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The structural proteins of several isolates of the Indiana serotype (Orsay, Glasgow, and San Juan) of vesicular stomatitis virus (VSV) were compared by sizing peptides generated by digestion with Staphylococcus aureus V8 protease or α-chymotrypsin. These digestions yielded nearly identical peptide patterns for all the VSV proteins except for the glycoprotein, indicating that the major surface antigen, even of these closely related isolates, is subject to change. When two serologically distinct VSV isolates (Indiana and New Jersey) were similarly examined, each protein had a peptide pattern characteristic of that serotype. The glycoprotein of the two serotypes had no peptides in common, while the internal viral proteins L, N, NS, and M showed various degrees of similarity. Digests of the L (polymerase) and M (membrane) protein, in particular, generated groups of small peptides with identical mobilities, suggesting that there may be core regions in the Indiana and New Jersey strain which are conserved. These conserved regions may in turn represent essential functions of rhabdovirus proteins and are promising targets for broad-spectrum antivirals.

Eleven temperature-sensitive mutants of Sindbis virus were classified according to their ability to complement other mutants in pairwise mixed infections at a non-permissive temperature. Five nonoverlapping complementation groups were thus defined. In two groups the temperature-sensitive function is required for viral RNA synthesis, while mutants of the other three groups are unable to carry out some essential step occurring after viral RNA synthesis. The progeny produced by complementation were found, in general, to include both the genomes used to initiate the mixed infection; these progeny particles were phenotypically mixed in a manner that suggests exclusion of mutationally altered protein from rescued particles.

In monolayers of chick embryo fibroblasts or BHK cells infected with Sindbis virus in the presence of actinomycin, host protein synthesis is inhibited by at least 80-90% between 2 and 3 hours after infection. Thereafter, Sindbis specified polypeptide chains form 60-70% of total protein synthesis. In particular, synthesis of the membrane protein of the virus constitutes 25% of total protein synthesis. Twelve to 16 polypeptides induced by virus infection have been identified in infected cells by acrylamide gel electrophoresis. The pattern of virus-specified proteins observed depends upon the length of the labeling period, the host cell, and mutations in the virus genome. The molecular weight of the membrane protein of the Sindbis virion has been estimated to be 53,000 and the core protein is estimated to be 30,000 in molecular weight.

The trypsin inhibitor TLCK ( N -α- p -tosyl- l -lysine chloromethyl ketone-HCI) has often been used to block proteolytic cleavages in experiments designed to detect precursor-product relationships of proteins. Recently it was reported that measles virus matrix (M) protein synthesized in the presence of TLCK has an apparent molecular weight 12000 daltons larger than that of standard M protein, (R. Vainionpaa, Arch. Virol. 60, 239–248, 1979). This suggested that TLCK was blocking proteolytic cleavage of a precursor M protein. In this report we show that TLCK did not function in this manner, but rather exerted some secondary affect, perhaps inducing a conformational change in the M protein. We also report here on TLCK induced alterations of the measles virus P protein and cellular actin. We conclude that since TLCK can alter the electrophoretic mobility of some proteins by a mechanism unrelated to inhibition of proteolytic cleavage, care should be taken in interpreting both past and future claims of precursor-product relationships in systems in which TLCK or related compounds are assumed to act by blocking proteolytic cleavages.

Two proteins of vesicular stomatitis virus (VSV), M (matrix) and NS (nucleocapsid-associated), each contained about the same proportion of phosphoserine and phosphothreonine, with phosphoserine as the principal phosphorylated amino acid. Essentially all phosphate (greater than 98%) present in the NS protein was in a typical phosphomonoester bond, while the M protein contained 10 to 15% of its phosphate in an undetermined linkage. Two forms of the NS protein, NS1, and the more highly phosphorylated NS2 form, were separated in SDS-polyacrylamide gels containing urea, and partially digested with chymotrypsin. A comparison of the chymotryptic peptides indicated that the same two phosphorylated forms, NS1 and NS2, were found in virions as well as in the cytoplasm of infected cells. These two forms were interconvertible in vitro . A conversion of NS2 to the lesser phosphorylated NSl form, presumably by a protein phosphatase activity, occurred in the presence of cytoplasm from either infected or uninfected Chinese hamster ovary (CHO) cells. However the amount of the NS1 species was greatly reduced upon acceptance of phosphate through the action of the virion-associated protein kinase. The products of the kinase reaction were analyzed in more detail by matching phosphopeptides from the partial protease digests of the products with NS and M proteins phosphorylated in vivo. In vitro , M protein was the major phosphate acceptor, while in vivo , NS protein had the greatest capacity to accept phosphate.