Production of host shutoff-defective mutants of herpes simplex virus type 1 by inactivation of the UL13 gene.
Two mutants of HSV-1(SC16) carrying disrupted UL13 genes have been generated independently by recombination of wild-type genomic DNA with a plasmid-cloned copy of the UL13 gene containing multiple stop codons. The two mutants were shown to be deficient in UL13 gene expression by Western blotting of infected cells. A revertant virus, in which UL13 expression was restored to a near-normal level, was generated by recombination of one of the UL13-negative mutants with a plasmid carrying the wild-type UL13 gene. The replication of the two UL13-negative viruses in cell culture was somewhat reduced compared to their wild-type parent, and the viruses were unable to produce shutoff of host protein synthesis. The replication of the revertant virus was intermediate between that of the UL13-negative and wild-type viruses, as was its ability to produce host shutoff. Cells infected with the UL13-negative mutants were shown to contain much lower levels than normal of the UL41 gene product, which is known to be required for virion host shutoff. However, there was no significant difference between levels of the UL41 gene product in wild-type and mutant virions. The UL13-negative viruses exhibited different patterns of protein phosphorylation from wild-type virus when infected cells were metabolically labeled with [32P]-orthophosphate and when lysates of infected cells and of virions were subjected to in vitro phosphorylation. However, the UL41 gene product could still be phosphorylated in lysates of UL13-negative virions. We conclude that the UL13 gene is necessary to produce the virion host shutoff effect, but it seems unlikely that the role of UL13 is simply to activate the UL41 gene product by phosphorylation.
Overton
H
H
Department of Molecular Virology, Roche Research Centre, Welwyn Garden City, Herts, United Kingdom.
McMillan
D
D
Hope
L
L
Wong-Kai-In
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004279:DNA, Viral; D003673:Defective Viruses / Q000235:genetics; D005814:Genes, Viral; D018259:Herpesvirus 1, Human / Q000235:genetics* / Q000502:physiology; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010766:Phosphorylation; D011494:Protein Kinases / Q000096:biosynthesis / Q000235:genetics*; D014407:Tumor Cells, Cultured; D014709:Vero Cells; D014779:Virus Replication / Q000235:genetics
D004279:DNA, Viral; D011494:Protein Kinases; C082380:UL13 protein, Simplexvirus