The peripheral matrix (M) protein of negative-strand viruses, such as vesicular stomatitis virus (VSV), plays a key role in virion assembly and transcription regulation. We propose to characterize the molecular basis for the membrane binding, nucleocapsid binding, and transcription-inhibition functions of the VSV M protein by the following chemical, immunological, genetic, and biophysical methods: 1. Peptides generated by cleavage with specific chemicals and proteases will be sequenced, mapped, and tested, along with synthetic oligopeptides, for their capacity to bind to phospholipid vesicles and nucleocapsids, and to inhibit viral transcription. 2. Monoclonal antibodies to M protein have been prepared and will be tested for their reactivity with specific peptides and for their capacity to inhibit the various biological functions of M protein and its peptides. 3. Complementation group III temperature-sensitive mutant M proteins, restricted in transcription-inhibition, and revertant M proteins will be tested for their capacity to bind to lipid vesicles, nucleocapsids, and specific monoclonal antibodies. 4. Recombinant DNA clones of mutant, revertant, and wild-type M-protein genes will be prepared and their cDNAs sequenced to identify the sites of lesions in M proteins defective in binding to vesicles and nucleocapsid and in transcription-inhibition. We eventually hope to prepare cDNA clones of M genes in expression vectors to make site-directed defective M proteins. 5. Biophysical studies with wild-type and mutant M proteins and their peptides will be performed to assay perturbations of membrane dynamics measured by fluorescence depolarization reacting with membranes as determined by circular dichroism spectroscopy. 6. M proteins of VSV-New Jersey and A/WSN/33 influenza virus and their monoclonal antibodies will also be examined by these methods for comparison with the functional and evolutionary relatedness of the M protein of VSV-Indiana.
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