Envelope proteins and replication of vesicular stomatitis virus: in vivo effects of RNA+ temperature-sensitive mutations on viral RNA synthesis

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.

supporting

confidence: 54%

mentioning

confidence: 99%

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

Gopalakrishna¹,

Lenard²

1985

“…Protein synthesis in VSV-infected Drosophila cells treated with actinomycin D. Drosophila cells, line 75E7, were pretreated with actinomycin D (0.1 pg/ml) 30 min before infection and then infected as in Fig. 2.…”

Section: Figmentioning

confidence: 99%

“…Finally, two different properties of G protein were recently suggested in mammalian cells: (i) its ability to cause early inhibition of cellular DNA and RNA synthesis (32); and (ii) a role in the equilibrium between viral genome replication and transcription (30). If this were also the case for Drosophila cells, G-protein synthesis control could be a way for insect cells to prevent inhibition of their own macromolecular synthesis and to counteract viral replication.…”

Section: A M;mentioning

confidence: 99%

Vesicular stomatitis virus growth in Drosophila melanogaster cells: G protein deficiency

Wyers¹,

Richard-Molard²,

Blondel³

et al. 1980

In cultured Drosophila melanogaster cells, vesicular stomatitis virus (VSV) established a persistent, noncytopathic infection. No inhibition of host protein synthesis occurred even though all cells were initially infected. No defective interfering particles were detected, which would explain the establishment of the carrier state. In studies of the time course of viral protein synthesis in Drosophila cells, N, NS, and M viral polypeptides were readily detected within 1 h of infection. The yield of G protein and one of its precursors; G1, was very low at any time of the virus cycle; the released viruses always contained four to five times less G than those produced by chicken embryo cells, whatever the VSV strain or serotype used for infection and whatever the Drosophila cell line used as host. Actinomycin D added to the cells before infection enhanced VSV growth up to eight times. G and G1 synthesis increased much more than that of the other viral proteins when the cells were pretreated with the drug; nevertheless, the released viruses exhibited the same deficiency in G protein as the VSV released from untreated cells. Host cell control on both G-protein maturation process and synthesis at traduction level is discussed in relation to G biological properties.

“…The M protein inhibits transcription of the viral genome both in vitro (1, 23) and in vivo (2,9). Much of the data concerning VSV M protein interaction with the nucleocapsid are derived from studies of transcription inhibition activity in vitro.…”

mentioning

confidence: 99%

Sequences of the vesicular stomatitis virus matrix protein involved in binding to nucleocapsids

Kaptur

Rhodes

Lyles

1991

The purpose of these experiments was to study the physical structure of the nucleocapsid-M protein complex of vesicular stomatitis virus by analysis of nucleocapsid binding by wild-type and mutant M proteins and by limited proteolysis. We used the temperature-sensitive M protein mutant tsO23 and six temperature-stable revertants of tsO23 to test the effect of sequence changes on M protein binding to the nucleocapsid as a function of NaCl concentration. The results showed that M proteins from wild-type, mutant, and three of the revertant viruses had similar NaCl titration curves, while the curve for M proteins from the other three revertants differed significantly. The altered NaCl dependence of M protein was correlated with a single amino acid substitution from Phe to Leu at position 111 compared with the original temperature-sensitive mutant and was not correlated with a substitution of Gly to Glu at position 21 in tsO23 and the revertants. To determine whether protease cleavage sites in the M protein were protected by interaction with the nucleocapsid, nucleocapsid-M protein complexes were subjected to limited proteolysis with trypsin, chymotrypsin, or Staphylococcus aureus V8 protease. The initial trypsin and chymotrypsin cleavage sites, located after amino acids 19 and 20, respectively, were as accessible to proteases when M protein was bound to the nucleocapsid as when it was purified, indicating that this region of the protein does not interact directly with the nucleocapsid. Furthermore, trypsin or chymotrypsin treatment released the M protein fragments from the nucleocapsid, presumably due to conformational changes following proteolysis. V8 protease cleaved the M protein at position 34 or 50, producing two distinct fragments. The M protein fragment produced by V8 protease cleavage at position 34 remained associated with the nucleocapsid, while the fragment produced by cleavage at position 50 was released from the nucleocapsid. These results suggest that the amino-terminal region of the M protein around amino acid 20 does not interact directly with the nucleocapsid and that conformational changes resulting from single-amino-acid substitutions at other sites in the M protein are important for this interaction.