Sites of in vivo phosphorylation of vesicular stomatitis virus matrix protein

Abstract

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

Interactions of normal and mutant vesicular stomatitis virus matrix proteins with the plasma membrane and nucleocapsids

“…The inability of MN15 protein to bind nucleocapsids would also be consistent with the suggestion by Kaptur et al (18) that proteolytic cleavage at the amino terminus may cause a conformational change that is disruptive to a downstream nucleocapsid binding region (26,32). Removal of critical phosphorylation sites in the amino terminus or the alteration of other phosphorylation sites in M protein due to a conformational change may affect nucleocapsid and/or membrane association (2,17).…”

Phosphorylation of Marburg virus matrix protein VP40 triggers assembly of nucleocapsids with the viral envelope at the plasma membrane

“…Phosphorylation of matrix proteins is not common to all members of the order Mononegavirales . Matrix proteins of Newcastle disease virus (Chambers and Samson, 1980) and mumps virus (Rima et al ., 1980) are not phosphorylated; however, matrix proteins of vesicular stomatitis virus, Sendai virus, human parainfluenza virus type 3 and respiratory syncytial virus have been found in a phosphorylated form in virions and/or in infected cells (Lamb and Choppin, 1977; Wechsler et al ., 1985; Lambert et al ., 1988; Kaptur et al ., 1992). Like with MARV VP40, in most cases, the phosphorylation of M proteins involves only a fraction of the matrix protein molecules.…”

“…Phosphoamino acid analysis of the [ 32 P]orthophosphate‐labelled Zaire Ebolavirus VP40 revealed a strong signal for phosphotyrosine as well (L. Kolesnikova, unpublished). The functional significance of matrix protein phosphorylation is currently unclear for most viruses of the order Mononegavirales (Kaptur et al ., 1992; 1995). Recently, Pei et al .…”

Architecture and regulation of negative-strand viral enzymatic machinery

“…81). Interestingly, phosphorylation of matrix has been observed for representative members of all nonsegmented NS RNA viral families encoding an inhibitory matrix protein, [82][83][84][85][86] and hyperphosphorylation of matrix has been associated with inhibition of VSV RNA synthesis, 87 indicating alternative mechanisms such as control of a posttranslational switch could determine when nonsegmented L proteins become template-locked and virion egress is favored. nonsegmented viruses of the Bornaviridae family), each encode alternative accessory proteins that downregulate polymerase activity and could maintain the conserved functional link between polymerase regulation and virion maturation (Fig.…”