Purification of Kaposi's sarcoma-associated herpesvirus (human herpesvirus 8) and analyses of the structural proteins

101

135

Murine gammaherpesvirus 68 (MHV68 [also known as ) is distinguished by its ability to replicate to high titers in cultured cells, making it an excellent candidate for studying gammaherpesvirus virion composition. Extracellular MHV68 virions were isolated, and abundant virion-associated proteins were identified by mass spectrometry. Five nucleocapsid protein homologues, the tegument protein homologue encoded by open reading frame (ORF) 75c, and envelope glycoproteins B and H were detected. In addition, gene products from MHV68 ORF20, ORF24, ORF28, ORF45, ORF48, and ORF52 were identified in association with virions, suggesting that these gammaherpesvirus genes are involved in the early phase of infection or virion assembly and egress.The herpesvirus virion is composed of an icosahedral nucleocapsid surrounded by a proteinacious layer of tegument, which in turn is enclosed by a glycoprotein-containing lipid envelope (50). The structure and protein composition of the nucleocapsid have been shown to be conserved among the three subfamilies (␣Ϫ, ␤Ϫ, and ␥Ϫ) of herpesviruses (11, 14, 62-64, 72, 74). The icosahedral nucleocapsid contains at least four integral structural proteins (the major capsid protein, triplex-1 protein, triplex-2 protein, and small capsid protein) surrounding a core of viral DNA (11,14,27,42,56,62,72,76). The other components of the virion, the envelope and the tegument in particular, are less well understood (38). The envelope contains viral glycoproteins critical for virion binding, entry, and signaling upon infection of a host cell (4,15,26,34,55,67). The tegument is the electron-dense component of the virion surrounding the capsid and interacting with the envelope (14,38,75). While the tegument component of alphaherpesviruses and betaherpesviruses is known to contain a number of gene products involved in assembly and egress of infectious virus (38) or modulation of the host cell environment upon initial infection (10,13,21,25,30,40), little is known about the protein composition of the gammaherpesvirus tegument nor about the functions of gammaherpesvirus tegument proteins immediately after infection of the cell.Study of the functions of tegument proteins in the two human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), is hampered by the lack of cell culture systems capable of supporting productive replication of these viruses. However, murine gammaherpesvirus 68 (MHV68, or ␥HV-68) is not constrained in this manner, replicating to high titers in conventional tissue culture systems. MHV68 is a model for studying de novo gammaherpesvirus infection and pathogenesis (16,20,36,66,73). The virus is found in wild murid rodents and is capable of infecting laboratory strains of mice (8,39,48). MHV68 establishes productive infection in lung epithelia and a latent infection in splenocytes, macrophages, dendritic cells, and lung epithelial cells (23,48,57,61,69).The MHV68 virion exhibits morphological similarity to the virion organization of other gammaherpe...

Section: Orf45 Is a Virion-associated Proteinmentioning

confidence: 53%

Identificationof Proteins Associated with Murine Gammaherpesvirus68Virions

Bortz

Whitelegge

Jia

et al. 2003

101

135

“…Apart from KS, KSHV is also associated closely with primary effusion lymphoma as well as multicentric Castleman's disease (MCD) (45,53,63). Like other herpesviruses, KSHV can establish latent infection in infected cells and can be reactivated to undergo lytic replication upon stimulation (52,76). It is believed that both viral latency and lytic reactivation contribute to pathogenesis mediated by KSHV (21).…”

mentioning

confidence: 99%

Cellular Corepressor TLE2 Inhibits Replication-and-Transcription- Activator-Mediated Transactivation and Lytic Reactivation of Kaposi's Sarcoma-Associated Herpesvirus

Liu

Liang

et al. 2010

Replication and transcription activator (RTA) encoded by open reading frame 50 (ORF50) of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential and sufficient to initiate lytic reactivation. RTA activates its target genes through direct binding with high affinity to its responsive elements or by interaction with cellular factors, such as RBP-J, Ap-1, C/EBP-␣, and Oct-1. In this study, we identified transducin-like enhancer of split 2 (TLE2) as a novel RTA binding protein by using yeast two-hybrid screening of a human spleen cDNA library. The interaction between TLE2 and RTA was confirmed by glutathione S-transferase (GST) binding and coimmunoprecipitation assays. Immunofluorescence analysis showed that TLE2 and RTA were colocalized in the same nuclear compartment in KSHV-infected cells. This interaction recruited TLE2 to RTA bound to its recognition sites on DNA and repressed RTA auto-activation and transactivation activity. Moreover, TLE2 also inhibited the induction of lytic replication and virion production driven by RTA. We further showed that the Q (Gln-rich), SP (Ser-Pro-rich), and WDR (Trp-Asp repeat) domains of TLE2 and the Pro-rich domain of RTA were essential for this interaction. RBP-J has been shown previously to bind to the same Pro-rich domain of RTA, and this binding can be subject to competition by TLE2. In addition, TLE2 can form a complex with RTA to access the cognate DNA sequence of the RTA-responsive element at different promoters. Intriguingly, the transcription level of TLE2 could be upregulated by RTA during the lytic reactivation process. In conclusion, we identified a new RTA binding protein, TLE2, and demonstrated that TLE2 inhibited replication and transactivation mediated by RTA. This provides another potentially important mechanism for maintenance of KSHV viral latency through interaction with a host protein.

“…In these studies, we found that the three-dimensional capsid structures and protein homolog compositions were strikingly similar (33, 64). Both KSHV and RRV exhibit structural features similar to those of other herpesviruses, including an icosahedral capsid (30,56,62) enclosing the viral genome, a proteinaceous tegument, and an envelope (36,44,59) decorated with glycoprotein spikes that facilitate virion-host cell interactions (1,8,9,22,68). These findings, coupled with the high yields of RRV in culture, have made RRV an attractive model system for the study of KSHV structure and assembly (2,30,33,47,64).…”

mentioning

confidence: 99%

Mass Spectrometric Analyses of Purified Rhesus Monkey Rhadinovirus Reveal 33 Virion-Associated Proteins

O’Connor

Kedes

2006

The repertoire of proteins that comprise intact gammaherpesviruses, including the human pathogen Kaposi's sarcoma-associated herpesvirus (KSHV), is likely to have critical functions not only in viral structure and assembly but also in the early stages of infection and evasion of the host's rapidly deployed antiviral defenses. To develop a better understanding of these proteins, we analyzed the composition of rhesus monkey rhadinovirus (RRV), a close phylogenetic relative of KSHV. Unlike KSHV, RRV replicates to high titer in cell culture and thus serves as an effective model for studying primate gammaherpesvirus structure and virion proteomics. We employed two complementary mass spectrometric approaches and found that RRV contains at least 33 distinct virally encoded proteins. We have assigned 7 of these proteins to the capsid, 17 to the tegument, and 9 to the envelope. Of the five gammaherpesvirus-specific tegument proteins, three have no known function. We also found three proteins not previously associated with a purified herpesvirus and an additional seven that represent new findings for a member of the gamma-2 herpesviruses. Detergent extraction resulted in particles that contained six distinct tegument proteins in addition to the expected capsid structural proteins, suggesting that this subset of tegument components may interact more directly with or with higher affinity for the underlying capsid and, in turn, may play a role in assembly or transport of viral or subviral particles during entry or egress.Kaposi's sarcoma remains the most common AIDS-related malignancy worldwide, and its causative agent is Kaposi's sarcoma-associated herpesvirus (KSHV), a member of the gamma subfamily of herpesviruses (7, 10). Unfortunately, KSHV grows to low titer in culture, making detailed structural and compositional studies of highly purified particles particularly challenging (4, 65). In contrast, rhesus monkey rhadinovirus (RRV), a nonhuman primate gamma-2 herpesvirus with high levels of homology to KSHV, grows to high titer in culture (2,33,47,64). We have previously characterized the capsids of both KSHV and RRV (30,33,56,64). In these studies, we found that the three-dimensional capsid structures and protein homolog compositions were strikingly similar (33, 64). Both KSHV and RRV exhibit structural features similar to those of other herpesviruses, including an icosahedral capsid (30,56,62) enclosing the viral genome, a proteinaceous tegument, and an envelope (36,44,59) decorated with glycoprotein spikes that facilitate virion-host cell interactions (1,8,9,22,68). These findings, coupled with the high yields of RRV in culture, have made RRV an attractive model system for the study of KSHV structure and assembly (2,30,33,47,64).Alpha-and betaherpesvirus studies provided the basis for early classification schemes for structural components of gammaherpesviruses. However, many structural proteins of the gamma subfamily demonstrate sufficient amino acid sequence divergence from alpha-and betaherpesviruses to suggest that ...