The aim of this research is to describe as completely as possible the action of M protein during that part of the infectious cycle commencing with virion budding from an infected cell, and concluding with the release of the genome-containing necleocapsid in a transcriptionally active form in the cytoplasma of an uninfected cell. Previously characterized temperature sensitive M protein mutants of VSV will be used in this study, and also VS virions made transcriptionally active by addition of melittin. The sequence alterations that confer altered function on the M protein mutants will be determined by sequence analysis of cDNA prepared using the mutant genome as template. The identity of the alteration(s) will be confirmed by sequencing revertants of these mutants. The infectivity of intact and melittin permeabilized VSV will be compared, using both mutant and wild type VSV. The proteins of melittin permeabilized VSV will be phosphorylated to determine whether this affects infectvity. The effect of phosphorylation of M on its nucleocapsid-binding and membrane-binding properties will be assessed, using melittin permeabilized VSV. M in different phosphorylated forms will be added to functioning nucleocapsids in detergent to assess the effect of phosphorylation on inhibition of transcription by M. The effect of M protein phosphorylation of G protein mobility following fusion will be assessed using fluorescence photobleaching recovery (FPR) on erythrocytes fused with VSV. FPR experiments will also be carried out using giant liposomes as target membranes; permeabilization of these with melittin might permit the study of G-M protein interactions in a completely reconstituted system. The thermolability of the highly thermolabile M mutant ts G31 will be investigated using melittin permeabilized virions to determine whether thermal inactivation blocks phosphorylation of M, thus blocking onset of transcription. If phosphorylation does alter the functional properties of M, phosphorylation sites will be identified by peptide analysis, and the protein kinase will be purified and characterized from virions. Attempts will be made to prepare fragments of M by limited proteolytic digestion; these will be assessed for nuecleocapsid and membrane binding activities, and as substrates for phosphorylation.
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