Transcriptional activation by the product of open reading frame 50 of Kaposi's sarcoma-associated herpesvirus is required for lytic viral reactivation in B cells

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) is a lymphotropic virus strongly linked to the development of KS, an endothelial cell neoplasm fre quent in persons with AIDS. Reactivation from latency in B cells is thought to be an important antecedent to viral spread to endothelial cells during KS pathogenesis. Earlier experiments have posited a role for the transcript ional activator encoded by KSHV open reading frame 50 (ORF50) in such react ivation, since ectopic overexpression of this protein induces reactivation in latently infected B cells. Here we have explored several aspects of the expression, structure, and function of this protein bearing on this role. T he ORF50 gene is expressed very early in lytic reactivation, before several other genes implicated as candidate regulatory genes in related viruses, a nd its expression can upregulate their promoters in transient assays. The p rotein is extensively phosphorylated in vivo and bears numerous sites for p hosphorylation by protein kinase C, activators of which are potent stimulat ors of lytic induction. The C terminus of the ORF50 protein contains a doma in that can strongly activate transcription when targeted to DNA; deletion of this domain generates an allele that expresses a truncated protein which retains the ability to form multimers with full-length ORF50 and functions as a dominant-negative protein. Expression of this allele in latently infe cted cells ablates spontaneous reactivation from latency and strikingly sup presses viral replication induced by multiple stimuli, including phorbol es ter, ionomycin, and sodium butyrate. These results indicate that the ORF50 gene product plays an essential role in KSHV lytic replication and are cons istent with its action as a putative molecular switch controlling the induc tion of virus from latency.