Analysis of the defects of temperature-sensitive mutants of vesicular stomatitis virus: intracellular degradation of specific viral proteins

Knipe, David M.; Lodish, Harvey F.; Baltimore, David

doi:10.1128/jvi.21.3.1140-1148.1977

Cited by 78 publications

(61 citation statements)

References 23 publications

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“…Such patch formation of a membrane protein was observed with paramyxoviruses (5). However, with VSV, M protein does not associate with nmembranes in the absence of functional nucleocapsids (25,26), and as shown above, M protein cannot be immunolabeled directly on membranes. Thus, such a scenario for assembly is less likely than other scenarios.…”

Section: Discussionmentioning

confidence: 67%

Stereo images of vesicular stomatitis virus assembly

Odenwald¹,

Arnheiter²,

Dubois‐Dalcq³

et al. 1986

J Virol

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Viral assembly was studied by viewing platinum replicas of cytoplasmic and outer plasma membrane surfaces of baby hamster kidney cells infected with vesicular stomatitis virus. Replicas of the cytoplasmic surface of the basilar plasma membrane' revealed nucleocapsids forming bullet-shaped tight helical coils. The apex of each viral nose cone was anchored to the memtrane and was free of uncoiled nucleocapsid, whereas tortuous nucleocapsid was attached to the base of tightly coiled structures. Using immunoelectron microscopy, we identified the nucleocapsid (N) viral protein as a component of both the tight-coil and tortuous nucleocapsids, whereas the matrix (M) protein was found only on tortuous nucleocapsids. The M protein was not found on the membrane. Using immunoreagents specific for the viral glycoprotein (G protein), we found that the amount of G protein per virion varied. The G protein was consistently localized at the apex of viral buds, whereas the density of G protein on the shaft was equivalent to that in the surrounding membrane. These observations suggest that G-protein interaction with the nucleocapsid, via its cytoplasmic domain may be necessary for the initiation of viral assembly. Once contact is established, nucleocapsid coiling proceeds with nose cone formation followed by formation of the helical cylinder. M protein may function to induce a nucleocapsid conformation favorable for coiling or may cross-link adjacent turns in the tight coil or both.

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Section: Discussionmentioning

confidence: 67%

Stereo images of vesicular stomatitis virus assembly

Odenwald¹,

Arnheiter²,

Dubois‐Dalcq³

et al. 1986

J Virol

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“…Following detergent solubilization of virions and cellular membranes, M protein was immunoprecipitated from all three fractions and analyzed by PAGE and autoradiography (Fig. 5) The group III ts M protein mutants are defective in virus assembly (15). However, M protein is synthesized at the nonpermissive temperature, associates with membranes in the absence of nucleocapsids (23), and is released from cells in particles devoid of nucleocapsids (29).…”

Section: Resultsmentioning

confidence: 99%

Sites of in vivo phosphorylation of vesicular stomatitis virus matrix protein

Kaptur

McCreedy

Lyles

1992

J Virol

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We mapped the in vivo phosphorylation sites for the matrix (M) protein of the Orsay and San Juan strains of vesicular stomatitis virus, Indiana serotype, using limited proteolysis and phosphoamino acid analysis. M protein was solubilized from 32P-labeled virions by using detergent and high-salt conditions, then treated with either trypsin or Staphylococcus aureus V8 protease, and analyzed by polyacrylamide gel electrophoresis and autoradiography to determine which fragments contained phosphate residues. The M protein fragment extending from amino acid 20 to the carboxy terminus contained approximately 70% of the control 32p label, while the fragment extending from amino acid 35 to the carboxy terminus had only trace amounts of label. These data indicate that the major phosphorylation site was between amino acids 20 and 34 in the Orsay strain M protein. Phosphoamino acid analysis of M protein by thin-layer electrophoresis showed the presence of phosphothreonine and phosphoserine and that phosphothreonine continued to be released after prolonged vapor-phase acid hydrolysis. These data identify Thr-31 as the primary in vivo phosphate acceptor for M protein of the Orsay strain of vesicular stomatitis virus. The San Juan strain M protein has serine at position 32, which may also be an important phosphate acceptor. In addition, phosphorylation at Ser-2,-3, or-17 occurs to a greater extent in the San Juan strain M protein than in the Orsay strain M protein. The subcellular distribution of phosphorylated M protein was investigated to determine a probable intracellular site(s) of phosphorylation. Phosphorylated M protein was associated primarily with cellular membranes, suggesting phosphorylation by a membrane-associated kinase. Virion M protein was phosphorylated to a greater extent than membrane-bound M protein, indicating that M protein phosphorylation occurs at a late stage in virus assembly. Phosphorylation of wild-type and temperature-sensitive mutant M protein was studied in vivo at the nonpermissive temperature. The data show that phosphorylated M protein was detected only in wild-type virus-infected cells and virions, suggesting that association with nucleocapsids may be required for M protein phosphorylation or that misfolding of mutant M protein at the nonpermissive temperature prevents

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“…Two groups of ts mutants were investigated. The first group of mutants, including /.yM301 (Knipe et al 1977a, b. Hale et al 1978) and /531 (Pringle 1970), fail to express M protein at the nonpermissive temperature (31°C) but do express G. Cells infected with these mutants were lysed by syngeneic VSV Indiana-immune cytotoxic T lymphocytes at both permissive and non-permissive temperatures (Table VI). However, lysis of target cells infected with /.J3 1 at the non-permissive temperature was definitely diminished, indicating that the lack of proper M alignment underneath the cell membrane may have an influence on the arrangement ofthe G protein on the cell surface and thus may influence target antigens indirectly (Zinkernagel et al 1978b).…”

Section: C) Virus Mutantsmentioning

confidence: 99%

“…The second group of mutants, /5M501 (Knipe et al 1977a, b. Hale et al 1978, and /.yO45 (Lafay 1974) failed to express G at the cell surface at the nonpermissive temperature. Target cells infected with these mutants resisted both virus specific cytotoxic T lymphocyte lysis and anti-VSV antibody plus C mediated lysis at the non-permissive temperature (Table VI).…”

Section: C) Virus Mutantsmentioning

confidence: 99%