Graduate Thesis Or Dissertation
 

Structure-function analysis of the vaccinia virus I7L proteinase

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https://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/z603r134m

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  • Vaccinia virus is the prototypic member of the Orthopoxvirus genus. It undergoes a complex replication process where a key step in the transition from immature virion to intracellular mature virion is the cleavage of the major core protein precursors. The product of the I7L open reading frame (ORF) is a protein with an apparent molecular weight of 47kDa and has been shown to be responsible for these cleavages. However, relatively little is known about the biochemistry of the cleavage reaction, the structural features which allow I7L to direct regulated catalysis, or mechanisms by which I7L activity is regulated. As one approach to answering these questions, a phenotypic analysis was conducted on a collection of six conditional-lethal mutants in which the mutation had been mapped to the I7L locus. Genomic sequencing showed all the mutants have single amino acid substitutions within the I7L ORF. The mutations fall into two groups: changes at three positions at the N-terminus between amino acids 29 and 37 and two different substitutions at amino acid 344, near the catalytic cysteine. Two of the mutants had the exact same change. Regardless of where the mutation occurred, mutants at the non-permissive temperature failed to cleave core protein precursors and had their development arrested after immature virion assembly but prior to core condensation. Thus, we propose that the two clusters of mutations may affect two different functional domains required for proteinase activity. In a second approach, by using a combination of immunoprecipitation, immunoblotting and mass spectrometry, we showed that I7L can form a homodimer and be cleaved into a product with a molecular weight of approximately 40kDa. Mass spectrometry analysis of proteins that co-immunoprecipitated with I7L failed to provide leads on potential I7L cofactors. I7L proteins with mutations matching those in the temperature-sensitive mutants described above were capable of forming dimers and being cleaved. Thus the mechanism by which the N-terminal mutants obtain their conditional-lethal phenotype remains unknown. Taken together, this suggests several possible new models for the regulation of I7L.
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