Phosphorylation of Vesicular Stomatitis Virus Proteins as a Possible Contributing Factor in Virion Uncoating

Abstract

SUMMARYThe relationship between the in vitro phosphorylation of vesicular stomatitis virus (VSV) proteins and virion uncoating was examined. Activation of the VSV virion kinase with low concentrations of melittin, the active peptide component of bee venom, in the presence of y-[32p]ATP resulted in the phosphorylation of virion proteins. Following the in vitro phosphorylation of VSV proteins in the presence of melittin and deoxyadenosine triphosphate, the virion envelope was disrupted based on the accessibility of the internal ribonucleoprotein core (RNP) to the heavy metal stain, uranyl acetate, as determined by electron microscopic observati~n. The RNP structure was not observed in unphosphorylated virions treated with melittin and uranyl acetate. Phosphorylated virions treated with uranyl acetate subsequently lost the capacity for transcription whereas unphosphorylated virions treated with the stain retained transcriptase activity. These observations suggest that phosphorylation of VSV proteins may contribute to virion uncoating by disrupting the virus envelope.

Effect of Melittin on Transcription by Vesiculovirus Mutant and Wild-Type Viruses

“…All three wildtype viruses exhibited a narrow optimal concentration range, concentrations above 20pg/0.1 ml being inhibitory. The concentra tion curves are similar to those presented by Witt et al [1981] for VSV Indiana, except that we determined the optimum concentration of melittin for in vitro transcription by VSV Indiana to be tenfold less than the value given by these authors.…”

“…The opti mum concentrations of melittin for in vitro transcription by purified virions of Chandipura (CHP) virus, VSV Indiana and VSV New Jersey were determined using the condi tions employed by Witt et al [1981], and the results are presented in figure 1. The opti mum concentration of melittin for in vitro transcription by all three vesiculoviruses was I0pg/0.1 ml reaction mixture.…”

“…One is the toxin melittin, a 26-amino acid peptide com ponent of bee venom, and the other pardaxin, a 162-amino acid component of a toxin from a Red Sea flatfish. Both proteins are hydrophobic in nature and both have been found to activate vesicular stomatitis virus (VSV) transcription in the absence of detergent [Pal et al, 1981: Roy et al, 1982Witt et al, 1981]. Examination of treated vi rions by electron microscopy revealed that the structure of the virion was unaffected [Witt et al, 1981], and that the viral envelope retained the appearance of a bilayered mem brane [Pal et al, 1981].…”

“…Both proteins are hydrophobic in nature and both have been found to activate vesicular stomatitis virus (VSV) transcription in the absence of detergent [Pal et al, 1981: Roy et al, 1982Witt et al, 1981]. Examination of treated vi rions by electron microscopy revealed that the structure of the virion was unaffected [Witt et al, 1981], and that the viral envelope retained the appearance of a bilayered mem brane [Pal et al, 1981]. Melittin and pardaxin render the viral membrane permeable to nu cleoside triphosphates, allowing the virion RNA to be transcribed.…”

Viral Aspects of Protein Phosphorylation

“…This proposal is based on the findings of experiments in which an endogenous protein kinase of VSV virions was activated by making it accessible to ATP in vitro, resulting in phosphorylation of the matrix M protein (which is not a DNA-binding protein) and disruption of the virion envelope (Witt et al, 1981). Similar experiments in vitro have led to proposals for a role for phosphorylation in the uncoating of an insect granulosis virus (Wilson & Consigli, 1985) and of poliovirus (Lackmann et al, 1987), both of which are non-enveloped viruses.…”