The Second-Site Mutation in the Herpes Simplex Virus Recombinants Lacking the γ 1 34.5 Genes Precludes Shutoff of Protein Synthesis by Blocking the Phosphorylation of eIF-2α

Abstract

ABSTRACT In cells infected with the herpes simplex virus 1 (HSV-1) recombinant R3616 lacking both copies of the γ 1 34.5 gene, the double-stranded protein kinase R (PKR) is activated, eIF-2α 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 γ 1 34.5 gene, infected-cell protein 34.5 (ICP34.5), binds protein phosphatase 1α and redirects it to dephosphorylate eIF-2α, thus enabling sustained protein synthesis. Serial passage in human cells of a mutant lacking the γ 1 34.5 gene yields second-site, compensatory mutants lacking various domains of the α47 gene situated next to the U S 11 gene (I. Mohr and Y. Gluzman, EMBO J. 15:4759–4766, 1996). We report the construction of two recombinant viruses: R5103, lacking the γ 1 34.5, U S 8, -9, -10, and -11, and α47 (U S 12) genes; and R5104, derived from R5103 and carrying a chimeric DNA fragment containing the U S 10 gene and the promoter of the α47 gene fused to the coding domain of the U S 11 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-2α 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-2α in vitro, whereas lysates of mock-infected cells or cells infected with HSV-1(F) or R5104 did not phosphorylate eIF-2α. We conclude that in contrast to wild-type virus-infected cells, which preclude the shutoff of protein synthesis by causing rapid dephosphorylation of eIF-2α, in cells infected with γ 1 34.5 − virus carrying the compensatory mutation, eIF-2α 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.