Minute virus of mice (MVM) is an autonomous parvovirus which exhibits teratogenic and tumor-suppressive activity in vivo. We propose to define precisely the mechanism by which MVM replicates its genome and to elucidate the functions that both viral and cellular proteins perform in the replication process. The primary gene products of MVM will be enumerated, and the specific transcripts coding for them identified, by in vitro translation of infected cell RNA purified by hybridization to cloned segments of the viral genome. The coding regions which are spliced together to produce these polypeptides will be determined by cloning and sequencing cDNA copies of each vira mRNA species. The unique and shared regions of each viral gene will then be located within the entire DNA sequence of MVM, which has recently been determined. Specific mutations will be introduced into unique coding sequences in an infectious viral genome which as been cloned into a bacterial plasmid. Similarly, mutations will be introduced into the terminal regions of the genome, known to contain all of the cis-acting functions essential for viral DNA replication and progeny virion maturation. Mutant plasmids will then be transfected into appropriate host cells and the biological functions of these regions in the viral life cycle analyzed. A parallel approach, designed also to identify cellular factors essential for viral DNA synthesis, will examine the components of viral DNA replication complexes. These will be isolated by both conventional purification schemes and by affinity chromatography on avidin-Sepharose following in vivo pulse-labelling with biotinyl nucleotides. The proteins present in these complexes will be separated and characterized as to genetic origin using immunological reagents, 2-D gel electrophoresis and peptide-mapping. In addition, each component of the complex will be evaluated for specific enzymatic activities. Finally, monoclonal antibodies against each will be prepared and used to purify the protein and further investigate its function. These purified components will be used to reconstruct the viral DNA replication machinery in vitro. Since parvoviruses have a limited genetic capacity and rely extensively upon host cell functions for their growth, we anticipate that our studies will also provide some basic insights into the mechanism of eukaryotic cell DNA replication.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI019973-05
Application #
3129438
Study Section
Virology Study Section (VR)
Project Start
1983-04-01
Project End
1988-03-31
Budget Start
1987-04-01
Budget End
1988-03-31
Support Year
5
Fiscal Year
1987
Total Cost
Indirect Cost
Name
Yale University
Department
Type
Schools of Medicine
DUNS #
082359691
City
New Haven
State
CT
Country
United States
Zip Code
06520
Wang, Z; Dey, S; Rosen, B P et al. (1996) Efflux-mediated resistance to arsenicals in arsenic-resistant and -hypersensitive Chinese hamster cells. Toxicol Appl Pharmacol 137:112-9
Pitluk, Z W; Ward, D C (1991) Unusual Sp1-GC box interaction in a parvovirus promoter. J Virol 65:6661-70
Gavin, B J; Ward, D C (1990) Positive and negative regulation of the minute virus of mice P38 promoter. J Virol 64:2057-63
Ahn, J K; Gavin, B J; Kumar, G et al. (1989) Transcriptional analysis of minute virus of mice P4 promoter mutants. J Virol 63:5425-39
Bodnar, J W; Hanson, P I; Polvino-Bodnar, M et al. (1989) The terminal regions of adenovirus and minute virus of mice DNAs are preferentially associated with the nuclear matrix in infected cells. J Virol 63:4344-53
Cotmore, S F; Tattersall, P (1988) The NS-1 polypeptide of minute virus of mice is covalently attached to the 5' termini of duplex replicative-form DNA and progeny single strands. J Virol 62:851-60
Bodnar, J W; Ward, D C (1987) Highly recurring sequence elements identified in eukaryotic DNAs by computer analysis are often homologous to regulatory sequences or protein binding sites. Nucleic Acids Res 15:1835-51
Welcher, A A; Torres, A R; Ward, D C (1986) Selective enrichment of specific DNA, cDNA and RNA sequences using biotinylated probes, avidin and copper-chelate agarose. Nucleic Acids Res 14:10027-44
Kasher, M S; Pintel, D; Ward, D C (1986) Rapid enrichment of HeLa transcription factors IIIB and IIIC by using affinity chromatography based on avidin-biotin interactions. Mol Cell Biol 6:3117-27
Chow, M; Bodnar, J W; Polvino-Bodnar, M et al. (1986) Identification and characterization of a protein covalently bound to DNA of minute virus of mice. J Virol 57:1094-104

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