The two non-structural proteins NS1 and NS2 of the autonomous parvovirus minute virus of mice (MVM) are critical for viral replication. Our overall goal is to understand how autonomous parvoviruses achieve lytic infection in permissive cells and persistence in animal populations. In the First Specific Aim we will characterize how NS1 achieves potent transactivation of the viral capsid-gene promoter P38. Our previous work has suggested that NS1 binding proximal to P38 likely increases the local concentration of both TBP and TFIIA at P38, thereby increasing the rate of pre- initiation complex formation and/or elongation. Subsequent to its binding NS1 may alter the local chromatin structure so that Sp1 can more efficiently engage P38 and activate the basal transcription machinery. To test this working model, and further explore the role of NS1 in P38 transactivation we propose the following. 1) because binding of NS1 to the promoter region is required for transactivation, we will characterize NS1 oligomerization and DNA binding in detail. 2) We will determine the minimal domain of NS1 so that can transactivate P38 in the context of the viral genome, thereby implicating the biochemical properties sufficient for this process. 3) We will evaluate the importance and consequences of NS1 interactions with SP1, TBP and TFIIA. 4) We will characterize the changes in P38 nucleosomal arrangement in response to NS1 in vivo and in vitro. MVM NS2 is required for viral replication in a host-range manner; it is required for viral replication in its normal murine host cell. NS2 mutants display two main phenotypes in murine cells; they do not generate progeny single-stand (ss) DNA, which can be attributed to a primary defect in capsid assembly, and are deficient in production of monomer replicative form (mRF) DNA. NS2 has important effects on the murine cell environment, and the lack of NS2 likely leads to a general effect that is at least in part responsible for premature abrogation of the viral life cycle. To further elucidate the role of NS2 we propose the following Second Specific Aim. 1) We will determine the role of NS2 in the accumulation of progeny ss and mRF. 2) We will determine the importance of phosphorylation and protein lability for NS2's role during infection. 3) We will determine the role of NS2 during infection of T-cell lines and in newborn mice. 4) We will analyze the effects of NS2 on cell-cycle progression and survival of infected cells. The completion of these experiments will further our understanding of the parvovirus life-cycle, and shed light on the function of the critical cellular proteins on which these viruses rely.
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