Suppression of the phenotype of γ134.5- herpes simplex virus 1: Failure of activated RNA-dependent protein kinase to shut off protein synthesis is associated with a deletion in the domain of the α47 gene

Earlier studies have shown that infection of human cells by herpes simplex virus 1 (HSV-1) results in the activation of RNA-dependent protein kinase (PKR) but that the α subunit of eIF-2 is not phosphorylated and that protein synthesis is unaffected. In the absence of the viral γ134.5 gene, eIF-2α is phosphorylated and protein synthesis is prematurely shut off (J. Chou, J. J. Chen, M. Gross, and B. Roizman, Proc. Natl. Acad. Sci. USA 92:10516-10520, 1995). A second recent paper reported the selection of second-site suppressor mutants characterized by near-wild-type protein synthesis in cells infected with γ134.5- mutants (I. Mohr and Y. Gluzman, EMBO J. 15:4759-4766, 1996). Here, we report the properties of the spontaneous HSV-1 suppressor mutant Sup-1, which is characterized by spontaneous deletion of 503 bp encompassing the domain of the α47 gene and junction with the inverted repeats flanking the unique short (U(s)) sequence of the HSV-1 DNA resulting in the juxtaposition of the α47 promoter to the coding domain of the U(s) 11 gene. This mutant does not exhibit the shutoff of protein synthesis characteristic of the γ134.5- virus. Specifically, Sup-1 in SK-N-SH human neuroblastoma cells (i) did not exhibit the function of the α47 gene characterized by a reduction in the transport of peptides across the endoplasmic reticulum of permealized cells consistent with the absence of α47 gene sequences, (ii) accumulated U(s)11 protein at levels analogous to those of the wild-type parent but the protein was made at earlier times after infection, as would be expected from a change in the promoter, and (iii) activated PKR like that of the parent, γ134.5- virus, but (iv) did not cause premature shutoff of protein synthesis and therefore was similar to the wild-type parent virus rather than the γ134.5- virus from which it was derived. We conclude that the mechanism by which Sup-1 blocks the shutoff of protein synthesis associated with phosphorylation of eIF-2α by the activated PKR is not readily explainable by a secondary mutation characterized by a deletion.