3-Herpesviruses (3-HVs) have developed a unique mode of interaction with their hosts to establish a life-long persistent infection, which frequently associates with the onset of various malignancies. One critical virulence factor involved in 3-HVs persistence and oncogenicity are the viral homologs of the Bcl-2 protein (referred to as vBcl-2) encoded by all 3-HVs. Alongside its well-characterized anti-apoptotic activity, our preliminary studies have established that the vBcl-2 of the 3-HV family effectively suppresses the anti-viral autophagy pathway ('self-eating', lysosome-dependent degradation and recycling of the intracellular components in response to stress), by directly targeting a key autophagy effector protein, Beclin1. Moreover, vBcl-2 has evolved enhanced anti-autophagic activity when compared to the host counterpart. Based on these findings, we hypothesize that the inhibition of autophagy by vBcl-2 constitutes a novel mechanism by which 3- HVs evade host immunity and confer persistent infection and pathogenesis. To test this hypothesis, we will focus primarily on the vBcl-2 of 3HV68 using well-established in vitro and in vivo systems. 3HV68 shares extensive genetic homology and biological similarity with EBV and KSHV. Infection of mice with 3HV68 provides a genetically tractable in vivo model for characterizing the chronic infection of 3HVs. Notably, the loss of vBcl-2 does not affect the lytic replication of 3HV68. Instead, it severely impairs the ability of 3HV68 to establish chronic infection in mice. In the first aim, we will dissect the molecular mechanism by which vBcl-2 antagonizes Beclin1-dependent autophagy. We have successfully identified the specific mutations that distinguish vBcl-2-mediated inhibition of autophagy from vBcl-2-mediated antagonism of apoptosis. In the second aim, we will investigate the specific roles of vBcl-2-mediated anti-autophagy in viral virulence in vivo. Insights gained from this study will reveal a novel paradigm for the roles of vBcl-2 in 3HVs infection, and establish autophagy as a fundamental host defensive mechanism against viral infections. PUBLIC HEALTH RELEVANCE: Autophagy has been increasingly recognized essential in host anti-viral defense, but its role in 3- herpesviruses infection remains largely unknown. The proposed study is targeted to understand the molecular mechanism of vBcl-2-mediated inhibition of autophagy, and its contribution to the persistent infection and/or pathogenesis of 3-herpesviruses. Insights gained from this study will establish a direct role for autophagy in viral virulence control and suggest strategy for much- needed anti-viral therapeutics.