The ORF45 protein of Kaposi's sarcoma-associated herpesvirus is associated with purified virions

Kaposi's sarcoma-associated herpesvirus (KSHV) ORF45 is encoded by an immediate-early gene in the KSHV genome. This protein was recently shown to interact with interferon regulatory factor 7 and inhibit virus-mediated alpha/beta interferon induction (Zhu et al., Proc. Natl. Acad. Sci. USA 99:5573-5578, 2002). ORF45 was characterized as a phosphorylated protein, and it is localized in the cytoplasm of infected cells. In this report, we provide evidence that ORF45 is associated with KSHV virions. (i) ORF45 was detected in gradient-purified virions by Western blotting along with known structural proteins of KSHV including gB, K8.1, and major capsid protein. In contrast, ORF50/Rta, K8alpha, and ORF59/PF8 were not detected in the same virion preparation. (ii) ORF45 comigrates with KSHV virions in sucrose gradient ultracentrifugation. (iii) Virion-associated ORF45 was resistant to trypsin digestion but became sensitive after the virions were treated with detergent which destroys the viral envelope. (iv) ORF45 remained associated with tegument-nucleocapsid complex when virion-specific glycoproteins were removed after detergent treatment. (v) An ORF45 protein band was visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of extensively purified KSHV virions and identified by mass spectrometry. (vi) By immunoelectron microscopy, virus-like structures were specifically stained by anti-ORF45 antibody. Based on the evidence, we conclude that ORF45 is associated with purified KSHV virions and appears to be a tegument protein. The presence of ORF45 in KSHV virions raised the possibility that this protein may be delivered to host cells at the start of infection and therefore have the opportunity to act at the very early stage of the infection, suggesting an important role of ORF45 in KSHV primary infection.     

Modulation of Kaposi's sarcoma-associated herpesvirus infection and replication by MEK/ERK, JNK, and p38 multiple mitogen-activated protein kinase pathways during primary infection

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma, a dominant AIDS-related tumor of endothelial cells, and several other lymphoproliferative malignancies. While activation of the phosphatidylinositol 3-kinase-protein kinase C-MEK-ERK pathway is essential for KSHV infection, we have recently shown that KSHV also activates JNK and p38 mitogen-activated protein kinase (MAPK) pathways during primary infection (J. Xie, H. Y. Pan, S. Yoo, and S.-J. Gao, J. Virol. 79:15027-15037, 2005). Here, we found that activation of both JNK and p38 pathways was also essential for KSHV infection. Inhibitors of all three MAPK pathways reduced KSHV infectivity in both human umbilical vein endothelial cells (HUVEC) and 293 cells. These inhibitory effects were dose dependent and occurred at the virus entry stage of infection. Consistently, inhibition of all three MAPK pathways with dominant-negative constructs reduced KSHV infectivity whereas activation of the ERK pathway but not the JNK and p38 pathways enhanced KSHV infectivity. Importantly, inhibition of all three MAPK pathways also reduced the yield of infectious virions during KSHV productive infection of HUVEC. While the reduction of infectious virions was in part due to the reduced infectivity, it was also the result of direct modulation of KSHV lytic replication by the MAPK pathways. Accordingly, KSHV upregulated the expression of RTA (Orf50), a master transactivator of KSHV lytic replication, and activated its promoter during primary infection. Furthermore, KSHV activation of RTA promoter during primary infection was modulated by all three MAPK pathways, predominantly through their downstream target AP-1. Together, these results indicate that, by modulating multiple MAPK pathways, KSHV manipulates the host cells to facilitate its entry into the cells and postentry productive lytic replication during primary infection.     

An Sp1 response element in the Kaposi's sarcoma-associated herpesvirus open reading frame 50 promoter mediates lytic cycle induction by butyrate

Kaposi's sarcoma-associated herpesvirus (KSHV) can be driven into the lytic cycle in vitro by phorbol esters and sodium butyrate. This report begins to analyze the process by which butyrate activates the promoter of KSHV open reading frame 50 (ORF50), the key viral regulator of the KSHV latency to lytic cycle switch. A short fragment of the promoter, 134 nucleotides upstream of the translational start of ORF50, retained basal uninduced activity and conferred maximal responsiveness to sodium butyrate. The butyrate response element was mapped to a consensus Sp1-binding site. By means of electrophoretic mobility shift assays, both Sp1 and Sp3 were shown to form complexes in vitro with the ORF50 promoter at the Sp1 site. Butyrate induced the formation of a group of novel complexes, including several Sp3-containing complexes, one Sp1-containing complex, and several other complexes that were not identified with antibodies to Sp1 or Sp3. Formation of all butyrate-induced DNA-protein complexes was mediated by the consensus Sp1 site. In insect and mammalian cell lines, Sp1 significantly activated the ORF50 promoter linked to luciferase. Chromatin immunoprecipitation experiments in a PEL cell line showed that butyrate induced Sp1, CBP, and p300 binding to the ORF50 promoter in vivo in an on-off manner. The results suggest that induction of the KSHV lytic cycle by butyrate is mediated through interactions at the Sp1/Sp3 site located 103 to 112 nucleotides upstream of the translational initiation of ORF50 presumably by enhancing the binding of Sp1 to this site.     

Octamer-Binding Sequence Is a Key Element for the Autoregulation of Kaposi's Sarcoma-Associated Herpesvirus ORF50/Lyta Gene Expression

The expression of the Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 50 (ORF50) protein, Lyta (lytic transactivator), marks the switch from latent KSHV infection to the lytic phase. ORF50/Lyta upregulates several target KSHV genes, such as K8 (K-bZip), K9 (vIRF1), and ORF57, finally leading to the production of mature viruses. The auto-upregulation of ORF50/Lyta is thought to be an important mechanism for efficient lytic viral replication. In this study, we focused on this autoregulation and identified the promoter element required for it. An electrophoretic mobility shift assay indicated that the octamer-binding protein 1 (Oct-1) bound to this element. Mutations in the octamer-binding motif resulted in refractoriness of the ORF50/Lyta promoter to transactivation by ORF50/Lyta, and Oct-1 expression enhanced this transactivation. These results suggest that the autoregulation of ORF50/Lyta is mediated by Oct-1.