Successful infection by the autonomous parvovirus minute virus of mice (MVM) requires the concerted action of its two nonstructural proteins NS1 and NS2. Both the relative ratio of NS2 to NS1, which is controlled by both alternative splicing and protein stability, and the interaction of NS2 with the nuclear export protein Crm1, are critical parameters of infection. Low relative levels of NS2, as well as mutations in NS2 that abolish its interaction with Crm1, allow generation of monomer replicative DNA forms (mRF), but not cell-associated or released, encapsidated progeny single-stranded DNA (ssDNA), indicating that NS2 may play a central role in the generation of ssDNA. In the absence of NS2 altogether, neither replicative form is produced in appreciable amounts in murine cells, suggesting that NS2 may play a role in maintaining a permissive cellular environment. NS2 interaction with Crm1 is also critical for virus egress from infected cells, and NS2:Crm1 interaction mutants are retained in the nucleus. Although NS2 is produced at high levels in permissive cells, it is also a very labile protein, suggesting that its lability is critical to its function in infection. NS2 is degraded by the proteasome in an ubiquitin-independent manner - an atypical, but important, mechanism of cellular protein degradation. To further characterize this critical effector of parvovirus replication we propose: I. To determine the role of NS2 in egress of assembled full virions from the nucleus of murine cells. II. To determine the role of NS2 in the production of progeny ssDNA and mRF. III. To determine how the lability of NS2 mediates its function during infection, and to characterize the mechanism governing the degradation of NS2. Completion of these experiments will help determine how the essential parvovirus protein NS2 functions in MVM replication. In addition, understanding how the NS2:Crml interaction facilitates MVM egress from the nucleus should help illuminate the process by which small non-enveloped viruses emerge from cells. Characterization of the ubiquitin-independent, proteasomal degradation of NS2 will further our understanding of this important cellular process, and finally, since the NS2 protein plays a critical role in both parvovirus assembly and progeny ssDNA production, these experiments will have the added benefit of helping to optimize the production and infectivity of parvovirus-based vectors.
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