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A modular study of the minute virus of mice non-structural protein 1: Activation, dimerization, and interaction

University of British Columbia

NS1, the major nonstructural parvoviral protein of the Minute Virus of Mice is a multifunctional protein responsible for many of the functions needed for viral replication. In this thesis, a modular approach was taken to resolve and isolate the mechanism for the activation of transcription, the oligomerization of NS1, and interactions with host cell cofactors. NS1 has been shown to transactivate the P38 promoter, used to express the structural protein, as well as its own strong promoter, P4. To identify the mechanism of activation, and to map the region of NS1 responsible for transactivation, NS1 and various deletions of NS1 were cloned inframe with the GAL4 DNA binding domain. These constructs were cotransfected into COS-7 or LA9 cells with a plasmid containing five GAL4 recognition sites and the EIB TATA box upstream of the chloramphenicol acetyl transferase reporter gene. Using this system, NS1 was shown to directly activate transcription through its 129 carboxyl-terminal amino acid residues. Any deletion from the carboxyl-terminal, including one with as few as 8 amino acids, completely abolished transactivation. The full length NS1 fused to GAL4 showed strong activation of the GAL4 promoter in LA9 cells while in COS-7 cells, deletion of residues 1 to 276 of NS1 was required to allow full transactivation by the GAL4DB-NS1 constructs. A yeast two-hybrid system was used to identify protein-protein interactions between NS1 and other proteins. Using this system, NS1 was shown to dimerize when expressed in yeast. Only an almost complete NS1 (1-638) bait was able to interact with the full length NS1, suggesting that there are multiple regions of the protein required for interaction, that the oligomerization domain is very large, or that the interaction domain is conformation dependent which is disrupted by the larger deletions. The two-hybrid system was also used to screen a HeLa cDNA library to identify host cell proteins that associate with NS1. A single partial HeLa cDNA clone was isolated that interacts with the amino-terminal of NS1 (1-276). More sequence was predicted from hEST data and the cDNA was estimated to be at least 2221 base pairs long. The cDNA has not previously been characterized but it contains four ribonucleoprotein (RNP) domains as well as a highly repetitive carboxyl-terminal region. A closely related mouse cDNA is predicted from murine EST data which encodes a protein with only a single amino acid residue change from the human protein. The protein is predicted to be a polynucleotide binding protein of approximately 60 kDa. There are many possible roles for this protein during MVM replication which further work will determine. [Scientific formulae used in this abstract could not be reproduced.]

Thesis/Dissertation

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2009-06-02

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http://hdl.handle.net/2429/8554

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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