The γ134.5 Protein of Herpes Simplex Virus 1 Has the Structural and Functional Attributes of a Protein Phosphatase 1 Regulatory Subunit and Is Present in a High Molecular Weight Complex with the Enzyme in Infected Cells

Abstract

The carboxyl-terminal domain of the ␥ 1 34.5 protein of the herpes simplex virus 1 binds to protein phosphatase 1␣ (PP1) and is required to prevent the shut-off of protein synthesis resulting from phosphorylation of the ␣ subunit of eIF-2 by the double-stranded RNA-activated protein kinase. The corresponding domain of the conserved GADD34 protein homologous to ␥ 1 34.5 functionally substitutes for ␥ 1 34.5. This report shows that ␥ 1 34.5 and PP1 form a complex in the infected cells, that fractions containing this complex specifically dephosphorylate eIF-2␣, and that both ␥ 1 34.5 and GADD34 proteins contain the amino acid sequence motif common to subunits of PP1 that is required for binding to the PP1 catalytic subunit. An oligopeptide containing this motif competes with ␥ 1 34.5 for binding to PP1. Substitution of Val 193 and Phe 195 in the PP1-binding motif abolished activity. These results suggest that the carboxyl-terminal domain of ␥ 1 34.5 protein has the structural and functional attributes of a subunit of PP1 specific for eIF-2␣, that it has evolved to preclude shut-off of protein synthesis, and that GADD34 may have a similar function.The ␥ 1 34.5 gene of herpes simplex virus 1 (HSV-1) 1 encodes two functions. The first enables the virus to replicate in vivo and particularly to multiply and spread in the central nervous system of experimental animal systems (1,2). This function appears to map throughout the coding domain of the gene (3, 4). The second blocks the shut-off of protein synthesis resulting from phosphorylation of the ␣ subunit of the translation initiation factor eIF-2 by the double-stranded RNA-activated protein kinase (PKR). This function maps in the 3Ј-terminal domain of the 263-codon gene (5, 6). Earlier studies have shown that in HSV-1-infected cells PKR is activated but that in cells infected with wild-type virus or virus carrying in-frame deletions of the 5Ј-terminal coding domain of the gene eIF-2␣ was not phosphorylated (7). In subsequent studies (8) we have shown that the ␥ 1 34.5 protein interacts with the protein phosphatase 1␣ (PP1). Indeed, infected cells contain a phosphatase activity that specifically dephosphorylates eIF-2␣ at a rate 3000-fold greater than that measured in uninfected cells. This phosphatase activity is inhibited by inhibitors of PP1. The hypothesis that emerged from these studies is that transcription of complementary sequences of the HSV-1 DNA results in the formation of double-stranded RNA, that PKR is activated in cells infected with both wild-type and mutant viruses, and that a domain of the ␥ 1 34.5 protein binds PP1 and redirects its activity to dephosphorylate eIF-2␣. This report centers on one aspect of this hypothesis: we show that in infected cells ␥ 1 34.5 protein is a component of a multi-protein, cytoplasmic complex containing PP1 and that the interacting domain of the ␥ 1 34.5 protein is near the carboxyl terminus of the protein and has an amino acid motif shared with accessory proteins or subunits interacting with the catalytic subunit of PP1...

The Direct Binding of the Catalytic Subunit of Protein Phosphatase 1 to the PKR Protein Kinase Is Necessary but Not Sufficient for Inactivation and Disruption of Enzyme Dimer Formation

“…A similar effect was observed using the positive control proteins P53 and SV40 T Ag (45). The specificity of PKR-PP1 C interaction was further demonstrated by the observation that the p53 protein did not bind PKR in this system, nor did we detect an interaction between PP1 C and eIF2␣, which is consistent with published observations (44,46). It is interesting to note that the catalytic subunit of PP2A (PP2A C ), the other major protein phosphatase, interacted with PKR very weakly in light of recent findings that PKR interacts with the regulatory subunit of PP2A (47).…”

“…Most of these bind to a small hydrophobic groove on the surface of PP1 C through a short conserved binding motif-the (R/K)(V/I/L)X(F/W/Y) motif, which is often preceded by further basic residues, although several putative targeting subunits do not possess an (R/K)(V/I/L)X(F/ W/Y) motif but nevertheless interact with the same region of PP1 C . In this regard, the herpes simplex virus type 1 (HSV-1)-encoded ␥1 34.5 protein contains such a motif, which interacts with PP1 C to redirect the phosphatase to dephosphorylate eIF2␣ (44,46). Selective dephosphorylation of eIF2␣ may be a clever strategy used by HSV-1 to circumvent the PKR-induced shut-off of protein synthesis while maintaining PKR activity for other biological functions that are essential to the virus life cycle.…”

The Human SNF5/INI1 Protein Facilitates the Function of the Growth Arrest and DNA Damage-inducible Protein (GADD34) and Modulates GADD34-bound Protein Phosphatase-1 Activity

“…ICP34.5 coexists in a high molecular weight complex with PP1 in cellular extracts derived from HSV1-infected HeLa cells (10). Mutations in either ICP34.5 or GADD34 which disrupt their binding to PP1 also impair PP1 activity and the protein malfolding response (4,12).…”

“…Recently both viral ICP34.5 and mammalian GADD34 proteins have been shown to regulate the activity of protein phosphatase 1 (PP1) in vitro (10,11). ICP34.5 coexists in a high molecular weight complex with PP1 in cellular extracts derived from HSV1-infected HeLa cells (10).…”

Viral activation of stress-regulated Rho-GTPase signaling pathway disrupts sites of mRNA degradation to influence cellular gene expression

“…56 When stress is resolved, the resumption of protein synthesis requires eIF2a dephosphorylation by a phosphatase complex comprised of a catalytic domain (protein phosphatase 1 [PP1]) and a regulatory domain (PPP1R15). 57 Two recent papers together indicate that G-actin associates with this complex and is required for efficient dephosphorylation of eIF2a. 57,58 Depletion of the G-actin pool with jasplakinolide causes PPP1R15A-PP1 complex destabilization, thereby extending the period of translation arrest.…”