Human cytomegalovirus (HCMV) infects a large proportion of the world's population, in most circumstances remaining latent without causing any disease. Loss of immune control can lead to reactivation of latent HCMV and severe disease manifestations. The ability of HCMV to survive the host immune response is due in large part to functions that permit evasion of innate and adaptive host defenses. Cell killing by apoptosis is one host-defense mechanism against viral infection. HCMV encodes several proteins that block apoptosis. One such protein, HCMV gpUL144, is a tumor necrosis factor receptor homologue. Preliminary data suggests that gpUL144 may block death receptor-mediated apoptosis. gpUL144 is dispensable for replication in vitro, but evolutionary conservation of gpUL144 in clinical isolates predicts an important role for gpUL144 in vivo. Cell culture methods will be used to define the extent to which, and mechanism by which, gpUL144 blocks different forms of receptor-mediated apoptosis. Native and epitope-tagged versions of gpUL144 will be stably expressed in cell types sensitive to different forms of receptor-mediated apoptosis and tested for the ability to block apoptotic signaling. The mechanism of blocking apoptosis will be probed by determining which proteins are recruited into death-inducing signaling complexes (DISC) following receptor ligation. The effect of gpUL144 expression on the subcellular localization of DISC components will be determined. Generation of gpUL144 specific antiserum will permit analysis of gpUL144 expression and function during infection. Because apoptotic pathways are highly conserved between mouse and humans, we will test if, and expect that gpUL144 will function in murine cells. Although outside the realm of this proposal, because murine cytomegalovirus (MCMV) lacks a UL144 homologue, infection of immune system knockout mice with a recombinant MCMV will permit analysis of gpUL144 function in vivo.
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