Herpes simplex virus 1 infection activates the endoplasmic reticulum resident kinase PERK and mediates eIF-2alpha dephosphorylation by the gamma(1)34.5 protein

Abstract

The gamma(1)34.5 protein of herpes simplex virus (HSV) plays a crucial role in virus infection. Although the double-stranded RNA-dependent protein kinase (PKR) is activated during HSV infection, the gamma(1)34.5 protein inhibits the activity of PKR by mediating dephosphorylation of the translation initiation factor eIF-2alpha. Here we show that HSV infection also induces phosphorylation of an endoplasmic reticulum (ER) resident kinase PERK, a hallmark of ER stress response. The virus-induced phosphorylation of PERK is blocked by cycloheximide but not by phosphonoacetic acid, suggesting that the accumulation of viral proteins in the ER is essential. Notably, the maximal phosphorylation of PERK is delayed in PKR+/+ cells compared to that seen in PKR-/- cells. Further analysis indicates that hyperphosphorylation of eIF-2alpha caused by HSV is greater in PKR+/+ cells than in PKR-/- cells. However, expression of the gamma(1)34.5 protein suppresses the ER stress response caused by virus, dithiothreitol, and thapsigargin as measured by global protein synthesis. Interestingly, the expression of GADD34 stimulated by HSV infection parallels the status of eIF-2alpha phosphorylation. Together, these observations suggest that regulation of eIF-2alpha phosphorylation by the gamma(1)34.5 protein is an efficient way to antagonize the inhibitory activity of PKR as well as PERK during productive infection.

FIG. 1.

HSV-1 infection activates PERK kinase. (A) HeLa cells or (B) mouse 10T1/2 cells were mock infected or infected with wild-type virus HSV-1(F) or the γ₁34.5 deletion mutant R3616 at 10 PFU per cell. At 24 h postinfection, cells were harvested and lysates were prepared. Samples were then electrophoretically separated on denaturing 12% polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were sequentially probed with antibodies against phosphorylated PERK (p-PERK), PERK, phosphorylated eIF2α (p-eIF-2α), and eIF-2α. The positions of the protein bands are indicated on the right.

FIG. 2.

(A) The effect of PAA on HSV-induced PERK activation. Monolayers of mouse NIH 3T3 cells were infected with the indicated viruses at 10 PFU per cell. At 1.5 h postinfection, the cells were either left untreated or treated with PAA (400 μg/ml). At 24 h postinfection, cells were harvested and processed for Western blot analysis with antibodies against phosphorylated PERK (p-PERK), PERK, phosphorylated eIF2α (p-eIF-2α), and eIF-2α in the same membrane. (B) The effect of PAA on gC expression. Cell lysates in panel A were processed for Western blot analysis with antibodies against gC, whose expression is dependent on viral DNA replication. (C) HSV-induced PERK activation requires viral protein synthesis. Assay conditions are similar to those described in panel A except the protein synthesis inhibitor cycloheximide (CHX; 50 mg/ml) instead of PAA was added to cells 30 min prior to virus infection and was present throughout infection. (D) The effect of cycloheximide on viral protein synthesis. Cell lysates in panel C were processed for Western blot analysis with antibodies against HSV antibodies. The positions of protein bands are indicated at right.

FIG. 3.

(A) Phosphorylation of PERK and eIF-2α in PKR^+/+ mouse embryonic fibroblasts. Cells were infected with wild-type virus HSV-1(F), the γ₁34.5 deletion mutant R3616, or H9813 in which Val¹⁹³ and Phe¹⁹⁵ were replaced by Glu and Leu, respectively, at 10 PFU per cell at 37°C. At different time points after infection, cells were harvested, lysed, and subjected to Western blot analysis with antibodies against phosphorylated PERK, phosphorylated eIF-2α, and eIF-2α. (B) Phosphorylation of PERK and eIF-2α in PKR^−/− mouse embryonic fibroblasts. Assays were carried out as described in panel A. The positions of protein bands are shown at right. (C) Quantitation of eIF-2α phosphorylation. Phosphorylated eIF-2α and total eIF-2α in panels A and B were quantitated by densitometry. Numbers were normalized to the total eIF-2α in each lane and expressed as eIF-2α phosphorylation relative to that in mock-infected cells at 3 h postinfection.

FIG. 4.

Growth properties of HSV-1(F), R3616, and H9813 in PKR^+/+ (A) and PKR^−/− (B) mouse embryo fibroblasts. Monolayers of cells were infected with indicated viruses at 0.05 PFU per cell at 37°C. At various times postinfection, cells were harvested and freeze-thawed three times, and virus yield was determined on Vero cells. Duplicate samples were analyzed in parallel at each time point.

FIG. 5.

The γ₁34.5 protein of HSV-1 alleviates the ER stress response mediated by DTT or TG. 293 HEK cells were transfected with either pGF9912 expressing the wild-type γ₁34.5 protein or pGF9913 expressing the γ₁34.5 mutant in which Val¹⁹³Glu and Phe¹⁹⁵Leu substitutions were made by Lipofectamine reagent, as suggested by the manufacturer (Invitrogen). Thirty-six hours after transfection, cells were treated with dimethyl sulfoxide, DTT (2 mM; Invitrogen), or TG (0.5 μM; Sigma) for 1 h. Cells were then labeled with [³⁵S]methionine (50 μCi/ml; ICN) in DMEM lacking methionine but supplemented with 5% fetal bovine serum for 15 min. Cell lysates were resolved on SDS-12% PAGE and subjected to ponceau S staining (A), autoradiography (B), and Western blot analysis with anti-γ₁34.5 antibody (C). (D) To measure eIF-2α phosphatase activity, cell lysates were prepared from the transfected cells and incubated with ³²P-labeled eIF-2 as described in Materials and Methods. The reaction mixtures were then separated by SDS-12% PAGE electrophoresis and subjected to autoradiography.

FIG. 6.

HSV-1 infection induces GADD34 expression. PKR^+/+ (A) and PKR^−/− (B) cells were mock infected or infected with indicated viruses at 10 PFU per cell. At 3, 7, and 24 h postinfection, cells were harvested, and total RNA from infected cells was extracted by using the RNeasy kit (QIAGEN Inc.). Equal amounts of RNA from each sample were then subjected to RT-PCR amplification of GADD34. As a control, the cellular 18s rRNA was included in the assay. The PCR products were separated on a 1.5% agarose gel, stained with ethidium bromide, and photographed with NucleoVision GelExpert system (Nucleotech Inc.).

FIG. 7.

A model depicting the role of the γ₁34.5 protein in inhibiting signaling through the integrated stress response pathway. HSV-1 infection triggers cellular responses involving PKR and PERK. The dsRNA produced by HSV early in infection activates PKR. In addition, viral proteins accumulated during infection activate PERK in the ER. Activation of PKR and PERK leads to eIF-2α phosphorylation and thereby shuts off protein synthesis. To block cellular responses, the γ₁34.5 protein recruits PP1, forming a complex that dephosphorylates eIF-2α. Phosphorylation of eIF-2α also activates the expression of GADD34, which serves as a feedback loop to mediate eIF-2α dephosphorylation by binding to PP1.