Sequence alterations in temperature-sensitive M-protein mutants (complementation group III) of vesicular stomatitis virus

Abstract

Sequences were determined of the coding regions of the M-protein genes of the Glasgow and Orsay strains of vesicular stomatitis virus (Indiana serotype) and of two group III (M-protein) mutants derived from each wild type. Synthetic primers were annealed with viral genomic RNA and extended with reverse transcriptase. The resulting high-molecular-weight cDNA was sequenced directly. Both Glasgow and Orsay wild types differed ip 13 bases from a clone of the San Juan strain sequenced by J. K. Rose and C. J. Gallione (J. Virol. 39:519-528, 1981). Six of these base changes caused amino acid changes in each wild type, whereas seven were degenerate. The Orsay aild Glasgow sequences resembled each other more closely than either resembled that of Rose and Gallione, differing in eight nucleotides and four amino acids. Each of the four mutants, however, differed from its pargnt wild type in only one or two point mutations. Every mutation caused a change either from or to a charged amino acid; the change for tsG3l was Lys (position 215) to Glu, the change for ts023 was Gly (position 21) 'to Glu, the change for ts089 was Ala (position 133) to Asp, the changes for tsG33 were Lys (position 204) to Thr and Glu (positioh 214) to Lys. The charge differences predicted from these amino acid changes was confirmed by nonequilibrium pH gradient electrophoresis for tsG31, tsG33, ts023, and the two wild types. These mutations affect residues spanning nearly 85% of the linear sequence, although the mutants possess nearly identical phenotypic properties.

Mapping regions of the matrix protein of vesicular stomatitis virus which bind to ribonucleocapsids, liposomes, and monoclonal antibodies

“…Comparison of this sequence with that derived from the cDNA clone of the M protein gene (47) indicated that the intermediate fragment had lost the first 18 amino acids from its amino terminus. A second fragment was also present in the preparation; this fragment had lost its first 19 amino acids, including the second lysine before the double arrow (Fig. 4B).…”

“…The tsO23 M protein was also found to have lost epitope 1 and will not bind monoclonal antibody to this epitope (39). Other group III temperature-sensitive mutants have also lost much of their ability to inhibit VSV transcription but have retained epitope 1 (39); two of these mutants have amino acid changes at the extreme carboxyl terminus of the M protein (19). These data seem to imply that mutations extending over 85% of the coding region of the J. VIROL.…”

“…Comparative nucleotide sequences of M genes derived from several group III temperature-sensitive mutants of VSV (19) provide important data that bear on these findings. Of particular interest is mutant ts023, the M protein of which has lost its ability to inhibit in vitro transcription of VSV (39).…”

“…Of particular interest is mutant ts023, the M protein of which has lost its ability to inhibit in vitro transcription of VSV (39). A single-point mutation at position 21, changing a glycine residue to a glutamate, is responsible for this phenotypic alteration (19). The tsO23 M protein was also found to have lost epitope 1 and will not bind monoclonal antibody to this epitope (39).…”

“…on July 4, 2020 by guest http://jvi.asm.org/ Downloaded from M-protein gene can lead to drastic alterations in secondary structure that affect the transcriptional inhibitory activity (19) and, to various degrees, the accessibility to monoclonal antibody of epitope 1 (39). Epitopes 2 and 3 have been identified by immunoblotting NCS-cleaved fragments of the M and MT proteins.…”