In vitro mRNA degradation system to study the virion host shutoff function of herpes simplex virus.
The virion host shutoff (vhs) gene of herpes simplex virus encodes a virion polypeptide that induces degradation of host mRNAs at early times and rapid turnover of viral mRNAs throughout infection. To better investigate the vhs function, an in vitro mRNA degradation system was developed, consisting of cytoplasmic extracts from HeLa cells infected with wild-type herpes simplex virus type 1 or a mutant encoding a defective vhs polypeptide. Host and viral mRNAs were degraded rapidly in extracts from cells productively infected with wild-type herpes simplex virus type 1 but not in extracts from mock-infected cells or cells infected with the mutant vhs1. In contrast, 28S rRNA was stable in all three kinds of extract. Accelerated turnover of host mRNAs was also observed in extracts from cells infected with wild-type virus in the presence of dactinomycin, indicating that the activity was induced by a structural component of the infecting virions. The in vitro vhs activity was inactivated by heat or proteinase K digestion but was insensitive to brief treatment of the extracts with micrococcal nuclease. It was not inhibited by placental RNase inhibitor, it exhibited a strong dependence upon added Mg2+, it was active at concentrations of K+ up to 200 mM, and it did not require the components of an energy-generating system. In summary, the in vitro mRNA degradation system appears to accurately reproduce the vhs-mediated decay of host and viral mRNAs and should be useful for studies of the mechanism of vhs action.
Krikorian
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Department of Microbiology, Stritch School of Medicine, Loyola University of Chicago, Maywood, Illinois 60153.
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D000818:Animals; D003593:Cytoplasm / Q000378:metabolism; D005814:Genes, Viral; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012339:RNA, Ribosomal, 28S / Q000235:genetics / Q000378:metabolism; D018139:Simplexvirus / Q000235:genetics* / Q000502:physiology; D014709:Vero Cells; D014771:Virion / Q000235:genetics* / Q000502:physiology
D012333:RNA, Messenger; D012339:RNA, Ribosomal, 28S; D008274:Magnesium