLF2 and BBLF1 Is Required for the Efficient Production of Infectious Epstein–Barr Virus Particles

“…Deleting HCMV UL94 is known to abolish virion production (Phillips and Bresnahan, 2012). MHV-68 and KSHV ORF33 are crucial for virion morphogenesis and egress (Guo et al, 2009;Wu et al, 2015); deletion of UL11 or UL16 in HSV-1 reduces secondary envelopment during viral assembly and causes the accumulation of unenveloped nucleocapsids in the cytoplasm Roizman, 1991, 1992;Starkey et al, 2014). Evidence suggests that the interaction between UL11 and UL16 provides a bridge between the nucleocapsid and the envelope during the final envelopment (Loomis et al, 2003;Meckes et al, 2010;Johnson and Baines, 2011).…”