AlaArg motif in the carboxyl terminus of the gamma(1)34.5 protein of herpes simplex virus type 1 is required for the formation of a high-molecular-weight complex that dephosphorylates eIF-2alpha.
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-2alpha). Here we show that Val193Glu and Phe195Leu substitutions in the PP1 signature motif of the gamma(1)34.5 protein abolished its ability to redirect PP1 to dephosphorylate eIF-2alpha 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-2alpha 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 eIF-2alpha 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-2alpha. 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.
Department of Microbiology and Immunology, College of Medicine, The University of Illinois at Chicago, Chicago, Illinois 60612, USA.
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D000409:Alanine / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002850:Chromatography, Gel; D004789:Enzyme Activation; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000502:physiology*; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D054645:Protein Phosphatase 1; D016415:Sequence Alignment; D017384:Sequence Deletion; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D015852:Eukaryotic Initiation Factor-2; D046911:Macromolecular Substances; D014764:Viral Proteins; C066469:gamma 34.5 protein, Human herpesvirus 1; D001120:Arginine; C019363:eIF-2 phosphatase; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D000409:Alanine