Hippo pathway control of KSHV latency

Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the infectious cause of Kaposi’s sarcoma, primary effusion lymphoma and multicentric Castleman’s disease. It is not known how KSHV causes cancer, but studies have shown that many viral gene products, including proteins and miRNAs, inactivate human tumour suppressor pathways. One putative KSHV oncoprotein, vGPCR, has been shown to inactivate the Hippo tumour suppressor pathway, but little is known about how this pathway might be controlled in the context of infection and expression of the full complement of viral gene products. Using an inducible model of KSHV lytic replication, I demonstrated that reactivation from latency and progression through the lytic cycle is associated with Hippo pathway activation. Silencing of the Hippo pathway transcription factor YAP1 increased reactivation from latency, lytic protein accumulation, and production of infectious virions. Accordingly, ectopic expression of the YAP1-deactivating kinase LATS1 also increased reactivation from latency. By contrast, LATS1 silencing reduced viral protein accumulation during late stages of lytic replication. Although the precise mechanism of Hippo-mediated control of the KSHV latent/lytic switch remains unclear, increased numbers of nuclear foci containing the viral latency-associated nuclear antigen (LANA) were observed in YAP1-silenced cells. LANA maintains latent viral episomes in the nucleus, suggesting that YAP1 deficiency may be linked to increased load of latency viral genomes, which may in turn influence the frequency of reactivation from latency. Finally, using a genetic screen to identify viral modulates of transcription of a luciferase reporter driven by the YAP1-binding protein TEAD, I demonstrated that several viral proteins can stimulate TEAD transcription, as well as two viral proteins, ORF21 and ORF71, that could inhibit it. Together, these findings suggest that KSHV latency is sensitive to perturbations in Hippo signal transduction, and that efficient lytic replication is aided by Hippo pathway activation. This presence of multiple Hippo pathway modulators in the KSHV genome suggests that the virus has evolved complex mechanisms to regulate this pathway.