Structure and function of the protein products encoded by the measles virusp cistron

The research work presented in this thesis involved an examination of the structure and function of the protein products of the measles virus (MV) P cistron. At least three protein products of the P cistron have been identified in MV infected cells. The largest of these is the P protein, which is translated in a canonical manner from genome faithful transcripts. The V protein is translated from a secondary population of edited mRNAs, and the C protein is generated via alternative translational initiation from both P- and V-specific mRNAs. Previous work in our laboratory had identified and characterized the cellular distribution of the MV C and V proteins in infected cells, but any insight into the function of these proteins was lacking. Contradictory results have been published as to the sub-cellular localization of the paramyxovirus C and V proteins, as well as their presence or absence in the virion.;We initiated this work with an examination of the zinc binding capacity of the V protein predicted from the primary sequence of the protein. In order to proceed with the zinc binding experiment, we overexpressed the MV P and V proteins in E. coli using the pGEX expression system. We used the glutathione-S-transferase (GST) fusion protein to demonstrate the specificity of the zinc binding activity in competition assays, as well as localizing the zinc binding domain to the carboxy-terminus of the protein.;We then used these and other GST fusion proteins to investigate the in vitro interactions of the MV P, V, and C proteins with other virally encoded proteins, as well as with cellular proteins. We identified three protein-protein interactions; P-L, P-NP, and P-P. The interaction domain of the P protein that binds the L protein, the NP protein, and the P protein was mapped to the carboxy-terminal half of the molecule that lies 3;In order to complete our examination of the protein interactions involved in the viral replication machinery, we proceeded to examine the interactions of the NP protein, again using both GST fusion protein analysis and the yeast two-hybrid system. An array of amino- and carboxy-terminal deletion mutants of the NP protein were expressed as GST fusions and used to map the NP interaction domains with the P protein and with itself.;During the course of our investigation of the P and V proteins, we unexpectedly discovered the existence of ribosomal frameshifting on the MV P mRNA. Deletion mutants were constructed in order to localize the frameshift site. The protein product of the construct with the largest deletion that still exhibited the frameshift phenotype was then purified and subjected to protein sequencing. The frameshift site was identified 5 codons upstream of the UAA stop codon that terminates the V reading frame. In addition, two-dimensional gel electrophoresis of anti-V immunoprecipitated viral protein provided strong circumstantial evidence for the existence of the novel protein product, which we have termed R, in infected cells.