Herpes simplex virus virion host shutoff protein requires a mammalian factor for efficient in vitro endoribonuclease activity.
The virion host shutoff protein (vhs) of herpes simplex virus (HSV) triggers global shutoff of host protein synthesis and accelerated mRNA turnover during virus infection and induces endoribonucleolytic cleavage of exogenous RNA substrates when it is produced in a rabbit reticulocyte (RRL) in vitro translation system. Although vhs induces RNA turnover in the absence of other HSV gene products, it is not yet known whether cellular factors are required for its activity. As one approach to addressing this question, we expressed vhs in the budding yeast Saccharomyces cerevisiae. Expression of vhs inhibited colony formation, and the severity of this effect varied with the carbon source. The biological relevance of this effect was assessed by examining the activity of five mutant forms of vhs bearing previously characterized in-frame linker insertions. The results indicated a complete concordance between the growth inhibition phenotype in yeast and mammalian host cell shutoff. Despite these results, expression of vhs did not trigger global mRNA turnover in vivo, and cell extracts of yeast expressing vhs displayed little if any vhs-dependent endoribonuclease activity. However, activity was readily detected when such extracts were mixed with RRL. These data suggest that the vhs-dependent endoribonuclease requires one or more mammalian macromolecular factors for efficient activity.
Lu
P
P
Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7.
Jones
F E
FE
Saffran
H A
HA
Smiley
J R
JR
D000818:Animals; D004722:Endoribonucleases / Q000378:metabolism*; D010641:Phenotype; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D012260:Ribonucleases; D012441:Saccharomyces cerevisiae / Q000254:growth & development; D018139:Simplexvirus / Q000502:physiology*; D014764:Viral Proteins / Q000502:physiology*
D012333:RNA, Messenger; D014764:Viral Proteins; C070722:virion host shutoff protein, Simplexvirus; D004722:Endoribonucleases; D012260:Ribonucleases