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Journal of Virology, August 2006, p. 7354-7363, Vol. 80, No. 15
0022-538X/06/$08.00+0     doi:10.1128/JVI.00479-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Resistance of mRNA Translation to Acute Endoplasmic Reticulum Stress-Inducing Agents in Herpes Simplex Virus Type 1-Infected Cells Requires Multiple Virus-Encoded Functions

Matthew Mulvey, Carolina Arias, and Ian Mohr*

Department of Microbiology, New York University School of Medicine, and NYU Cancer Institute, New York, New York 10016

Received 7 March 2006/ Accepted 15 May 2006


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Via careful control of multiple kinases that inactivate the critical translation initiation factor eIF2 by phosphorylation of its alpha subunit, the cellular translation machinery can rapidly respond to a spectrum of environmental stresses, including viral infection. Indeed, virus replication produces a battery of stresses, such as endoplasmic reticulum (ER) stress resulting from misfolded proteins accumulating within the lumen of this organelle, which could potentially result in eIF2{alpha} phosphorylation and inhibit translation. While cellular translation is exquisitely sensitive to ER stress-inducing agents, protein synthesis in herpes simplex virus type 1 (HSV-1)-infected cells is notably resistant. Sustained translation in HSV-1-infected cells exposed to acute ER stress does not involve the interferon-induced, double-stranded RNA-responsive eIF2{alpha} kinase PKR, and it does not require either the PKR inhibitor encoded by the Us11 gene or the eIF2{alpha} phosphatase component specified by the {gamma}134.5 gene, the two viral functions known to regulate eIF2{alpha} phosphorylation. In addition, although ER stress potently induced the GADD34 cellular eIF2{alpha} phosphatase subunit in uninfected cells, it did not accumulate to detectable levels in HSV-1-infected cells under identical exposure conditions. Significantly, resistance of translation to the acute ER stress observed in infected cells requires HSV-1 gene expression. Whereas blocking entry into the true late phase of the viral developmental program does not abrogate ER stress-resistant translation, the presence of viral immediate-early proteins is sufficient to establish a state permissive of continued polypeptide synthesis in the presence of ER stress-inducing agents. Thus, one or more previously uncharacterized viral functions exist to counteract the accumulation of phosphorylated eIF2{alpha} in response to ER stress in HSV-1-infected cells.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Through careful monitoring of conditions within the lumen of the endoplasmic reticulum (ER), eukaryotic cells are able to detect the accumulation of misfolded polypeptides and activate the unfolded protein response (UPR). As a direct consequence of the UPR, translation is arrested through the activation of the ER resident type I membrane protein PERK, a kinase that phosphorylates eIF2 on its alpha subunit and consequently inactivates this critical translation initiation factor. Phosphorylation of eIF2{alpha} by activated PERK temporarily halts the cotranslational flow of proteins destined to be secreted into the ER, induces the production of cellular chaperones, and allows additional time for processing ER client proteins (reviewed in reference 13). Recovery from this stress-induced translational arrest involves GADD34, a cellular eIF2{alpha} phosphatase subunit (33). This quality control pathway operates in reaction to a diverse assortment of physiological stresses, including viral infection.

To complete their productive replication cycle and in turn propagate the infection to neighboring cells, viruses must produce the components necessary to assemble and release a new generation of progeny particles. In the case of many different viruses enveloped with cellular membranes studded with virus-encoded glycoproteins, this involves introducing an enormous burden of client proteins into the ER lumen over the course of the replicative cycle (20). Whereas some aspects of the UPR, such as the induction of chaperones, are conceivably beneficial and might aid viral replication by providing new resources for folding viral glycoproteins, the unabated inhibition of viral mRNA translation by activated PERK would be devastating, effectively obstructing the lytic growth program. Indeed, it has become clear that the biology of many enveloped viruses can trigger ER stress, which unchecked can result in apoptosis (7, 21, 31, 32, 36, 41, 43). Many viruses, however, are equipped to effectively counter components of the UPR.

Among the characterized functions capable of preventing ER stress-induced eIF2{alpha} phosphorylation in virus-infected cells, each also counteracts PKR, the interferon-induced eIF2{alpha} kinase activated by double-stranded RNA (dsRNA). The vaccinia virus K3L gene product and the HCV E2 protein are both pseudosubstrates capable of precluding the activated eIF2{alpha} kinases PKR and PERK from engaging the bona fide translation initiation factor substrate (34, 35, 42, 46). Influenza virus, however, appears to harness the activity of the cellular protein p58(IPK), normally found in an inactive complex with Hsp40 (24, 44). Once released from Hsp40, p58(IPK) can bind to both PKR and PERK and prevent their activation (51). Yet another set of strategies operates in herpesvirus-infected cells, as human cytomegalovirus has recently been shown to activate the UPR in a controlled manner, inducing a limited set of responses that foster viral replication but preventing the accumulation of phosphorylated eIF2{alpha} (17, 18). Finally, the {gamma}134.5 gene product encoded by herpes simplex virus type 1 (HSV-1) binds the cellular protein phosphatase 1{alpha} (PP1{alpha}), forming a holoenzyme capable of removing phosphate from eIF2 in response to a broad spectrum of eIF2{alpha} kinases, including PKR and PERK (2, 16). While all of these virus-encoded functions effectively counteract a broad spectrum of cellular eIF2{alpha} kinases, none of them appear to be dedicated UPR antagonists. This contrasts sharply with the multitude of virus-encoded functions that specifically antagonize the dsRNA-responsive eIF2{alpha} kinase PKR, a critical component of the cellular antiviral defense circuitry.

Despite its potential to neutralize a variety of eIF2{alpha} kinases by acting downstream of eIF2{alpha} phosphorylation, the {gamma}134.5 protein is not sufficient alone to adequately prevent eIF2{alpha} phosphorylation and protect HSV-1-infected cells from a panoply of host stress responses. In addition to the {gamma}134.5 gene product, the dsRNA binding protein encoded by Us11 is required to prevent PKR activation together with eIF2{alpha} phosphorylation, promote normal translation rates, and engender full resistance of virus-infected cells to type 1 interferon. Surprisingly, Us11 mutant viruses do not synthesize proteins at wild-type (WT) rates and are sensitive to alpha interferon even though they express WT levels of the {gamma}134.5 phosphatase component (29, 30). The requirement for Us11 to specifically antagonize PKR and maintain normal translation rates in HSV-1-infected cells implies that recruiting PP1{alpha} may not be sufficient to prevent the accumulation of phosphorylated eIF2{alpha} in HSV-1-infected cells. A kinase-specific antagonist such as Us11 produced during the late phase might serve to alleviate the load on the {gamma}134.5-PP1{alpha} holoenzyme. Our understanding of how both the Us11 and {gamma}134.5 gene products act to coordinately control eIF2{alpha} phosphorylation catalyzed by PKR raises the prospect that the ability of the {gamma}134.5-PP1{alpha} complex to adequately counter other eIF2{alpha} kinases might likewise be limited as well. Perhaps just as the Us11 protein specifically antagonizes the dsRNA-activated eIF2{alpha} kinase PKR and works in conjunction with the {gamma}134.5 protein to preserve supplies of active eIF2, heretofore-unidentified HSV-1 functions might target eIF2{alpha} kinases other than PKR to specifically prevent the accumulation of phosphorylated eIF2 in response to a diverse array of potential stresses. In support of this hypothesis, we now present evidence that mRNA translation in cells infected with an HSV-1 mutant deficient in both {gamma}134.5 and Us11, the only characterized regulators of eIF2{alpha} phosphorylation, remains resistant to a variety of treatments that induce acute ER stress. This phenotype does not appear to involve the induction of GADD34, a cellular gene induced in response to ER stress that promotes recovery from the UPR. Instead, it requires the expression of a viral gene or genes, defining a new HSV-1 function or functions capable of regulating eIF2{alpha} phosphorylation in response to ER stress-inducing agents.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and viruses. Vero cells were propagated in Dulbecco's modified Eagle's medium supplemented with 5% calf serum, 2 mM glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. PKR0/0 cells were grown in identical medium except that 10% fetal bovine serum was used in place of the calf serum.

The {Delta}34.5 (SPBg5e) and {Delta}34.5-(IE)Us11 (SUP1), {Delta}Us11, and {Delta}US11{Delta}34.5 viruses have been previously described (27, 30). The following viruses were generous gifts from the indicated investigators: vhs{Delta}Sma (38) was provided by G.S. Read (University of Missouri, Kansas City); R2621 (vhs:lacZ; see reference 37), R2622 ({Delta}34.5 vhs:lacZ; see reference 37), and R3616 ({Delta}34.5) were provided by B. Roizman (University of Chicago); and n12 ({Delta}ICP4; see reference 5) was provided by Neal DeLuca (University of Pittsburgh School of Medicine). Virus (1 x 107 PFU/ml of HSV-1) was inactivated by exposure of 2.5 ml in a 10-cm dish to UV light (150 mJ) in a Stratagene Stratalinker.

Antibodies and chemicals. Polyclonal antiserum directed against the {gamma}134.5 protein was described previously (30). The anti-eIF2{alpha} phospho-Ser51-specific antibody was purchased from either Research Genetics (catalog no. RG001) or StressGen. The mouse monoclonal antibody that detects both phosphorylated and unphosphorylated eIF2{alpha} was generated by the late Edward Henshaw and graciously provided by Michael Clemens (St. George's Hospital Medical School, London, United Kingdom). The following rabbit polyclonal antibodies were kind gifts from other laboratories: anti-GADD34 (provided by David Ron and Heather Harding, New York University School of Medicine), anti-ICP27 (provided by Saul Silverstein, Columbia University College of Physicians and Surgeons), and anti-TK (provided by Bernard Roizman, University of Chicago). Mouse monoclonal antibodies directed against the ICP4 (catalog no. 1114) and ICP0 proteins (catalog no. 1110) were purchased from Goodwin Institute. Dimethyl sulfoxide (DMSO), actinomycin D (ActD), thapsigargin (Tg), and tunicamycin were purchased from Sigma-Aldrich. Phosphonoacetic acid (PAA) was from Calbiochem.

35S Labeling of newly synthesized proteins following treatment with ER stress-inducing agents. Cells were infected with wild-type or mutant derivatives of HSV-1 at the indicated multiplicity of infection (MOI). At 12 h postinfection (p.i.), cultures were mock treated with either DMSO-containing medium or 1 µM Tg for 30 min and then immediately radiolabeled with [35S]cysteine-methionine in the presence of Tg for an additional 30 min as described previously (27). Alternatively, cells treated with 2.5 µg/ml tunicamycin (Tm) at 16.5 h p.i. for the indicated times and immediately radiolabeled in the continued presence of Tm for an additional 30 min. Total cellular protein was subsequently solubilized in 250 µl sample buffer (62.5 mM Tris-HCl [pH 6.8], 2% sodium dodecyl sulfate [SDS], 10% glycerol, 0.7 M ß-mercaptoethanol), boiled for 3 min, and fractionated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Labeled proteins were visualized by exposing the fixed, dried gel to X-ray film. Alternatively, where indicated, proteins were transferred to nitrocellulose following SDS-PAGE. Immunoblots were processed, incubated with primary antibody, and developed using ECL reagent according to the manufacturer's instructions (Amersham).

In some experiments, cells were pretreated for 1 h in medium containing ActD (5 µg/ml), DMSO (5 µl/ml), or PAA (300 µg/ml) and subsequently infected for 1.5 h in the continued presence of these agents. At the indicated times, infected cultures were exposed to Tg for 30 min in the continued presence of these reagents and subsequently radiolabeled for an additional 30 min as described above.

Cycloheximide block and actinomycin chase procedure. Cells were pretreated for 1 h in cycloheximide (CHX) (100 µg/ml) and subsequently mock infected or infected at a high MOI with the indicated virus for 1.5 h in the presence of CHX as originally described (6). After 1.5 h, the inoculum was removed and cells were refed with medium plus CHX. At 5 h p.i., the CHX was removed and the cultures were washed twice with medium containing either DMSO (5 µl per ml) or ActD (5 µg/ml). Following an additional 5 h incubation, cultures were either mock treated or treated with Tg for 30 min and immediately radiolabeled as described above.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
A novel HSV-1 function(s) prevents the inhibition of translation in response to ER stress-inducing agents. To evaluate whether HSV-1 indeed encodes functions that could prevent the inhibition of translation in response to ER stress, we examined the effects of adding compounds that induce ER stress on translation rates in HSV-1-infected cells. In the ER lumen, the folding of HSV-1 glycoproteins is thought to proceed through an association with the Ca2+-dependent chaperone calnexin (50). Tg causes the efflux of Ca2+ stores from the ER, promoting the accumulation of unfolded proteins within the ER lumen, as the ER chaperones calnexin and calreticulin require Ca2+ for their activity (47). Treatment of uninfected or mock-infected cells with Tg thereby activates the UPR, resulting in eIF2{alpha} phosphorylation and the inhibition of translation (Fig. 1). However, HSV-1-infected cells were relatively resistant to the effects of Tg treatment compared to uninfected cells (Fig. 1). As the HSV-1 {gamma}134.5 and Us11 gene products are both known to regulate translation by modulating eIF2{alpha} phosphorylation (29, 30), and the {gamma}134.5 protein by virtue of its association with PP1{alpha} could potentially reverse the actions of multiple eIF2{alpha} kinases (2, 16), we examined the Tg sensitivity of a panel of HSV-1 mutants deficient for either one or both of these functions. PKR-deficient cells were required in this experiment, as eIF2{alpha} phosphorylation irreversibly inhibits translation and viral replication in nonpermissive cells infected with an HSV-1 {Delta}34.5 mutant (4). Whereas the Us11 mutant ({Delta}Us11) expresses a functional {gamma}134.5 protein (30), a suppressor mutant ({Delta}34.5IEUs11) lacks the {gamma}134.5 gene and expresses Us11, normally produced late in the replicative cycle, as an immediate-early (IE) protein (27, 28). The double mutant {Delta}34.5-{Delta}Us11 is deficient in all currently known HSV-1 functions that can regulate eIF2{alpha} phosphorylation (30). Significantly, each of these mutant viruses retains the Tg resistance (TgR) phenotype (Fig. 1), demonstrating that the TgR determinant(s) is specified by a gene(s) other than Us11 or {gamma}134.5. Finally, as PKR-deficient cells exhibited the TgR phenotype, the eIF2{alpha} kinase PKR was therefore not required.


Figure 1
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  		FIG. 1. Translation in HSV-1-infected cells is resistant to ER stress. PKR–/– cells were mock infected or infected with either a {gamma}134.5-Us11 double mutant ({Delta}34.5{Delta}Us11), the suppressor mutant ({Delta}34.5IEUs11), a Us11 mutant ({Delta}Us11), or WT HSV-1 at an MOI of 5. At 12 h p.i., the indicated samples were treated with 1 µM Tg for 30 min and immediately subjected to a 30-min pulse with 35S-labeled amino acids. Total protein was subsequently isolated and fractionated by SDS-PAGE. The fixed, dried gel was exposed to X-ray film, and the migration of molecular mass standards (in kilodaltons) appears on the left.

 
To discount the possibility that the TgR phenotype results entirely from its effects on Ca2+ signaling, the sensitivity of viral translation to another compound that induces ER-stress by an entirely unrelated mechanism was examined. By inhibiting N-linked protein glycosylation within the ER, Tm interferes with glycoprotein processing and proper protein folding, allowing misfolded proteins to accumulate in the ER. Inhibiting N-linked glycosylation prevents the association of viral glycoprotein precursors (gB, gC, and gD) with the ER chaperone calnexin (50), interfering with protein folding and creating ER stress without altering Ca2+ concentration. Whereas translation in mock-infected cells is extremely sensitive to Tm treatment, translation in cells infected with either WT HSV-1 or a {gamma}134.5-deficient mutant is highly resistant to the presence of Tm. The effectiveness of the Tm treatment is evident in the altered mobility of many viral glycoproteins upon SDS-PAGE, consistent with the accumulation of the high-level mannose precursor forms (Fig. 2). Thus, while cellular translation is exquisitely sensitive to ER stress induced by Tg and Tm, translation in HSV-1-infected cells is highly resistant. Moreover, the resistance of viral translation to these ER stress-inducing agents does not require any of the known virus-encoded functions that regulate eIF2{alpha} phosphorylation.


Figure 2
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  		FIG. 2. Translation in HSV-1-infected cells is resistant to tunicamycin-induced ER stress. Vero cells were mock infected or infected (MOI = 5) with either {Delta}34.5 or WT HSV-1. At 7 h postinfection, the noted cultures (+) were treated with Tm for 0.5, 1.5, or 3 h and immediately radiolabeled with 35S-labeled amino acids for 30 min. Total protein was then isolated and fractionated by SDS-PAGE, and the fixed, dried gel was exposed to X-ray film. Molecular mass markers (in kilodaltons) appear to the left.

 
Resistance of HSV-1 to ER stress involves an uncharacterized mechanism to prevent the accumulation of phosphorylated eIF2{alpha}. ER stress induced by Tg treatment in uninfected cells inhibits translation due to the accumulation of phosphorylated eIF2{alpha} (14). As the {Delta}34.5-{Delta}Us11 double mutation does not encode either of the previously described HSV-1 functions capable of modulating eIF2{alpha} phosphorylation, the phosphorylation state of eIF2{alpha} was directly evaluated in Tg-treated cells following infection with HSV-1. PKR-deficient cells were once again utilized in this experiment to rule out the potential involvement of this dsRNA-dependent eIF2{alpha} kinase. While the total amount of eIF2{alpha} remained constant, a marked increase in the abundance of phosphorylated eIF2{alpha} was observed in mock-infected cells following Tg treatment (Fig. 3). Strikingly, the accumulation of phosphorylated eIF2{alpha} in cells infected with the {Delta}34.5-{Delta}Us11 double-mutant virus was markedly attenuated upon Tg treatment (Fig. 3). Thus, HSV-1 appears able to prevent the accumulation of phosphorylated eIF2{alpha} following treatment with the ER stress-inducing agent Tg and is thereby able to sustain translation. This mechanism does not involve inhibition of the dsRNA-dependent eIF2{alpha} kinase PKR or require the previously characterized products of the Us11 or {gamma}134.5 genes.


Figure 3
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  		FIG. 3. A function other than the Us11 or {gamma}134.5 gene product prevents eIF2{alpha} phosphorylation triggered by ER stress in HSV-1-infected cells. PKR–/– cells were mock infected or infected with {Delta}34.5{Delta}Us11 (MOI = 5). At 12 h postinfection, the indicated cultures were treated with Tg for 30 min. After an additional 30 min, total protein was isolated, fractionated by SDS-PAGE, and transferred to a membrane support. The bottom strip was probed with an antibody that detects total eIF2{alpha}, while the top strip was probed with antiserum that specifically recognizes eIF2{alpha} phosphorylated on Ser-51.

 
In uninfected cells, eIF2{alpha} phosphorylation promotes expression of GADD34, a component of the cellular eIF2{alpha} phosphatase and an important mediator of programmed recovery from translational repression (33). We next considered whether the accumulation of this cellular component played a role in the resistance of HSV-1 translation to ER stress-inducing agents. For example, induction of the GADD34 protein would target PP1{alpha} to phosphorylated eIF2{alpha}, thereby increasing the available supply of unphosphorylated, active eIF2 and allowing sustained translation in the presence of Tg. Indeed, it has been reported previously that the mRNA for GADD34 is induced transiently in HSV-1-infected cells, although the accumulation of the protein product was not investigated (2). While GADD34 is markedly induced in mock-infected cells after Tg treatment, it is not detectable in PKR–/– cells infected with a {gamma}134.5 deletion mutant at either 0.5 or 7.5 h following Tg treatment (Fig. 4). As translation rates in the preceding experiments were measured in the 30 min immediately after treatment with Tg, GADD34 induction cannot account for the Tg-resistant translation we observed in virus-infected cells. Taken together, these results suggest that HSV-1 encodes a previously uncharacterized function(s), distinct from the products of the {gamma}134.5 and Us11 genes, which mediates resistance to multiple ER stress-inducing compounds.


Figure 4
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  		FIG. 4. GADD34 is not induced in HSV-1-infected cells. PKR–/– cells were either mock infected or infected with {Delta}34.5 (MOI = 5). At 7 h postinfection, the indicated cultures were treated with Tg (+) or DMSO (–) for 30 min and incubated for an additional 0.5 or 7.5 h. Total protein was then fractionated by SDS-PAGE, and GADD34 was identified by immunoblotting. Molecular mass standards (in kilodaltons) appear on the left.

 
Resistance of HSV-1 to ER stress-inducing agents requires viral gene expression. We next sought to define the phase of the viral life cycle during which the TgR phenotype manifests itself. In particular, it was critical to learn how virus binding to the cell surface, virus entry into the cell, virion components, or, alternatively, expression of viral genes might contribute to the development of Tg resistance. To determine whether viral entry was sufficient for Tg resistance, cells were infected with a preparation of UV-inactivated {Delta}34.5 virus. UV inactivation allows virion entry and tegument deposition into the cytosol but eliminates expression of the viral genome. While cells infected with a {Delta}34.5 mutant virus were resistant to Tg treatment, cells infected with a UV-inactivated preparation of virus were unable to prevent the cessation of translation in response to Tg, suggesting that binding of HSV-1 to the cell surface and viral entry were not sufficient to confer resistance to ER stress (Fig. 5). However, as we were unable to rule out the possibility that UV irradiation had somehow nonspecifically damaged the virions, we followed up this observation with an experiment to evaluate whether viral gene expression was required to confer Tg resistance. When actinomycin D is used to prevent transcription, binding, entry, and tegument deposition would occur normally without any prior manipulation of the virus preparation. Importantly, these experiments required the use of vhs mutant strains, as our assay involved measuring translation rates in the absence of viral gene expression and vhs encodes an eIF4F-associated RNase (8, 10, 11). In strains with a wild-type vhs gene, release of the vhs polypeptide from the tegument into the cytosol upon viral entry results in the indiscriminate destruction of viral and cellular mRNA (9), making it impossible to assess the effects of Tg on translation in the absence of mRNA synthesis. A vhs null mutant in the strain F background (vhs:lacZ; kindly provided by B. Roizman) was sensitive to Tg only in the presence but not the absence of ActD (Fig. 6). Identical results were obtained with vhs {Delta}sma, an independent vhs mutant (kindly provided by G.S. Read) constructed using the KOS strain (data not shown). To rule out the possibility that Tg resistance observed in the absence of ActD was in any way due to the expression of the {gamma}134.5 gene product, we made use of a {gamma}134.5-vhs double mutant ({Delta}34.5 vhs:lacZ; generously provided by B. Roizman). This double mutant behaved similarly to the two vhs single-mutant viruses (Fig. 6). Thus, we conclude that the TgR phenotype is sensitive to actinomycin D and prior exposure of viral particles to UV irradiation. This suggests that the ability of HSV-1 to counter ER stress is not triggered by viral entry or encoded by a tegument function but requires gene expression. Furthermore, this analysis brings the total of HSV-1 strains we evaluated to three (Patton, KOS, and F), all of which have the inherent ability to prevent the cessation of protein synthesis in response to ER stress. This establishes that the TgR phenotype is in no way specific to a particular individual strain.


Figure 5
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  		FIG. 5. Translation in cells infected with UV-inactivated HSV-1 is sensitive to ER stress. PKR–/– cells were mock infected or infected (MOI = 5) with a {gamma}134.5 mutant stock that was either UV inactivated ({Delta}34.5UV) or untreated ({Delta}34.5). At 3 h postinfection, the indicated samples were treated with Tg for 30 min and immediately subjected to a 30-min pulse with 35S-labeled amino acids. Total protein was isolated and fractionated by SDS-PAGE, and the fixed, dried gel was exposed to X-ray film. Molecular mass standards (in kilodaltons) appear on the left.

 

Figure 6
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  		FIG. 6. Resistance to ER stress requires HSV-1 gene expression in infected cells. Vero cells were either mock infected or infected (MOI = 5) with a {gamma}134.5-vhs double mutant ({Delta}34.5 vhs:lacZ), a {gamma}134.5 mutant ({Delta}34.5), or a vhs mutant (vhs:lacZ) in the presence or absence of ActD. At 7.5 h postinfection, the designated cultures (+) were treated with Tg for 30 min and immediately radiolabeled with 35S-labeled amino acids for an additional 30 min. Total protein was isolated and fractionated by SDS-PAGE, and the fixed, dried gel was exposed to X-ray film. Molecular mass markers (in kilodaltons) are shown on the left.

 
Resistance of HSV-1 to ER stress-inducing agents does not require viral DNA synthesis. Having determined that viral gene expression is required to establish the TgR phenotype, the contribution of different kinetic classes of genes was next investigated. To further define when the TgR phenotype becomes evident in infected cells, Tg sensitivity was evaluated in the presence of PAA, an inhibitor of viral DNA replication. Blocking viral DNA synthesis prevents entry into the late phase of the productive cycle, inhibiting transcription of the viral true late {gamma}2 genes. While mock-infected cells remain Tg sensitive in the presence or absence of PAA, cells infected with a virus deficient for both Us11 and {gamma}134.5 genes remain TgR in the presence and absence of PAA (Fig. 7). The difference in the pattern of translated polypeptides in virus-infected cells reflects the fact that PAA-treated cells only express IE, E, and {gamma}1 proteins whereas untreated cells can produce {gamma}2 proteins. We conclude from these observations that the onset of the TgR phenotype requires viral gene expression but not viral DNA replication. Thus, the products of viral "true late" {gamma}2 genes are not absolutely required for detection of TgR protein synthesis in HSV-1-infected cells.


Figure 7
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  		FIG. 7. Expression of Tg resistance occurs prior to viral DNA synthesis. PKR–/– cells mock infected or infected (MOI = 5) with {Delta}34.5{Delta}Us11 were maintained in the presence or absence of PAA (300 µg/ml) and treated with Tg (1 µM) or DMSO for 30 min at 12 h postinfection. After being labeled with 35S-labeled amino acids for an additional 30 min, total protein was isolated and fractionated by SDS-PAGE. The fixed, dried gel was exposed to X-ray film. Molecular mass markers (in kilodaltons) are shown on the left.

 
Establishing the TgR state in HSV-1-infected cells occurs very early in the viral developmental program. To further delineate the onset of the TgR phenotype, (PKR–/–) cells were infected with an ICP4 mutant virus. In the absence of the ICP4 gene product, most viral early genes are not expressed and the replicative cycle arrests after the production of the IE polypeptides (5, 6). While incorporation of 35S-labeled amino acids was exquisitely sensitive to Tg in mock-infected cells, mRNA translation proceeded effectively in cells infected with an ICP4 null mutant at MOIs ranging from 5 to 25 (Fig. 8A). As the ICP4 mutant strain used in the experiment contained a WT {gamma}134.5 gene, the possibility that the {gamma}134.5 protein might accumulate under these conditions was considered, as this had never before been investigated. Indeed, analysis of total protein isolates by immunoblotting revealed the accumulation of a cross-reacting protein in an MOI-dependent manner (Fig. 8B). It is likely that this protein represents the {gamma}134.5 gene product, as it is not produced in a {gamma}134.5 null mutant strain. In complete agreement with earlier published studies, the ICP4 polypeptide was not detected in cells infected with the ICP4 null mutant, and the ICP0 protein was overproduced relative to a virus with an intact ICP4 gene (Fig. 8B). Significantly, the {gamma}134.5 protein is not the first non-IE protein reported to be produced in cells infected with an ICP4 mutant. Prior studies from multiple laboratories have clearly demonstrated that the product of the early ICP6 gene also accumulates in cells infected with ICP4 mutants (5, 6, 12). While the ICP6 gene is thought to directly respond to the ICP0 transactivator (12), detailed analysis of the delayed-early, leaky late {gamma}134.5 promoter in cells infected with ICP4 mutants has not been performed. The {gamma}134.5 gene product is reportedly also a virion component, and a spectrum of viral mRNAs packaged into the HSV-1 virion have the potential to be translated upon delivery to the cytosol (15, 40). However, infected cells treated with actinomycin D are not TgR, arguing that transcription is required to produce this phenotype. Regardless of the mechanism by which the {gamma}134.5 gene product accumulates in cells infected with an ICP4 mutant virus, an alternate strategy was required to evaluate whether IE gene expression played a role in establishing the TgR phenotype.


Figure 8
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  		FIG. 8. Cells infected with an ICP4-deficient virus exhibit the TgR phenotype but contain the {gamma}134.5 protein. (A) Vero cells were either mock infected or infected with the n12 ICP4 null mutant ({Delta}ICP4). At 7 h postinfection, Tg was added to the designated cultures (+) for 30 min followed immediately by labeling with 35S-labeled amino acids for 30 min. Total protein was then isolated and fractionated by SDS-PAGE. Molecular mass markers (in kilodaltons) are shown on the left. (B) Vero cells were infected as described for panel A. Cells infected with the {gamma}134.5 null mutant R3616 ({Delta}34.5) were also included in this analysis. Following Tg treatment of the designated cultures (+), total protein was isolated, fractionated by SDS-PAGE, transferred to a membrane support, cut into strips, and probed with antisera directed against ICP4, ICP27, or {gamma}134.5.

 
To investigate whether IE gene expression was important for establishing the TgR phenotype, the classic technique of infecting cells during a CHX block followed by their release into an ActD chase was enlisted (6). Infecting cells in the presence of CHX prevents the synthesis of new proteins but allows for the accumulation of IE mRNAs whose transcription is stimulated by tegument-derived VP16 acting in concert with host cell transcription factors. Washing out the CHX and releasing the infected cells into medium containing ActD attenuates further transcription, allowing translation of only those mRNAs allowed to accumulate during the CHX block. In addition to the IE mRNAs with VP16-responsive promoter elements, any mRNAs brought in as components of the infecting virion could conceivably be translated as well, provided of course that they are sufficiently stable. After allowing time for the mRNAs synthesized during the CHX block to be translated, PKR–/– cells were subsequently exposed to Tg, and viral translation rates were evaluated. Since our assay measures translation in the presence of ActD, an inhibitor of transcription, it was again necessary to execute this experiment in a vhs mutant background in order to prevent the complete destruction of the existing mRNA population. To completely rule out a role for the {gamma}134.5 gene product in this process, R2622, a {gamma}134.5 null vhs double mutant, was used. The effectiveness of the CHX block-ActD chase protocol in confining gene expression to the IE phase of the viral life cycle was evaluated by immunoblotting (Fig. 9A). Whereas the IE gene products ICP4 and ICP27 accumulate in cells infected with either R2622 or its vhs-positive parent strain R3616, the TK early gene product is only observed when ActD is omitted from the chase medium. Thus, the CHX block-ActD chase protocol effectively allows for the production of IE gene products but not the production of the early TK gene product. In all cases the intensity of the immunoreactive bands is not affected by Tg treatment. Finally, the reduction in IE protein accumulation observed in ActD-treated R3616-infected cells compared to R2622-infected cells reflects the action of the functional vhs nuclease acting on viral mRNA in R3616-infected cells.


Figure 9
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  		FIG. 9. Establishment of the TgR phenotype coincides with the accumulation of viral immediate-early proteins. After a 1 h preincubation in CHX, PKR–/– cells were either mock infected (MOCK) or infected with either the {gamma}134.5-vhs double mutant R2622 ({Delta}34.5 vhs:lacZ) or the {gamma}134.5 deletion mutant R3616 ({Delta}34.5). During this time, viral immediate-early mRNAs accumulate without being translated (CHX block). At 5 h postinfection, the cycloheximide was washed out and replaced with media containing ActD for an additional 5 h to allow translation of the accumulated mRNA but prevent further transcription (ActD chase). Alternatively, DMSO-containing medium was applied after the cycloheximide was washed out as a control (DMSO chase). (A) Total protein was isolated, fractionated by SDS-PAGE, transferred to a membrane support, cut into strips, and probed with antisera against viral immediate-early (IE or {alpha}) antigens (ICP4 or ICP27) or a viral early (ß) antigen (thymidine kinase [tk]). (B) Tg was added to the designated cultures (+) for 30 min. After being labeled with 35S-labeled amino acids for 30 min, total protein was isolated and fractionated by SDS-PAGE. Molecular mass markers (in kilodaltons) are shown on the left.

 
To determine whether the limited set of viral proteins produced in R2622-infected cells subjected to the CHX block-ActD chase protocol was sufficient to establish the TgR phenotype, total radiolabeled protein was isolated and fractionated by SDS-PAGE. Whereas mock-infected cells subjected to the CHX block-ActD chase protocol remained Tg sensitive, R2622-infected cultures exhibited the TgR phenotype (Fig. 9B). R3616-infected cultures were sensitive to Tg during the ActD chase, as they accumulated significantly fewer IE polypeptides due to the activity of the Vhs protein. As R2622 contains a complete deletion of the {gamma}134.5 gene, the {gamma}134.5 gene product can in no way contribute to the TgR phenotype in this experiment. Thus, we conclude that the limited subset of gene products produced during the CHX block-ActD chase protocol is sufficient to at least initially establish the TgR state in infected cells. This is consistent with one or more of the viral IE gene products acting on their own or in concert with any of the virion components delivered into the infecting cell upon entry.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A critical integration point for a variety of signals, the phosphorylation state of eIF2{alpha} mediates the response of the cellular translational machinery to numerous environmental stresses. While much has been learned regarding viral tactics for countering the dsRNA-dependent eIF2{alpha} kinase PKR, an interferon-induced gene product important in antiviral defense (reviewed in reference 25), how the activity of the remaining three known eIF2{alpha} kinases, each of which responds to a discrete form of stress, might be controlled in infected cells remains relatively unexplored. One of these kinases is PERK, an ER membrane-spanning protein kinase activated by the accumulation of misfolded polypeptides within the lumen of this organelle. Although PERK is reportedly activated in HSV-1-infected cells, the viral {gamma}134.5 protein, a PP1{alpha}-regulatory subunit that directs the phosphatase catalytic activity to eIF2{alpha}, functions like the cellular GADD34 protein to prevent the accumulation of phosphorylated eIF2 in response to ER stress-inducing agents (2). In this report, we provide evidence that translation in HSV-1-infected cells is resistant to treatment with a variety of ER stress-inducing agents known to promote eIF2{alpha} phosphorylation; moreover, we demonstrate that this phenotype does not require the products of the Us11 and {gamma}134.5 genes, the only virus-encoded functions known to regulate eIF2{alpha} phosphorylation. As the resistance of translation in infected cells to ER stress-inducing agents requires viral gene expression, this study establishes the existence of one or more novel HSV-1-encoded functions that prevent the inhibition of translation in response to ER stress.

Many aspects of infection, including the countless ways by which viruses gain control over cellular functions, impose multiple, independent forms of stress on their eukaryotic host cells. Introduction of copious quantities of glycoproteins into the secretory pathway, replication on internal membranes, and assembly of enveloped virions are all aspects of viral replication that potentially challenge the capacity of the ER and may at times trigger the UPR (20, 45). Indeed, infection of cells with rotavirus (49), paramyxovirus SV5 (36, 48), or several flaviviruses (19, 43) stimulates synthesis of BiP, a chaperone induced by the cellular UPR. In addition, BiP is also induced in cells infected with neuroinvasive strains of HSV-1 (23), suggesting that HSV-1 infection triggers at least some aspects of the cellular UPR. While the arm of the UPR that extends the folding capacity of the organelle may be hugely beneficial to viral replication, attenuating translation is likely to pose a significant impediment. Some viruses, such as Japanese encephalitis virus, bovine viral diarrhea virus, and several pathogenic murine retroviruses, are highly sensitive to PERK-induced apoptosis (7, 19, 21, 31, 43). To bypass PERK-induced apoptosis, the HCV E2 glycoprotein doubles as a broad-spectrum eIF2{alpha} kinase pseudosubstrate, foiling the efforts of both PERK and PKR to inactivate eIF2. In other cases, by restricting vesicular stomatitis virus replication PERK blocks excessive virus-induced apoptosis (1). Larger DNA viruses may have even more elaborate strategies for manipulating the UPR. Human cytomegalovirus makes use of the beneficial aspects of the UPR, such as ATF4 induction, but prevents the undesirable events such as the accumulation of phosphorylated eIF2{alpha} by activated PERK (17, 18). While replication of African swine fever virus in the cytosol does not activate PERK, it actively prevents activation of CHOP, a proapoptotic transcription factor, in response to multiple stimuli (32). The vaccinia virus K3L gene encodes an eIF2{alpha} kinase pseudosubstrate, and the HSV-1 {gamma}134.5 gene product specifies an eIF2{alpha}-targeting subunit for the PP1{alpha} holoenzyme. Both K3L and {gamma}134.5 act downstream of activated eIF2{alpha} kinases and can counteract a variety of eIF2{alpha} kinases activated by diverse environmental stresses (2, 42). Strangely, vaccinia virus and HSV-1 also encode dedicated antagonists of the dsRNA-dependent protein kinase PKR (29, 30, 39). Surprisingly, even though it can oppose multiple eIF2{alpha} kinases, the {gamma}134.5 gene product is unable to completely counteract PKR and the effects of interferon in infected cells, highlighting the importance of upstream kinase-specific antagonists in regulating eIF2{alpha} phosphorylation (29, 30). The new HSV-1-specified function that prevents the inhibition of translation in response to ER stress-inducing agents described in this report could conceivably be an antagonist of PERK, the upstream eIF2{alpha} kinase. Such a PERK-specific antagonist might work in conjunction with the {gamma}134.5-eIF2{alpha} phosphatase subunit in much the same way as the PKR inhibitor encoded by the Us11 gene. Further work is clearly required to identify the gene or genes responsible for this activity and to characterize the mechanisms through which their protein products act to regulate eIF2{alpha} phosphorylation.

While viral gene expression is clearly required to produce the TgR phenotype, the number of genes required to produce this phenotype is not yet known. The onset of the TgR phenotype occurs during the IE phase of the viral life cycle and does not require true late gene expression. However, this does not necessarily mean that redundant gene products expressed during the early, {gamma}1 or {gamma}2 phases of the life cycle are not involved in this process and may in fact be critical to maintain the TgR phenotype late in the viral replicative program. Given that two distinct viral genes products act at discrete times in the viral developmental life cycle to properly control the accumulation of phosphorylated eIF2{alpha} in response to activated PKR and that both are required for full resistance to interferon treatment (29, 30), it is not at all unreasonable to suggest that a similar scenario may be involved in controlling PERK and the elaborate UPR.

Recently, it was reported that the GADD34 cellular mRNA was induced transiently in HSV-1-infected cells, suggesting that the GADD34 polypeptide might play a role in regulating the UPR in HSV-1-infected cells (2). Certainly, a variety of independent mechanisms operate to ensure that host mRNA translation is radically impaired in HSV-infected cells (reviewed in reference 26). Notably, a virus-specified RNase encoded by the vhs gene associates with eIF4F, resulting in enhanced turnover of most but not all host and viral mRNAs. Consistent with the well-documented mechanisms that repress host polypeptide synthesis, we are clearly unable to detect the accumulation of the GADD34 protein in HSV-1-infected cells, making it unlikely that the GADD34 polypeptide is involved in producing the TgR phenotype. Perhaps the transient induction of GADD34 mRNA is related to observations regarding the aberrant accumulation of normally repressed transcripts, such as embryonic alpha globin mRNA, in HSV-1-infected cells (3).

While the TgR gene or genes remain to be identified, it is worth considering a few potential mechanisms that might possibly contribute to the development of this phenotype. One potential strategy could conceivably reduce the load of new, incoming ER client proteins. As ER stress sensors such as PERK respond when the burden of client proteins exceeds the capacity of resident ER chaperones to properly fold them, restricting the abundance of ER clients is an important regulator of ER homeostasis. Thus, the potent HSV-1-induced host cell shutoff (reviewed in reference 26) resulting from the combined reduction in host mRNA transcription, inhibition of mRNA splicing, and accelerated mRNA turnover might severely limit the synthesis of new ER client proteins and thereby act to limit ER stress. Unfortunately, this process is complex, requiring the concerted action of multiple IE gene products together with tegument-provided activities. Certainly, another obvious potential target is the cellular eIF2{alpha} kinase PERK itself. A transmembrane ER resident protein, the luminal domain of PERK is normally held in an inactive state bound to the chaperone BiP (reviewed in reference 13). As unfolded or malfolded proteins accumulate in the ER, BiP is released from PERK, allowing PERK monomers to multimerize within the plane of the ER membrane. Dimerization allows the cytosolic catalytic domains of each subunit to phosphorylate its homodimeric partner, and this activated kinase subsequently phosphorylates eIF2{alpha}. Indeed, it has already been reported that PERK is activated in HSV-1-infected cells (2). Thus, one possibility is that the viral mediator of the TgR phenotype somehow prevents activated PERK from transducing its signal to eIF2{alpha}. Therefore, there may be a viral eIF2{alpha} pseudosubstrate involved in producing the TgR phenotype. One need not invoke the existence of a dedicated gene product to accomplish this task. On the contrary, given the multifunctional nature of viral proteins, it is likely that a small number of residues in a virus-encoded, secreted polypeptide would be sufficient to establish this type of inhibitory activity. Indeed, the HCV E2 glycoprotein has been adapted to incorporate such a module (34). HSV-1 encodes many more glycoproteins, some of which already have disparate immunomodulatory functions and serve as Fc receptors (22). Alternatively, a viral protein might physically associate with activated PERK and inhibit its eIF2{alpha} kinase activity. While it is likely that a pseudosubstrate would at least in part reside in the cytosol, as this is the location of the PERK catalytic domain, a virus-encoded PERK inhibitor could reside within the ER and perhaps modulate PERK activity through an association with the luminal domain of the kinase. Further studies will be required to first identify the viral gene product(s) involved in producing the TgR phenotype before embarking on an investigation of its mechanism of action. Irrespective of the mechanism involved, the HSV-1 TgR determinant(s) represents a new, previously uncharacterized function that controls eIF2{alpha} phosphorylation in response to ER stress-inducing agents, revealing yet another unexpected layer of complexity with respect to how the activity of eIF2 is regulated in HSV-1-infected cells.


   ACKNOWLEDGMENTS
 
We are most grateful to Neal DeLuca, Bernard Roizman, and Sully Read for their generous gifts of viral mutants and to Heather Harding along with David Ron for helpful discussions.

This work was supported by grants from the National Institutes of Health and the Irma T. Hirschl Charitable Trust to I.M.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, NYU School of Medicine, MSB214, 550 First Avenue, New York, NY 10016. Phone: (212) 263-0415. Fax: (212) 263-8276. E-mail: ian.mohr{at}med.nyu.edu. Back


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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Virology, August 2006, p. 7354-7363, Vol. 80, No. 15
0022-538X/06/$08.00+0     doi:10.1128/JVI.00479-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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