Protein Synthesis and Endoplasmic Reticulum Stress Can Be Modulated by the Hepatitis C Virus Envelope Protein E2 through the Eukaryotic Initiation Factor 2{alpha} Kinase PERK

The hepatitis C virus envelope protein, E2, is an endoplasmicreticulum (ER)-bound protein that contains a region of sequencehomology with the double-stranded RNA-activated protein kinasePKR and its substrate, the eukaryotic translation initiationfactor 2 (eIF2). We previously reported that E2 modulates globaltranslation through inhibition of the interferon-induced antiviralprotein PKR through its PKR-eIF2 ${alpha}$ phosphorylation site homologydomain (PePHD). Here we show that the PKR-like ER-resident kinase(PERK) binds to and is also inhibited by E2. At low expressionlevels, E2 induced ER stress, but at high expression levels,and in vitro, E2 inhibited PERK kinase activity. Mammalian cellsthat stably express E2 were refractory to the translation-inhibitoryeffects of ER stress inducers, and E2 relieved general translationinhibition induced by PERK. The PePHD of E2 was required forthe rescue of translation that was inhibited by activated PERK,similar to our previous findings with PKR. Here we report theinhibition of a second eIF2 ${alpha}$ kinase by E2, and these resultsare consistent with a pseudosubstrate mechanism of inhibitionof eIF2 ${alpha}$ kinases. These findings may also explain how the viruspromotes persistent infection by overcoming the cellular ERstress response.

INTRODUCTION

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Introduction

Hepatitis C virus (HCV) is a major public health problem, withat least 2.7 million persons infected in the United States andmore than 170 million infected worldwide. Most HCV infectionsbecome persistent, which often leads to chronic liver disease,including cirrhosis and hepatocellular carcinoma. Viral persistencemay be partially attributable to the ability of the virus toevade the host immune responses. Viral variants that escapeinnate or acquired immunity are likely to also be importantin allowing the virus to establish persistence. For instance,many strains of HCV are resistant to alpha interferon (IFN)therapy and may also be resistant to endogenous IFN. Severalmechanisms for IFN resistance have been proposed, includinginteractions of both the E2 envelope protein and NS5A with theIFN-induced double-stranded RNA-activated protein kinase PKR(7, 23). HCV E2 protein is one of two envelope glycoproteinsand contains a region of sequence homology with a region ofPKR that contains autophosphorylation sites that affect theactivation of the kinase (23, 24). Adjacent to this region ofPKR homology lies a sequence identical to the phosphorylationsite in the alpha subunit of the eukaryotic translation initiationfactor 2 (eIF2 ${alpha}$ ) targeted by PKR for translational inhibitionduring virus infection. This region in E2, termed the PKR-eIF2 ${alpha}$ phosphorylation site homology domain (PePHD), is required forinhibition of PKR by E2 (23). HCV E2 containing PePHD sequencesfrom IFN-resistant strains of virus inhibits PKR, confirmingthat these sequences are important for the interaction of E2with PKR.

The presence of the PePHD in the E2 protein suggests that E2may serve as a substrate or pseudosubstrate for PKR. Therefore,we set out to examine the possible effects of E2 on the activityof another eIF2 ${alpha}$ kinase. The PKR-like endoplasmic-reticulum (ER)-residentkinase (PERK) (8), also known as pancreatic eIF2 kinase (19),is a part of the host cell antiviral response (12). PERK maybe involved in the down-regulation of protein synthesis duringviral infection due to ER stress because the ER is overloadedwith proteins (17). ER stress is characterized by cellular responsesthat attempt to reduce the accumulation of aggregated and misfoldedproteins. Signal transduction that results in the upregulationof chaperone gene expression and an arrest in protein synthesisare two features that help the cell cope with ER stress.

PERK is a type I transmembrane protein that attenuates proteinsynthesis during ER stress by phosphorylating serine 51 on thealpha subunit of the heterotrimeric translation initiation factoreIF2. A ternary complex of eIF2, methionyl-tRNA, and GTP recruitsthe 40S ribosome to the mRNA and is required for general translationinitiation. Phosphorylated eIF2 entraps the guanine nucleotideexchange factor, eIF2B, thus preventing subsequent rounds oftranslation initiation (10). In this way PERK acts to limitprotein synthesis during ER stress events, such as virus infection.

Although the kinase domain of PERK is cytoplasmic, the lumenalportion contains the regulatory domain, comprised of an oligomerizationdomain and a chaperone-binding domain (15). PERK is held inan inactive, monomeric state by association with the chaperoneGRP78 (BiP) and GRP94, which are released upon ER stress (1,15). Release of BiP enables oligomerization, autophosphorylation,and activation of PERK, resulting in attenuation of translation(1). ER stress can be induced by misfolded E2, which binds toBiP, thus activating both grp78 and grp94 promoters (14), resultingin higher levels of BiP. BiP is thought to entrap stable HCVglycoprotein aggregates, and this complex is involved in a nonproductivefolding pathway (2). Because overexpression of BiP leads tothe inactivation of PERK, the two proteins are presumably relatedthrough a negative-feedback mechanism involving competitivebinding (1). Here we show that E2 can serve as a pseudosubstrateinhibitor of PERK and a potential viral regulator of the ERstress response.

MATERIALS AND METHODS

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Materials and Methods

Cells and transfections. Human embryonic kidney (HEK 293) cells were maintained in Dulbecco'smodified Eagle medium (DMEM) supplemented with fetal bovineserum (10%). Transfections were performed as described previously(22). Stable cell lines were constructed by transfecting pTreor pTre-E2 into a HeLa tetracycline (TET)-off (TET removed)cell line (Clontech) selected in DMEM with 100 mg of G418 (Invitrogen)/ml,0.4 mg of hygromycin (Gibco-Invitrogen)/ml, and 1 µg ofTET/ml. Stable cell lines were measured for growth by seeding1.4 x 10⁵ cells per 6-cm dish. At 24-h intervals, cells weretrypsinized and counted (Beckman Coulter Inc.). Each valuesis expressed as the mean of three samples. E2 expression wasinduced by the removal of TET. Cells were treated with castanospermine(CST) at a concentration of 1 or 2 mM for 2 h or thapsigargin(Tg) at 1 µM or tunicamycin (Tu) at 2.5 µg/ml for3 h at 37°C. Cells were metabolically labeled by depletionin DMEM minus methionine for 30 min and addition of [³⁵S]methionine(30 µCi/ml) for an additional 30 min during CST treatment.

Plasmids and mutagenesis. The E2 gene was cloned by PCR from the HCV genotype 1a cDNA(HCV-1; E2L-pcDNA3) or HCV genotype 1b (pcDNAEF-E2) and includedthe signal leader peptide as described previously (18, 23).The eIF2 ${alpha}$ homology region in the PePHD was mutated in E2L-pcDNA3by the PCR fusion technique as described previously (23) togenerate the following changes: nucleotides 2319 to 2330 (Ser-Glu-Leu-Ser)were changed to Gly-Gln-Gln-His, generating the mutant E2-clu1.E2- ${Delta}$ C was constructed by deletion of nucleotides 1910 to 2526in E2L-pcDNA3 as described previously (23). The constructionof GST-E2, E2-flag, and hnRNPK has been described by Tayloret al. (23). GST-PERK consists of the kinase domain of PERKfused to glutathione S-transferase (GST). The construction ofthe GST-PERK, PERK-c-myc-pcDNA1, and PERK K/A plasmids has beendescribed by Harding et al. (8). To create pTre-E2, a 900-bpEcoRI fragment containing the E2 coding sequence was isolatedfrom pE2 (23) and ligated into the EcoRI polylinker site ofplasmid pTre (Clontech).

Binding assay. The expression of GST fusion proteins was induced by isopropyl-ß-D-thiogalactopyranosidein BL21(DE3) bacterial cells. Proteins were purified from sonicatedlysates by incubation with glutathione-coated beads at 4°Cfor 2 h. The beads were washed with phosphate-buffered salinecontaining 0.3% NP-40. The in vitro-translated, ³⁵S-labeledE2 or hnRNP K proteins were then incubated with the beads for2 h at 4°C in binding buffer (0.04 M HEPES-K [pH 7.5], 0.1M KCl, 0.1% NP-40, 0.02 M ß-mercaptoethanol). Thebeads were washed five times with binding buffer at 0.3% NP-40.Radiolabeled, bound proteins were eluted with sodium dodecylsulfate (SDS) sample buffer and boiled before SDS-polyacrylamidegel electrophoresis (PAGE) analysis.

Immunoprecipitation. HEK 293 cells were transfected with plasmids containing a wild-typePERK-pcDNA1 (8) and/or E2 (E2L-pcDNA3 or pcDNAEF-E2) by thecalcium phosphate method as described previously (22) or withFugene 6 (Roche) as per the manufacturer's specifications. c-Myc-taggedPERK was immunoprecipitated from cytoplasmic extracts with monoclonalanti-c-Myc antibody (Zymed) and protein A Sepharose as describedpreviously (22).

Western blotting. HeLa cells were stably transfected with pTRE, pTre-E2, or pTre-E2 ${Delta}$ Cand treated with Tg or Tu as appropriate. Cell lysates wereresolved on 8 to 16% polyacrylamide-SDS gels. Proteins weretransferred to nitrocellulose by electrophoresis and probedwith anti-eIF2 ${alpha}$ or anti-phospho-eIF2 ${alpha}$ antibodies (Cell Signaling).

Kinase assay. GST and GST-E2 were purified from isopropyl-ß-D-thiogalactopyranoside-inducedBL21(DE3) cells with glutathione Sepharose 4B beads (PharmaciaBiotech). c-Myc-tagged PERK was immunoprecipitated with anti-c-Mycantibody bound to protein-A Sepharose beads, which were incubatedwith purified GST or GST-E2 protein for 30 min at 30°C.Kinase reactions were conducted by adding [ ${gamma}$ -³²P]ATP in kinasebuffer according to the method of Taylor et al. (22) followedby incubation for 20 min at 30°C. Histone H2a (3 µg;Sigma) or eIF2 ${alpha}$ (3 µg) was then added for another 15 minat 30°C.

CAT assay. Chloramphenicol acetyltransferase (CAT) enzyme activity wasmeasured from cytoplasmic fractions of transfected HEK 293 cellsas described previously (23).

RESULTS

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Results

E2 and PERK associate in vitro and in cells. To determine whether E2 protein interacts with PERK in vitro,we performed an in vitro binding assay with a purified fusionprotein expressed in Escherichia coli containing the C-terminal,kinase domain of PERK fused to GST. GST-PKR and GST proteinwere included as controls for specificity of binding (Fig. 1A).Radiolabeled unglycosylated E2 protein bound specifically towild-type GST-PKR and to GST-PERK in vitro, but not to GST alone.Radiolabeled, in vitro translated full-length PERK or hnRNPKwere incubated with GST or GST-E2 fusion proteins, bound toglutathione beads. Only PERK bound to GST-E2, suggesting thatPERK binds specifically to E2 in vitro (Fig. 1B). HEK 293 cellswere transfected with PERK-c-myc-pcDNA1, and lysates were incubatedwith GST or GST-E2 bound to glutathione Sepharose beads. Afterprobing with anti-c-Myc antibodies, PERK was detected in thesuspension that contained GST-E2 (Fig. 1C). In a coimmunoprecipitationassay (Fig. 1D), where Flag-tagged E2 and c-Myc-tagged PERKwere overexpressed in HEK 293 cells, PERK coimmunopurified withE2 anti-Flag antibodies (anti-Flag M2 beads; Sigma) bound toE2 protein. PERK was visualized by Western blot analysis withanti-c-Myc antibodies. Conversely, we used anti-c-Myc antibodiesto immunopurify c-Myc-tagged PERK and visualized ³⁵S-metabolicallylabeled, unglycosylated E2 (data not shown). These data suggestthat PERK and E2 interact in vitro and in cells, similar toour previous results demonstrating that the unglycosylated formof E2 coimmunoprecipitates with PKR (18).

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FIG. 1. E2 binds to PERK in vitro. (A) In vitro-translated, ³⁵S-labeled E2 was incubated with GST, GST-PKR, or GST-PERK fusion protein bound to glutathione beads. Bound proteins were separated by SDS-PAGE and visualized by autoradiography. (B) In vitro-translated, ³⁵S-labeled full-length PERK (P) or hnRNPK (h) was incubated with GST or GST-E2 fusion proteins bound to glutathione beads. Input represents 10% of the input volume of in vitro translation products used for the binding reactions. hnRNPK (small nuclear RNA binding protein K) was used as a negative binding control. (C) HEK 293 cells were transfected with PERK-c-Myc. The cell lysate was incubated with GST- or GST-E2 bound to glutathione beads. Beads were washed, and bound proteins were eluted and separated by SDS-PAGE. Western blot transfers were probed with anti-c-Myc antibodies, and proteins were visualized by chemiluminescence. (D) Coimmunoprecipitation assay. HEK 293 cells were transfected with PERK-c-Myc, E2-flag, or both PERK-c-Myc and E2-flag. Cell lysates were immunoprecipitated with anti-Flag M2 agarose beads (Sigma) for immunoprecipitation of E2 and separated by SDS-PAGE. Western blot transfers were probed with anti-c-Myc antibodies for detection of coimmunoprecipitated PERK, and proteins were visualized by chemiluminescence. Mouse heavy chain of the antibody used for immunoprecipitation is shown as a gel loading control.

E2 inhibits PERK kinase activity. To assess the effect of E2 on PERK kinase activity in vitro,we performed kinase assays with wild-type PERK that was immunopurifiedfrom PERK-transfected HEK 293 cells. The kinase assay was performedwith the immunoprecipitates in the presence of the purifiedGST or GST-E2 fusion proteins, using histone H2a as a substrate(Fig. 2A). Incubation with GST-E2, but not GST, inhibited bothautophosphorylation of PERK and histone phosphorylation. Catalyticallyinactive PERK (PERK K/A) was not significantly phosphorylated.While some phospho-PERK K/A can be seen, this may be due toself-association with endogenous wild-type PERK. H2A phosphorylationwas not observed in these assays, confirming that PERK K/A isnot active. Phosphorylation of eIF2 ${alpha}$ was also inhibited by GST-E2(Fig. 2B). The presence of GST-E2 (M_r= 66) did not result inthe appearance of any additional phosphorylated protein, indicatingthat GST-E2 was not phosphorylated by PERK in vitro. These resultssuggest that E2 serves as an inhibitor of PERK kinase activityin vitro.

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FIG. 2. E2 inhibits PERK kinase activity in vitro. (A) Protein kinase assay of immunoprecipitated PERK. PERK-c-Myc was transfected into HEK 293 cells, and at 48 h posttransfection cells were harvested, lysed, and immunoprecipitated with anti-c-Myc antibody. No added protein (0), GST (2.5 and 5 µg of protein), or GST-E2 (2 and 5 µg of protein) was incubated with the immunoprecipitates before the kinase reaction began. Histone H2a was added as a substrate after an initial incubation with [ ${gamma}$ -³²P]ATP. PERK K/A was used as a negative control for histone phosphorylation. PERK serves as an autophosphorylation substrate. (B) Immunoprecipitation kinase assay with transfected PERK-c-Myc and eIF2 ${alpha}$ used as a substrate.

E2 reverses PERK-mediated translational repression. Since E2 repressed PERK kinase activity in vitro, we predictedthat E2 would enhance translation in mammalian cells. CAT-expressingplasmids were cotransfected with PKR- or PERK-expressing plasmids,which resulted in reduced CAT activity (Fig. 3), suggestingthat translation was inhibited by overexpression of these kinases,which induces their autoactivation (24). Furthermore, the translationalinhibition by PERK was seen only when the catalytically activekinase was used and not when PERK K/A was used (Fig. 3B). Inreporter assays, exogenously expressed PERK was used to separatethe effects of E2 from those of endogenous PKR and PERK. Increasingamounts of E2, transfected into these cells, rescued the negativeeffects of PERK (Fig. 4A). The extent of translational enhancementby E2 correlated with the amount of transfected E2 plasmid (Fig.4A). We previously showed that enhanced CAT activity in thepresence of E2 was due to increased translation and not transcription(23). Low levels of transfected E2 caused an enhancement ofPERK's inhibitory effect (Fig. 4B), suggesting that inhibitionof translation by PERK was exacerbated when E2 was present atlow protein concentrations. At higher concentrations (e.g.,1:1 ratio of PERK to E2), translation was rescued (Fig. 4A).An E2 truncation mutant, which lacks most of the C terminusincluding the PePHD (E2- ${Delta}$ C), was not capable of enhancing translationeven at high concentrations (Fig. 4C). Furthermore, the E2 variantcontaining the PePHD sequence derived from the generally IFN-sensitiveHCV genotypes 2 and 3 (E2-clu1) (Fig. 4D) also showed a reducedability to enhance translation, and at low concentrations bothvariants exacerbated the inhibitory effects of PERK. Amountswere chosen to maximize the ratios, and the ratios can be usedto compare experiments. Absolute protein ratios were not analyzedin these experiments however. Taken together, these data suggestthat E2 induces an ER stress response at low expression levelsand exacerbates the negative effect on translation caused byoverexpression of PERK. E2 can also enhance translation of cellulargenes at high expression levels by inhibition of PERK throughinteraction with the PePHD region.

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FIG. 3. Overexpression of PERK inhibits protein synthesis in mammalian cells. (A) HEK 293 cells were transfected with equal amounts of total plasmid DNA using pcDNA3 as the carrier. Samples contained CAT-pcDNA3 (0.3 µg) and pcDNA3, PERK-pcDNA1 (0, 3, or 8 µg), or PKR-pcDNA3 (0, 5, or 10 µg). Cell lysates were assayed for CAT enzyme activity 48 h after transfection. Transfections were performed in triplicate, and the values were obtained from means of $>=$ 3 separate experiments each (± standard deviation). CAT activity is expressed in relative units, where the pcDNA3 control (0) was 100%. (B) HEK 293 cells were cotransfected with 0.3 µg of CAT-pcDNA3 and 0, 3, or 10 µg of PERK K/A.

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FIG. 4. (A) E2 enhances protein synthesis in mammalian cells. CAT enzyme activity from cells transfected with CAT-pcDNA3 (0.3 µg), PERK-c-Myc (8 µg), and increasing amounts of pcDNA3-E2 (0 to 8 µg) is shown. (B) Low levels of E2 exacerbate the negative effects of overexpressed PERK on translation. CAT activity in cells transfected with CAT-pcDNA3 (0.3 µg), PERK-c-Myc (0 to 8 µg), and E2 (0 to 3 µg) is shown. (C and D) Mutant E2 inhibits translation at low levels but does not stimulate translation at high levels. CAT activity from cells transfected with CAT-pcDNA3 (0.3 µg), PERK (0 to 4 µg), and E2- ${Delta}$ C (C) or E2-clu1 (D) (0 to 6.5 µg) is shown.

E2 confers resistance to ER stress. To study the effects of ER stress in the presence of E2, weestablished a stable HeLa cell line with inducible E2 expressiontargeted to the ER. E2 is glycosylated at the ER membrane (Fig.5A), and its increased gel mobility after endoglycosidase Htreatment (Fig. 5A), which removes carbohydrate residues, demonstratesthat E2 is being expressed. Little difference in cell growthwas observed between the different cell lines, although E2-expressingcells grew to larger numbers, suggesting a slight enhancementof cell growth (Fig. 5B). To examine the effect of E2 duringER stress, these cell lines were treated with CST, an inhibitorof ${alpha}$ -glucosidase (Fig. 5C). CST interferes with the ability ofcalnexin to associate with glycoproteins in the ER and resultsin the induction of ER stress and inhibition of protein synthesis.Calnexin binds specifically to E2 and plays a role in foldingof HCV glycoproteins (2). Cell lines expressing either E2 orthe E2- ${Delta}$ C mutant were refractory to CST treatment, demonstratedby increased incorporation of [³⁵S]methionine into proteins(Fig. 5C). This shows that E2 can evade the ER stress responseinduced by CST. The effect may be independent of the C-terminalportion of E2. To determine if E2 was evading the effects ofER stress specifically through inhibition of PERK, we analyzedeIF2 ${alpha}$ phosphorylation under ER stress conditions in the E2-expressingcell lines. Tg, which promotes the depletion of lumenal calciumstores, and Tu, which blocks N glycosylation, were used to induceER stress. In the presence of E2, decreased eIF2 ${alpha}$ phosphorylationwas observed in cells that were treated with Tg or Tu (Fig.5D, lanes 5 and 6). Interestingly, in the presence of TET, forcontrol cells and those that express E2 (lanes 1 and 4), eIF2 ${alpha}$ was phosphorylated in the absence of Tg and Tu, perhaps dueto the presence of the TET-off inducible plasmid or becausethe cells were stressed due to a high population density, whichmay result in PERK activation as well (P. R. Romano, unpublishedobservations). Taken together, these results demonstrate thatE2 can reverse ER stress-mediated eIF2 ${alpha}$ phosphorylation and theresulting inhibition of translation by inhibiting PERK.

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FIG. 5. E2-expressing cells are resistant to ER stress transducers. (A) E2-expressing cells were grown in the absence of TET (-) or in the absence of TET, followed by endoglycosidase H treatment (endo H), and in the presence of TET (+). Arrowheads denote full and partially glycosylated forms of E2; the open arrow denotes unglycosylated E2. Cell lysates were separated by SDS-PAGE and analyzed by Western blotting, probed with the monoclonal anti-E2 antibody H52 (3). (B) Cell growth of stable cell lines in the absence of TET. Cell lines are designated by ${diamondsuit}$ for pTre-E2, ${triangleup}$ for pTre-E2 ${Delta}$ C, and ${square}$ for pTre vector alone. (C) Cell lines grown in the absence of TET and treated with 0, 1, and 2 mM CST and metabolically labeled with [³⁵S]methionine. Cytoplasmic extracts were separated by SDS-PAGE. (D) Cells were grown in the presence of (lanes 1 and 4) or absence of TET and treated with Tg (lanes 2 and 5) or Tu (lanes 3 and 6). Lysates were separated by SDS-PAGE, analyzed by Western blotting, and probed with anti-eIF2 ${alpha}$ antibody or anti-phospho-eIF2 ${alpha}$ antibody.

DISCUSSION

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Discussion

These results combined suggest that the E2 protein of HCV genotype1 binds to and inhibits PERK in vitro and in mammalian cells.These effects require a region within the E2 protein which bearssequence homology to the eIF2 ${alpha}$ phosphorylation site, althoughwe cannot exclude the possibility that other sequences in E2may be important for its interaction with PERK. Taken together,these results suggest that in addition to providing the relativeresistance of HCV genotype 1 to IFN, E2 could provide the viruswith an ability to escape the negative translational effectsof ER stress.

E2 is localized to the perinuclear region of the cytoplasm andcontains a transmembrane domain in the last 29 amino acids ofits C terminus that retains E2 at the ER membrane (3). The signalpeptide leader sequence is also required for the proper folding,localization, and processing of E2 proteins. Interestingly,PKR also localizes to the perinuclear region of the cytoplasmand the nucleolar region (11), and both of these proteins colocalizein cells (23). This is consistent with the finding that PKRis closely associated with ribosomes through binding to theL18 protein (13), including most likely ER-bound ribosomes.PKR interacts with the abundant unglycosylated form of E2 onthe cytoplasmic side of the ER, and overexpression of PKR leadsto an accumulation of this unprocessed form of E2 (18). Therefore,E2 and PERK also likely interact with each other on the cytoplasmicside of the ER as the kinase domain of PERK is cytoplasmic.Because BiP is activated during E2 expression, the ER overloadresponse or unfolded-protein response are probably activated.Misfolding of E2 probably occurs (2), leading to an accumulationof unglycosylated E2 that gets retained in the cytoplasm. Thesesignals may be detected by BiP and mediated by the recognitionof hydrophobic residues in E2, which would be exposed in itsmisfolded state but concealed in its properly folded state.

Cells have the ability to detect foreign, nonhost molecules,such as viruses. PKR acts as an antiviral protein by detectingdouble-stranded RNA, produced during viral replication. PERK,however, senses misfolded or overexpressed proteins and possiblyadditional signals that induce ER stress (e.g., Ca²⁺ flux).Both kinases regulate translation through phosphorylation ofeIF2 ${alpha}$ . The ER overload response occurs during viral infectionwhere an abundance of viral glycoproteins and excessive membranetraffic result in ER stress (17). A cytopathic strain of bovineviral diarrhea virus, another member of the flaviviridae family,induces ER stress-mediated apoptosis (12). Consequently, persistentinfection with noncytopathic strains of bovine viral diarrheavirus may result from a viral strategy that allows it to evadethe ER stress response.

HCV E2 can induce ER stress, leading to the transactivationof BiP (14), which is activated during the unfolded-proteinresponse and mediates the folding of newly synthesized proteinsin the ER lumen (for a review, see reference 13). PERK is heldat the ER in an inactive, monomeric form by BiP and possiblyby GRP94 (1, 15) (Fig. 6A). During ER stress, including stressinduced by viral infection (12), the latent PERK-BiP heterodimerdissociates, whereby PERK oligomerizes and becomes active, resultingin phosphorylation of eIF2 ${alpha}$ and the inhibition of both cap-dependentand internal ribosome entry site (IRES)-dependent translationinitiation (Fig. 6A). Overexpression of PERK in mammalian cellscan also lead to autophosphorylation and activation of the kinase(8). In our model (Fig. 6B), HCV E2 is multiply glycosylatedand probably induces ER stress by protein overexpression, ERoverload, or underglycosylation and, thus, misfolding. At firstthis leads to ER stress, but as E2 is expressed, PERK bindsto and possibly recognizes E2 as a pseudosubstrate, and PERKthen may be sequestered by E2 from its substrate eIF2, allowingtranslation to proceed (Fig. 6B). Similarly, PERK is inhibitedthrough a pseudosubstrate mechanism by the vaccinia virus proteinK3L, which is also a PKR inhibitor (20). The finding that PERKactivity increases upon dissociation from BiP, and BiP activityincreases in the presence of E2, provides a basis for how E2can induce the ER stress response, thus allowing for properfolding of viral proteins while maintaining translation throughinhibition of at least two eIF2 ${alpha}$ kinases.

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FIG. 6. Model of ER stress and PERK activation and inhibition. (A) Classical ER stress induces the dissociation of the latent BiP-PERK heterodimer, leading to PERK oligomerization. PERK oligomerization results in activation of the kinase, which phosphorylates eIF2 ${alpha}$ , leading to the inhibition of translation. (B) E2 stimulates ER stress, leading to PERK oligomerization, but blocks the kinase through binding and prevents productive activation, preventing eIF2 ${alpha}$ phosphorylation, thus allowing translation to proceed. KD, kinase domain; LD, lumenal domain.

ER stress mediated by glucose deprivation stimulated translationfrom the cellular IRES of cat-1 mRNA (5). Although cat-1 translationincreased in a PERK/phospho-eIF2 ${alpha}$ -independent manner, BiP IRES-dependenttranslation was not stimulated by ER stress (6). Furthermore,expression of HCV subgenomic replicons that contain only nonstructuralgenes and lack the structural genes (e.g., E2) induces ER stressand stimulates HCV IRES activity in a PERK/BiP-independent pathway(21). In fact, lower levels of eIF2 ${alpha}$ phosphorylation correlateswith increased HCV IRES activity (21; D. R. Taylor, unpublishedresults) and BiP IRES activity (21). Thus, HCV may benefit fromER stress inducers that are unrelated to PERK.

It is further noted that loss of PERK in mice (9) and humans(4) resulted in the destruction of pancreatic beta cells, characterizedby insulin-dependent diabetes mellitus. HCV infection has beenassociated with diabetes mellitus (reference 16 and referencestherein), and it will be interesting to find out if the inhibitionof PERK by E2 plays a role in its etiology.

Our model (Fig. 6) is consistent with previous findings forHCV E2, PERK, and BiP and suggests that HCV E2 may help to circumventthe coordinated antiviral response that includes the ER stressresponse pathways in addition to the IFN-induced antiviral pathways.These findings suggest that E2 may play a role in HCV persistentinfection by overcoming the cellular ER stress response andmay point to new directions for the development of therapeuticsfor the treatment of HCV.

ACKNOWLEDGMENTS

We thank Heather Harding and David Ron for the PERK constructsand Glen Barber for purified eIF2 ${alpha}$ protein. The H52 antibodywas a gift from J. Dubuisson. We also thank Michael Lai forhelpful discussions in the early part of this work.

This work was supported by a fellowship to D.R.T. from the OakRidge Institute. N.P. is a fellow of the Howard Hughes MedicalInstitute.

FOOTNOTES

* Corresponding author. Mailing address: 8800 Rockville Pike, HFM-448, Bethesda, MD 20892. Phone: (301) 827-1878. Fax: (301) 496-1810. E-mail: taylord{at}cber.fda.gov.

Present address: INSERM U370, Faculte' de Medecine Necker, 75730Paris Cedex 15, France.

REFERENCES

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