The second-site mutation in the herpes simplex virus recombinants lacking the gamma134.5 genes precludes shutoff of protein synthesis by blocking the phosphorylation of eIF-2alpha.
In cells infected with the herpes simplex virus 1 (HSV-1) recombinant R3616 lacking both copies of the gamma134.5 gene, the double-stranded protein kinase R (PKR) is activated, eIF-2alpha is phosphorylated, and protein synthesis is shut off. Although PKR is also activated in cells infected with the wild-type virus, the product of the gamma134.5 gene, infected-cell protein 34.5 (ICP34.5), binds protein phosphatase 1alpha and redirects it to dephosphorylate eIF-2alpha, thus enabling sustained protein synthesis. Serial passage in human cells of a mutant lacking the gamma134.5 gene yields second-site, compensatory mutants lacking various domains of the alpha47 gene situated next to the US11 gene (I. Mohr and Y. Gluzman, EMBO J. 15:4759-4766, 1996). We report the construction of two recombinant viruses: R5103, lacking the gamma134. 5, US8, -9, -10, and -11, and alpha47 (US12) genes; and R5104, derived from R5103 and carrying a chimeric DNA fragment containing the US10 gene and the promoter of the alpha47 gene fused to the coding domain of the US11 gene. R5104 exhibited a protein synthesis profile similar to that of wild-type virus, whereas protein synthesis was shut off in cells infected with R5103 virus. Studies on the wild-type parent and mutant viruses showed the following: (i) PKR was activated in cells infected with parent or mutant virus but not in mock-infected cells, consistent with earlier studies; (ii) lysates of R3616, R5103, and R5104 virus-infected cells lacked the phosphatase activity specific for eIF-2alpha characteristic of wild-type virus-infected cells; and (iii) lysates of R3616 and R5103, which lacked the second-site compensatory mutation, contained an activity which phosphorylated eIF-2alpha in vitro, whereas lysates of mock-infected cells or cells infected with HSV-1(F) or R5104 did not phosphorylate eIF-2alpha. We conclude that in contrast to wild-type virus-infected cells, which preclude the shutoff of protein synthesis by causing rapid dephosphorylation of eIF-2alpha, in cells infected with gamma134.5(-) virus carrying the compensatory mutation, eIF-2alpha is not phosphorylated. The activity made apparent by the second-site mutation may represent a more ancient mechanism evolved to preclude the shutoff of protein synthesis.
The Marjorie B. Kovler Viral Oncology Laboratories, The University of Chicago, Chicago, Illinois 60637, USA.
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Cassady
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D000818:Animals; D002213:Capsid / Q000096:biosynthesis / Q000235:genetics; D036022:Capsid Proteins; D002454:Cell Differentiation; D002522:Chlorocebus aethiops; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D017353:Gene Deletion; D005814:Genes, Viral; D005838:Genotype; D006367:HeLa Cells; D018259:Herpesvirus 1, Human / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D016254:Mutagenesis, Insertional; D009154:Mutation; D010750:Phosphoproteins / Q000096:biosynthesis / Q000235:genetics; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D011995:Recombination, Genetic; D014709:Vero Cells; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*
D036022:Capsid Proteins; D015852:Eukaryotic Initiation Factor-2; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; C109087:US10 protein, Herpes simplex virus type 1; C074546:US11 protein, herpesvirus; D014764:Viral Proteins; C066469:gamma 34.5 protein, Human herpesvirus 1; D011494:Protein Kinases; D010744:Phosphoric Monoester Hydrolases