Gammaherpesviruses are associated with the development of lympho-proliferative disorders and lymphoma, particularly in immunosuppressed individuals. Indeed, half of the lymphomas that arise in HIV infected individuals are associated with either EBV or KSHV infection. Perturbation of host cell cycle is a common strategy employed by DNA viruses to achieve a cellular environment conducive to viral growth. Adenovirus, polyoma virus, papilloma virus, and many herpesviruses encode genes that directly alter the host cell cycle, or interact with host gene products to the same end. Rhadinoviruses (?2-herpesviruses), such as Kaposi sarcoma-associated herpesvirus (KSHV), herpesvirus saimiri (HVS), and murine gammaherpesvirus-68 (MHV68), encode a homolog of mammalian D-type cyclins. We have previously shown that the MHV68 v-cyclin is required for: (i) efficient acute replication in the lungs of mice; and (ii) reactivation from latently infected macrophages and B cells. We have recently identified a tissue culture model that recapitulates the replication defect observed with v-cyclin null and v-cyclin CDK binding mutants in vivo. Further characterization of MHV68 replication in this tissue culture model has identified a profound defect in the egress of v-cyclin null and v-cyclin CDK binding mutants from infected cells. In this new R21 application, we propose to investigate the role that the MHV68 v-cyclin plays in virus egress/release from lung epithelial cells.
The specific aims are as follows:
Aim 1. Characterization of the v-cyclin null mutant MHV68 egress phenotype: 1.a Localization of virions in wt and v-cyclin mutant infected cells; 1.b Cellular localization of v-cyclin during virus infecton; 1.c Assess egress phenotype of a KSHV v-cyclin null mutant in HUVECs.
Aim 2. Analysis of v-cyclin functions in the absence of MHV68 infection: 2.a Generate and characterize inducible v-cyclin expressing cell lines; 2.b Identify v-cyclin interacting partners.
The gamma-2 herpesviruses (also known as rhadinoviruses) all encode a homolog of the D type cyclins. The murine gammaherpesvirus 68 (MHV68) v-cyclin plays roles in both acute virus replication in the lungs and virus reactivation from latency. We have identified a tissue culture model which mimics the replication defect observed in vivo with MHV68 v-cyclin mutants. Notably, this model has revealed a previously unknown role for a v-cyclin in virus egress from permissively infected cells. The proposed studies will investigate the basis for this replication defect, focusing on observed differences in cellular cytoskeleton organization in infected cells.
