Herpes simplex virus 1 infection activates the endoplasmic reticulum resident kinase PERK and mediates eIF-2α dephosphorylation by the γ134.5 protein

Virus infection of mammalian cells elicits cellular responses that limit or inhibit viral replication. A well-characterized host response involves the double-stranded RNA (dsRNA)-dependent protein kinase (PKR), which is a critical player of the interferon system (16). During virus infection, PKR is activated to phosphorylate the α subunit of translation initiation factor eIF-2 (eIF-2α). Phosphorylation of eIF-2α increases its affinity for guanine nucleotide exchange factor eIF-2B, thus sequestering eIF-2B in an inactive complex with phosphorylated eIF-2 and GDP (13, 14). Consequently, eIF-2B is not available to catalyze nucleotide exchange on nonphosphorylated eIF-2, which leads to the inhibition of protein synthesis. As a countermeasure, viruses have evolved a variety of mechanisms to inhibit the PKR response. For example, adenovirus VAI RNA, influenza virus-induced cellular p58, and hepatitis C virus NS5A bind to and block the activation of PKR (16). Poliovirus degrades PKR, whereas vaccinia virus E3L serves as a decoy of dsRNA and thus prevents the activation of PKR (16, 32). Unlike other viruses, herpes simplex virus 1 (HSV-1) infection results in activation of PKR (7, 20). However, only in cells infected with γ134.5 null mutants is eIF-2α phosphorylated (7, 23). Therefore, replication of γ134.5 null mutants triggers translation inhibition (7, 9, 10). It has been documented that in wild-type virus-infected cells, the γ134.5 protein binds to protein phosphatase 1 (PP1), forming a high-molecular-weight complex that specifically dephosphorylates eIF-2α (5, 22, 23). This activity is linked to HSV resistance to interferon (4). Apparently, HSV has evolved a unique strategy to evade the antiviral action of PKR by targeting eIF-2α. As viral inhibition of PKR seems more economic before its activation, the question arises as to what biological advantage derives from the γ134.5 protein's acting on eIF-2α rather than on PKR directly. The γ134.5 protein encoded by HSV-1(F) consists of 263 amino acids with a large amino-terminal domain, a linker or swivel region containing repeats of three amino acids (Ala-Thr-Pro), and a carboxyl-terminal domain (11, 12). The carboxyl domain is required to recruit PP1 and block translation shutoff during HSV-1 infection (3, 10, 21). This portion of the γ134.5 protein is homologous to the corresponding domain of cellular GADD34, which is expressed under conditions of DNA damage, growth arrest, differentiation, and apoptosis (24, 26, 39). GADD34 facilitates apoptosis induced by ionizing radiation or methyl methanesulfonate, and this activity is negatively regulated by Src kinase Lyn (17, 24). In addition, GADD34 negatively modulates transforming growth factor β signaling (33). Importantly, GADD34 controls stress-induced translation inhibition as well as gene expression under stress conditions in the endoplasmic reticulum (ER) (27, 29, 30). In light of these observations, it is notable that the carboxyl terminus of GADD34 functionally substitutes for the corresponding domain of the γ134.5 protein in the context of the HSV-1 genome (21, 23). Thus, the conserved carboxyl-terminal domains represent a functional module that may perform a common function in cellular processes, such as ER stress response. The ER is a signal-transducing organelle continuously sensing intracellular changes triggered by diverse stimuli, which include heat shock, hypoxia, and virus infection. A key component is the ER resident kinase PERK (19, 34), which regulates the inhibition of translation initiation through phosphorylation of eIF-2α and the induction of genes encoding chaperones (18, 19, 27, 29, 30). These events are collectively defined as the integrated stress response (27). Moreover, PERK mediates ER stress-induced growth arrest (2). Obviously, viruses that use the ER as a part of their replication apparatus must cope with the ER stress response. Several RNA viruses have been reported to interfere with the ER stress response through different mechanisms, such as Japanese encephalitis virus, bovine viral diarrhea virus, and hepatitis C virus (25, 31, 36, 37). In this report, we show that HSV infection activates PERK that is dependent on viral protein production. Activation of PERK leads to eIF-2α phosphorylation in PKR+/+ and PKR−/− cells. However, in PKR+/+ cells the maximal PERK activation is delayed, and hyperphosphorylation of eIF-2α appears stronger with an early kinetics. Further, the γ134.5 protein blocks virus and dithiothreitol (DTT)- and thapsigargin (TG)-induced inhibition of translation involving PERK. Intriguingly, HSV-1-induced GADD34 expression parallels the status of eIF-2α phosphorylation. These data suggest that HSV infection triggers and then disarms cellular responses by the action of the γ134.5 protein in order to maximize viral replication.