AlaArg motif in the carboxyl terminus of the γ134.5 protein of herpes simplex virus type 1 is required for the formation of a high-molecular-weight complex that dephosphorylates eIF-2α

The gamma (1)34.5 protein of herpes simplex virus (HSV) type 1 functions to prevent the shutoff of protein synthesis mediated by the double-stranded-RNA-dependent protein kinase PKR. This is because gamma (1)34.5 associates with protein phosphatase 1 (PP1) through its carboxyl terminus, forming a high-molecular-weight complex that dephosphorylates the alpha subunit of translation initiation factor eIF-2 (eIF-2 alpha). Here we show that Val(193)Glu and Phe(195)Leu substitutions in the PP1 signature motif of the gamma (1)34.5 protein abolished its ability to redirect PP1 to dephosphorylate eIF-2 alpha and replication of mutant viruses was severely impaired. The gamma (1)34.5 protein, when expressed in Sf9 cells using a recombinant baculovirus, was capable of directing specific eIF-2 alpha dephosphorylation. Deletions of amino acids 258 to 263 had no effect on activity of gamma (1)34.5. However, deletions of amino acids 238 to 258 abolished eTF-2 alpha phosphatase activity but not PP1 binding activity. Interestingly, deletions in the AlaArg motif of the carboxyl terminus disrupted the high-molecular-weight complex that is required for dephosphorylation of eIF-2 alpha. These results demonstrate that gamma (1)34.5 is functionally active in the absence of any other HSV proteins. In addition to a PP1 binding domain, the carboxyl terminus of gamma (1)34.5 contains an effector domain that is required to form a functional complex.