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

Abstract

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.

FIG. 1

Amino acid sequence alignment of the carboxyl-terminal domains of the γ₁34.5 proteins of HSV-1 (10) and HSV-2 (32) and of GADD34 proteins of human (25), mouse (30), and hamster (41).

FIG. 2

Replication of wild-type HSV-1(F) and the γ₁34.5 mutants R3616 and H9813 in SK-N-SH cells. Confluent monolayers of cells were infected with viruses at 0.01 PFU per cell and incubated at 37°C. At various times postinfection, the cells were harvested, freeze-thawed three times, and titrated on Vero cells. Duplicate samples were analyzed in parallel at each time point, and the data represent assays from three experiments.

FIG. 3

(A) Expression of the γ₁34.5 protein. HeLa cells were mock infected or infected with HSV-1(F), R3616 (in which the coding region of the γ₁34.5 gene was deleted), or H9813 (in which Vla¹⁹³Glu and Phe¹⁹⁵Leu substitutions were made in the γ₁34.5 gene) at 10 PFU per cell). At 15 h postinfection, cells were harvested, washed with phosphate-buffered saline, and resuspended in disruption buffer containing 50 mM Tris-HCl (pH 7.0), 5% 2-mercaptoethanol, 2% SDS, and 2.75% sucrose. Samples were then electrophoretically separated on denaturing 12% polyacrylamide gels and transferred to a nitrocellulose membrane. The blot was probed with anti-γ₁34.5 antibody (1). (B) eIF-2α phosphatase activity in HeLa cells which were mock infected or infected with the indicated viruses. ³²P-labeled eIF-2α, prepared as described in Materials and Methods, was incubated with lysates of HeLa cells which were mock infected or infected with indicated viruses at 34°C. After incubation for 2 min, the reaction was stopped by the addition of disruption buffer, and samples were separated electrophoretically on a denaturing 12% polyacrylamide gel, transferred to a nitrocellulose membrane, and subjected to autoradiography (21). Lanes 1, ³²P-labeled eIF-2α and GST-PKR not reacted with cell lysates; lanes 2 to 5, ³²P-labeled eIF-2α reacted with lysates of cells which were mock infected or infected with the indicated viruses. (C) Immunoblot of the autoradiogram in panel B.

FIG. 4

(A) eIF-2α phosphatase activity in Sf9 cells which were mock infected or infected with recombinant baculoviruses. Sf9 cells (10⁷ cells) were either mock infected or infected at 5 PFU per cell with GF9909, which expresses the wild-type γ₁34.5 protein, GF2003, which expresses the mutant γ₁34.5 protein with Val¹⁹³Glu and Phe¹⁹⁵Leu substitutions, GF9910, which expresses PP1, or GF9911, which expresses glucuronidase. At 48 h postinfection, cells were harvested and lysates were prepared as described in Materials and Methods. Aliquots of the lysates were then reacted with ³²P-labeled eIF-2 at 34°C. After a 2-min incubation, the reaction was stopped by adding disruption buffer, and samples were processed as described for Fig. 3B. Lane 6, ³²P-labeled eIF-2α and GST-PKR not reacted with cell lysates; lanes 1 to 5, ³²P-labeled eIF-2α and GST-PKR reacted with lysates of cells which were mock infected or infected with the indicated recombinant baculoviruses. (B) Immunoblot of the nitrocellulose membrane in panel A.

FIG. 5

(A) Schematic diagram of recombinant baculoviruses expressing wild-type and mutant forms of the γ₁34.5 protein. Line 1, domain structure of the wild-type γ₁34.5 protein. (ATP)₁₀ represents the triplet repeats of AlaThrPro, which connects the amino-terminal domain and the carboxyl-terminal domain. Numbers on the top denote the amino acid positions. Line 2, wild-type γ₁34.5 protein. Line 3, mutant γ₁34.5 protein with Val¹⁹³Glu and Phe¹⁹⁵Leu substitutions. Vertical lines indicate the positions of substitutions. Lines 4 to 8, deletion mutants expressing different-length segments of the γ₁34.5 protein. Numbers on the top of each line indicate the first and last amino acids contained in each construct. (B) Expression of wild-type and mutants of the γ₁34.5 protein. Cell lysates of Sf9 cells which were mock infected or infected with the indicated viruses were subjected to electrophoresis on a denaturing 12% polyacrylamide gel, transferred to a nitrocellulose membrane, and reacted with anti-His tag antibody (Qiagen Inc).

FIG. 6

(A) Activity of the γ₁34.5 mutants in Sf9 cells. Sf9 cells (10⁷ cells) were mock infected (lane 2) or infected with the indicated recombinant baculoviruses (lanes 3 to 9) at 5 PFU per cells at 27°C. At 48 h after infection, cells were harvested and lysates were prepared as described in Materials and Methods. Aliquots of each lysate were then incubated with ³²P-labeled eIF-2α, and samples were processed for autoradiography as described for Fig. 3B. (B) Quantitation of the phosphorylated eIF-2α. Phosphorylated eIF-2α in each lane in panel A was quantitated after eIF-2α phosphatase assays with a PhosphorImage SI system (ImageQuant software). The numbers indicate the percentages of phosphorylated eIF-2α remaining after incubation with the cell lysates relative to that of unreacted eIF-2α.

FIG. 7

Interaction of PP1 with the γ₁34.5 mutants. Sf9 cells infected with baculovirus expressing wild-type or mutant forms of the γ₁34.5 protein were harvested at 48 h postinfection and lysed in buffer containing 50 mM HEPES (pH 7.6), 150 mM NaCl, 10 mM MgCl₂, and 1% Triton X-100. After 30 min on ice, the lysates were precleared with GST beads and then incubated with GST-PP1 bound to beads at 4°C overnight. After washing three times, the protein complexes were solubilized in disruption buffer, electrophoretically separated on denaturing 12% polyacrylamide gel, and transferred to a nitrocellulose sheet. The blot was probed with anti-His tag antibody (Qiagen Inc).

FIG. 8

(A) Superdex 200 gel filtration analysis of cytoplasmic extracts from Sf9 cells infected with GF9909, expressing the wild-type γ₁34.5 protein. Sf9 cells were infected with 5 PFU of GF9909 per cell at 27°C. At 48 h postinfection, cells were harvested and lysates were prepared and assayed for eIF-2α phosphatase activity as described in Materials and Methods. The dashed line represents ³²P-labeled eIF-2α remaining after incubation with aliquots of indicated fractions, measured as described in Materials and Methods. The size of the eIF-2α phosphatase complex (340,000) was estimated with reference to the elution position of the following protein size markers: horse spleen apoferritin (465,000), aldolase (150,000), bovine serum albumin (69,000), ovalbumin (45,000), and rabbit reticulocyte thioredoxin (11,600). (B) Immunoblot of fractions from Superdex 200 column chromatography with anti-γ₁34.5 antibody. Lysates of Sf9 cells infected with GF9909 or GF2022 were chromatographed as described for panel A. Aliquots of fractions 16 to 27 from each were separated on a SDS–12% polyacrylamide gel, transferred to a nitrocellulose membrane, and reacted with anti-γ₁34.5 antibody as described in Materials and Methods.