The major objective of this proposal is to determine the mechanism(s) of autonomous parvovirus transcription, at the molecular and enzymatic level, using the fibrotropic (p) and lymphotropic (i) variants of Minute Virus of Mice (MVM) and the human B19 virus as model systems. These viruses show a remarkable target cell specificity for their replication, both in vivo and in vitro. In the case of MVM(p) and MVM(i), which are reciprocially restricted in their ability to replicate in fibroblasts and lymphocytes, recent studies have shown that the replication block in restrictive host cells occurs at the level of transcriptional initiation and demonstrated a requirement for a differentiation- specific intra-nuclear """"""""factor"""""""" for the establishment of active viral transcription templates. To further define the transcription processes of these viruses a combination of genetic, biochemical and immunological approaches will be taken to identify both the genomic sequences and the viral or cellular proteins involved. Particular attention will be paid to the characterization of the various molecular forms of the MVM non-structural proteins NS-1 and NS-2, since one or more of these polypeptides, derived by transcription off the P-4 promoter, functions as a transactivator of the P-38 promoter. Conventional and affinity chromatographic techniques will be applied to the purification of those cellular factors from murine and human cell so identified, and antibody or oligonucleotide probes will be used to clone the genes for these factors. Mutational analysis of viral and cellular genes will be done so as to identify the functional domains of each gene product. Such studies should hopefully provide further insights into the regulation of viral gene expression in infected eukaryotic cells.
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