Autonomous parvoviruses are small, single-stranded DNA viruses which are incapable of inducing resting cells to enter S-phase and thus replicate exclusively in dividing cell populations. They show complex tissue specificities which operate at multiple steps in the infectious process, and the proposed research is aimed at understanding the molecular basis of these tropisms and the mechanisms by which parvoviruses initiate productive or abortive life cycles in their hosts. While many cell types share appropriate cell surface receptors, viral gene expression is restricted to a subset of cells which express a specific, as yet unidentified, intracellular receptor capable of interacting with a mutable element on the viral capsid termed the allotropic determinant. The crystal structure of the lymphotropic variant of minute virus of mice (MVMi) will be completed, and the structures of both virions and empty capsids will be compared with host range variants differing by just one or two amino acids. Capsid structure will be probed genetically and serologically to explore cell ligands and the disposition of elements disordered in the crystals. Strategies to identify the high abundance, high affinity viral receptor on the surface of human T-cells will be developed. Monoclonal antibodies raised against virions will be screened for their ability to recognize the allotropic determinant, in an attempt to place this on the structural map. Antibodies directed against specific features on the virion surface will be electroporated into the cytosol of target cells, in order to explore the process of penetration. Non-replicative functions of NS1, the major viral non-structural protein, will be studied by analyzing non-cytostatic cytocidal mutants. parameters of the growth of MVMi in cycling and quiescent human T-cells will be examined, so as to determine the fate and state of the viral genome in non-cycling cells. These studies will exploit the recent development of MVM-Tar, a chimeric MVMi derivative which is dependent upon the HIV-1 tat transactivator protein for its own replication. The roles of the NS proteins in the synthesis and assembly of the capsid proteins will be studied. A random sequence PCR technique will be employed to determine the consensus DNA binding site for NS1. The importance of this NS1:DNA interaction in the assembly of viral nucleoprotein and in transcriptional transactivation of the viral P38 promoter will be assessed. The knowledge gained will be used to construct a synthetic promoter for the development of cell lines for packaging MVM-based expression vectors. New mutants, expressor cell lines and recombinant vaccinia viruses will be used to explore the involvement of the NS2 family of minor NS proteins in the synthesis and assembly of capsids. Finally, viral recombinants will be constructed with dual utility, both as transient expression vectors for human cells, and as tools for dissecting the viral packaging mechanism. Since transformed cells are exceptionally susceptible to killing by parvoviruses, understanding their complex target cell specificity, the means whereby the usurp cellular control and how they generate infectious particles, may allow development of these viruses as rugged, potentially injectable vectors for targeting disseminated transformed cells in neoplastic disease.
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