Phosphorylation of Vesicular Stomatitis Virus In Vivo and In Vitro

The structural protein, NS, of purified vesicular stomatitis virus (VSV) is a phosphoprotein. In infected cells phosphorylated NS is found both free in the cytoplasm and as part of the viral ribonucleoprotein (RNP) complex containing both the 42S RNA and the structural proteins L, N, and NS, indicating that phosphorylation occurs as an early event in viral maturation. VSV contains an endogenous protein kinase activity, probably of host region, which catalyzes the in vitro phosphorylation of the viral proteins NS, M, and L, but not of N or G. The phosphorylated sites on NS appear to be different in the in vivo and in vitro reactions, and are differentially sensitive to alkaline phosphatase. After removal of the membrane components of purified VSV with a dextran-polyethylene glycol two-phase separation, the kinase activity remains tightly associated with the viral RNP. However, viral RNP isolated from infected cells shows only a small amount of kinase activity. The protein kinase enzyme appears to be a cellular contaminant of purified VSV because an activity from the uninfected cell extract can phosphorylate in vitro the dissociated viral proteins NS and M. The virion-associated activity may be derived either from the cytoplasm or the plasma membrane of the host cell since both of these cellular components contain protein kinase activity similar to that found in purified VSV.     

Inhibition of Protein Synthesis in L Cells Infected with Vesicular Stomatitis Virus

The inhibition of protein synthesis in L cells by vesicular stomatitis virus (VSV) requires the synthesis of new protein subsequent to virus infection. However, two mechanisms may be involved in the inhibition of cell protein synthesis by VSV: an initial, multiplicity-dependent, ultraviolet-insensitive inhibition and a progressive, ultraviolet-sensitive inhibition.     

Effects of Psoralen-Derivatized Oligonucleoside Methylphosphonates on Vesicular Stomatitis Virus (VSV) Protein Synthesis In Vitro and in VSV-Infected Cells

Abstract

Oligodeoxyribonucleoside methylphosphonates (16-mers) targeted to VSV mRNAs were derivatized with 4′-{[N-(aminoethyl)amino]methyl}-4,5′8-trimethylpsoralen. These oligomers specifically inhibit translation of their targeted mRNAs in vitro following UV irradiation of the oligomer-mRNA complexes. Psoralen-derivatized oligonucleoside methylphosphonates are stable in cells and can inhibit VSV protein synthesis in culture following UV-irradiation of VSV-infected cells.

     

Temperature-Sensitive Mutants of Vesicular Stomatitis Virus: In Vitro Studies of Virion-Associated Polymerase

The temperature dependence of the virion-associated polymerase activity of six temperature-sensitive (ts) mutants of vesicular stomatitis virus (tsW10, 11, 14, 16B, 28, and 29) has been examined in vitro and compared to the heat-resistant parent (HR). The polymerase of five of the mutants (tsW10, 11, 14, 16B, and 28) appears to be significantly more ts than that of HR. Because certain pairs of these five mutants can complement each other's in vitro polymerase activity, it appears that in vitro some components involved in the polymerase of one virion can be utilized by another virion. Examination of 19 revertants of tsW11 and tsW16B which had regained their ability to replicate at 38 C showed that their in vitro polymerase activity had also become less ts. Furthermore, it was found that the pairs of mutants which showed in vitro complementation of polymerase activity at 38 C were those which had shown complementation in yielding infectious progeny in mixedly infected cells. These two observations suggest that the ts behavior of the in vitro polymerase activity of the five mutants is related to their inability to replicate at the nonpermissive temperature.     

In Vitro Synthesis of Proteins by Membrane-Bound Polyribosomes from Vesicular Stomatitis Virus-Infected HeLa Cells

Membrane-bound polysomes from vesicular stomatitis virus (VSV)-infected HeLa cells synthesize predominantly three proteins in an in vitro protein synthesizing system. These three proteins have different molecular weights than the viral structural proteins, i.e., 115,000, 88,000, and 72,000. Addition of preincubated L or HeLa cell S10 or HeLa cell crude initiation factors stimulates amino acid incorporation and, furthermore, alters the pattern of proteins synthesized. Stimulated membrane-bound polysomes synthesize predominantly viral protein G and lesser amounts of N, NS, and M. In vitro synthesized proteins G and N are very similar to virion proteins G and N based on analysis of tryptic methionine-labeled peptides. Most methionine-labeled tryptic peptides of virion G protein contain no carbohydrate moieties, since about 90% of sugar-labeled peptides co-chromatograph with only about 10% of methionine-labeled peptides. Sucrose gradient analysis of the labeled RNA present in VSV-infected membrane-bound polysomes reveals a relative enrichment in a class of viral RNA sedimenting slightly faster than the total population of the 13 to 15S mRNA, as compared to a VSV-infected crude cytoplasmic extract. A number of proteins, other than the viral structural proteins, are synthesized in the cytoplasm of five lines of VSV-infected cells. One of these proteins has the same molecular weight as the major in vitro synthesized protein, P(88). In vitro synthesized protein P(88) does not appear to be a precursor of viral structural proteins G, N, or M based on pulse-chase experiments and tryptic peptide mapping. Nonstimulated membrane-bound polysomes from uninfected HeLa cells synthesize the same size distribution of proteins as nonstimulated VSV-infected membrane-bound polysomes.     

Protein Kinase and Phosphoproteins of Vesicular Stomatitis Virus

Protein kinases of similar but not identical activity were found associated with vesicular stomatitis (VS) virions grown in mouse L cells, primary chicken embryo (CE) cells, and BHK-21 cells, as well as being present in VS virions grown in HeLa and Aedes albopictus cells. The virion kinase preferentially phosphorylated the nucleocapsid NS protein in vitro and to a lesser extent the envelope M protein. Other virion proteins were phosphorylated in vitro only after drastic detergent treatment. Partial evidence that the virion kinase is of cellular origin was obtained by finding reduced enzyme activity in virions released from cells pretreated with actinomycin D and cycloheximide. Selective detergent and detergent-salt fractionation of VS virions revealed that the kinase activity was present in the envelope but not the spikes. The virion kinase activity in a Triton-salt-solubilized envelope fraction could be separated from M and G proteins and partially purified by phosphocellulose column chromatography. Virions released from L, CE, and BHK-21 cells infected in the presence of [(32)P]orthophosphate were labeled almost exclusively in the NS protein. Both soluble and nucleocapsid-associated NS phosphoprotein were present in cytoplasmic extracts of VS viral-infected L cells. The origin and function of the NS phosphoprotein remain to be elucidated.     

Model for Vesicular Stomatitis Virus

Vesicular stomatitis virus contains single-stranded ribonucleic acid of molecular weight 3.6 x 10(6) and three major proteins with molecular weights of 75 x 10(3), 57 x 10(3), and 32.5 x 10(3). The proteins have been shown to be subunits of the surface projections, ribonucleoprotein, and matrix protein, respectively. From these values and from estimates of the proportions of the individual proteins, it has been calculated that the virus has approximately 500 surface projections, 1,100 protein units on the ribonucleoprotein strand, and 1,600 matrix protein units. Possible models of the virus are proposed in which the proteins are interrelated.     

Entry of Vesicular Stomatitis Virus into L Cells

Early stages of the entry of vesicular stomatitis (VS) virus into L cells were followed by electron microscopy with the aid of ferritin antibody labeling. Cells which were infected at 0 C and incubated for 10 min at 37 C were reacted first with antiviral-antiferritin hybrid antibody and then with ferritin or fluorescein-labeled apoferritin. Extensive ferritin labeling of the cell surface was detected by both electron and fluorescence microscopy. The labeled regions of the cell surface were continuous with and indistinguishable from the rest of the host cell membrane, suggesting incorporation of viral antigens into the cell surface during viral penetration. Fusion of parental viral membrane with host cell membrane was further demonstrated by examining the localization of (3)H-labeled viral structural proteins in cells infected at 0 C and incubated for short periods at 37 C. Viral nucleoprotein was found in a soluble fraction of the cells which was derived primarily from the cytoplasm, whereas a particulate fraction from the cells was enriched in viral envelope proteins. Cytoplasmic membrane was isolated from these cells, and this membrane contained viral envelope proteins. These results suggest that penetration by VS virus occurs by fusion of the viral and cellular membranes followed by release of nucleo-protein into the cytoplasm.     

Structure of the Vesicular Stomatitis Virus L Protein in Complex with Its Phosphoprotein Cofactor

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