Mutational analysis of the herpes simplex virus virion host shutoff protein: evidence that vhs functions in the absence of other viral proteins.
Herpes simplex virus (HSV) virions contain one or more factors that trigger rapid shutoff of host protein synthesis and accelerated decay of cellular and viral mRNAs in infected cells. HSV isolates bearing mutations at the virion host shutoff (vhs) locus (gene UL41) are defective for both processes, indicating that the vhs protein is required; however, it is not clear whether the role of vhs in shutoff is direct or indirect and if other virion components are also necessary. We therefore used a transient-cotransfection assay to determine if the vhs protein displays activity in the absence of other viral gene products. We found that a vhs expression vector strongly suppressed expression of a cotransfected lacZ reporter gene and that this effect was eliminated by the vhs1 point mutation that abolishes virion-induced host shutoff during HSV infection. Further evidence for the biological relevance of the transfection assay came from the demonstration that five vhs in-frame linker insertion mutations yielded concordant results when assayed in cotransfected cells and following transfer into the viral genome: three mutations eliminated activity in both assays, while two had no effect. On the basis of these results, we conclude that the vhs protein can trigger host shutoff in the absence of other HSV proteins. The cotransfection assay was used to rapidly assess the activities of a panel of linker insertion mutants spanning the vhs polypeptide. All mutations that mapped to regions conserved among the vhs homologs of alphaherpesvirus inactivated function; in contrast, four of five mutations that mapped to regions that are absent from several vhs homologs had no effect. These results further support the biological relevance of the transfection assay and begin to delineate functional domains of the vhs polypeptide.
Jones
F E
FE
Pathology Department, McMaster University, Hamilton, Ontario, Canada.
Smibert
C A
CA
Smiley
J R
JR
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis; D015967:Gene Expression Regulation, Viral / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D010641:Phenotype; D012260:Ribonucleases; D018139:Simplexvirus / Q000235:genetics / Q000502:physiology*; D014162:Transfection; D014709:Vero Cells; D014764:Viral Proteins / Q000235:genetics / Q000502:physiology*; D014771:Virion / Q000235:genetics / Q000502:physiology; D014779:Virus Replication
D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; C070722:virion host shutoff protein, Simplexvirus; D012260:Ribonucleases