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Hepatitis C virus envelope proteins regulate CHOP via induction of the unfolded protein response

Shiu-Wan Chan^*,,1 and Philip Anthony Egan^*

^* Faculty of Life Sciences, The University of Manchester, Manchester, UK; and
Manchester Royal Infirmary, Manchester, UK

¹Correspondence: Faculty of Life Sciences, Jackson’s Mill, The University of Manchester, P. O. Box 88, Sackville St., Manchester M60 1QD, UK. E-mail: shiu-wan.chan{at}manchester.ac.uk

SPECIFIC AIMS

It is widely believed that unfolded protein response (UPR) contributes to the pathogenesis of a number of human diseases. Despite growing evidence to suggest induction of the UPR in virus infections, the role of viral envelope proteins in the pathogenesis of diseases is only just emerging. Therefore, we investigated regulation of the UPR indicator CCAAT/enhancer-binding protein-homologous protein (CHOP) and engagement of the ER-associated degradation (ERAD) in response to expression of the hepatitis C virus (HCV) envelope proteins E1 and E2 because these proteins target and mature in the ER with a tendency to form malfolded aggregates and hence may play a role in the pathogenesis of HCV-related diseases.

PRINCIPAL FINDINGS

1. HCV envelope proteins activate CHOP promoter
Using a transient transfection system with HeLa cells and luciferase reporter gene driven by the full-length 954bp CHOP promoter, we found that expression of E1 and E2 caused significant activation of the CHOP promoter over a time course of 16, 48, and 72 h (Fig. 1 A). Induction of the CHOP promoter was maximum at 48 h posttransfection, concomitant with the kinetics of E1 and E2 expression during cotransfection and E2 expression on its own but displaying a delayed kinetics in relation to E1 expression on its own (Fig. 1B ). Similar findings were observed in a hepatocyte cell line HepG2. The specificity of CHOP activation by ER-targeting HCV envelope proteins was confirmed by 1) activation of the CHOP promoter by a very low level of E1 and E2; 2) the dose-dependent activation of the CHOP promoter; 3)the activating effect of HCV envelope protein on another UPR gene promoter BiP; 4) the inability of the HCV envelope proteins to activate a non-UPR gene promoter SV40; 5) the inability of ER-targeting green fluorescent protein to activate the CHOP promoter; and 6) the inability of cytosolic-targeting envelope proteins to activate the CHOP promoter. Induction of CHOP was also observed at the mRNA and protein levels.

View larger version (19K): [in this window] [in a new window]   Figure 1. E1 and/or E2 activate CHOP and BiP promoters. A) HeLa cells seeded in 12-well plates were transfected with 0.2 µg of CHOP or BiP promoter-luciferase reporter plasmid, 0.1 µg of internal normalization control pCMV-ßGal and 0.2 µg of one of the following: 0.2 µg empty vector (vector), 0.1 µg E1+0.1 µg empty vector (E1), 0.1 µg E2+0.1 µg empty vector (E2), or 0.1 µg E1+0.1 µg E2 (E1+E2). Transfection efficiencies were in the range of 30%. The cells were harvested at 16, 48, and 72 h posttransfection and assayed for luciferase and ß-galactosidase activities. The promoter levels as measured by the luciferase activities were normalized against ß-galactosidase activities and expressed as fold induction relative to the vector controls at each time points, which were set as 1. All values represent mean ± SEM of 3–6 independent experiments performed in triplicate. *Significance of difference. B) HeLa cells seeded in 6-well plates were transfected with one of the following: 0.75 µg E1+0.75 µg empty vector (E1), 0.75 µg E2+0.75 µg empty vector (E2), or 0.75 µg E1+0.75 µg E2 (E1+E2). Protein lysates were harvested at 16 h, 48 h, and 72 h posttransfection and used in immunoblots with antibodies specific for E2 (H52), E1 (Lau-E1-159), or ß-tubulin. The E2 proteins migrated as multiple species.

2. HCV E1 and/or E2 activate both ERSE and the ATF4 element in the CHOP promoter
Two responsive elements have been identified in the CHOP promoter, the ER stress element (ERSE) and the activating transcription factor 4 (ATF4) element. To assess the relative contribution of these responsive elements in CHOP regulation in the context of HCV protein expression, we employed deletion promoter constructs encompassing the ERSE and/or the ATF4 element. Deletion of both the ERSE and ATF4 element resulted in complete abolition of the CHOP promoter activities. Either the ERSE or the ATF4 element alone was sufficient for eliciting full promoter activity in response to HCV envelope protein expression.

3. PERK is essential for CHOP induction by HCV E1 and/or E2
Using mouse embryonic stem cells deficient in PKR-like ER-resident kinase (PERK) it has been shown that PERK is absolutely required for CHOP induction in response to common ER stress inducers tunicamycin, thapsigargin, and dithiothreitol (DTT). To evaluate the role of PERK in HCV envelope protein-activated UPR, we made use of mouse embryonic fibroblasts (MEFs) deficient in the ER stress kinase PERK. Significant elevation of the CHOP promoter activities by expression of the E1 and/or E2 proteins was observed in wild-type Perk+/+ MEFs whereas CHOP promoter activities were completely abolished in Perk–/– MEFs, suggesting that PERK is necessary and sufficient for CHOP activation by E1 and/or E2. Another eIF2 kinase, the mammalian GCN2, induces CHOP gene expression upon amino acid starvation. CHOP induction in response to E1- and/or E2-transfection was unimpaired in Gcn2–/– cells compared with that of the wild-type Gcn2+/+ cells, confirming that CHOP induction by E1 and/or E2 is specific to the ER stress kinase PERK.

4. HCV E1 and/or E2 activate the XBP1-ERAD pathway
In mammalian cells UPR is regulated not only by the ER stress kinase PERK but also by the transcription factors ATF6 and XBP1. Using RT-PCR and specific primers to distinguish between the unspliced (inactive) and spliced (active) forms of XBP1 transcripts we detected XBP1 splicing in HeLa cells at 16 h but not 48 h posttransfection with HCV E1 and/or E2 (Fig. 2 ). To further illustrate the significance of XBP1 splicing, we examined the effect of E1 and E2 expression on a downstream target of XBP1. The unfolded protein response element (UPRE) in the promoters of ERAD genes is a unique XBP1 binding site. Using a luciferase reporter gene driven by six copies of the UPRE very high levels of the UPRE promoter activity were detected in E1- and E2-expressing HeLa and HepG2 cells. Despite the low levels of XBP1 splicing in these cells, the magnitude of UPRE activation was similar to that induced by the potent UPR inducer tunicamycin, confirming the significance of XBP1 splicing in downstream signaling in E1- and E2-expressing cells.

View larger version (38K): [in this window] [in a new window]   Figure 2. E1 and/or E2 expression induces XBP1 splicing. A) Ethidium bromide-stained gels showing the unspliced (US) and spliced (S) XBP1 bands amplified from RT-PCR using specific primer pairs. The upper panels show two sets of control: 1) HeLa cells treated with the solvent control DMSO or 2 µg/mL tunicamycin (Tm) for 16 h, and 2) HeLa cells treated with 2 µg/mL tunicamycin (Tm) over a time course of 16 h. The lower panels show HeLa cells transfected for 16 h or 48 h with one of the following: 1.5 µg empty vector (vector), 0.75 µg E1+0.75 µg empty vector (E1), 0.75 µg E2+0.75 µg empty vector (E2), or 0.75 µg E1+0.75 µg E2 (E1+E2). The bands were quantified using ImageQuant 5.0. The levels of the transcripts were normalized against GAPDH mRNA and expressed as fold increase relative to the DMSO solvent control, the 0 h tunicamycin treatment or the vector controls, which were set as 1. B) Prolonged exposure of the gel from 16 h transfection to show that XBP1 was clearly spliced in E1- and E2-expressing cells but not in empty vector-transfected cells.

CONCLUSIONS AND SIGNIFICANCE

HCV infection can be involved in a spectrum of diseases involving cell death and cell growth. This includes hepatitis, fibrosis, cirrhosis, and ultimately hepatocellular carcinoma (HCC). In this study we showed that the HCV E1 and E2 proteins activated the CHOP and IRE1-XBP1 pathways, which should allow a better understanding of pathogenesis in HCV-related diseases. BiP, CHOP, IRE1, and XBP1 play different and sometimes opposing roles in the UPR in the regulation of cell death and cell survival. It can be envisaged that the immediate cytopathic effect of ER stress as a result of CHOP and IRE1 activation may be the underlying cause of hepatitis. In an attempt to rescue cells from ER stress, XBP1 is activated to trigger the two cellular adaptive responses, UPR and ERAD. Thus, XBP1 activation may indirectly contribute to HCC via up-regulation of its target gene BiP. On the other hand, XBP1 may contribute directly to HCC. XBP1 is also essential for hepatocyte growth. Thus, chronic activation of XBP1 may have conferred a proliferative advantage to the stressed cells, which will ultimately lead to tumor formation.

Within the CHOP promoter there are two responsive elements, the ERSE and the ATF4 element. It has been suggested that during ER stress ATF6 and XBP1 are responsible for transactivation of the ERSE whereas a pathway dependent on PERK-mediated eIF2 phosphorylation and ATF4 translation is responsible for transactivation of the ATF4 element. We showed that both responsive elements were activated by HCV envelope protein expression and that PERK is necessary and sufficient for the induction of CHOP. This implies the presence of other signaling pathways downstream of PERK that are independent of ATF4 in CHOP induction and highlights the potential of pathway "cross-talk" between the PERK pathway and other stress-induced pathways.

We also obtained evidence of ERAD engagement in cells transfected with E1 and/or E2 by the demonstration of XBP1 splicing and UPRE induction in these cells. The IRE1-XBP1 pathway has been shown to be responsible for UPRE-mediated transcriptional induction of the ER degradation-enhancing -mannosidase-like protein (EDEM), which is directly involved in the recognition of malfolded proteins for degradation. Therefore, the detection of XBP1 splicing in E1- and/or E2-transfected cells leads us to speculate on a role in the induction of ERAD in response to HCV envelope protein expression. A summary of the interpretations of the findings is depicted in Fig. 3 .

View larger version (15K): [in this window] [in a new window]   Figure 3. Signaling mechanisms for HCV envelope protein-mediated UPR and ERAD. Retention of HCV envelope proteins E1 and E2 in the endoplasmic reticulum induces unfolded protein response (UPR) by activating both the ER stress element (ERSE) and the activating transcription factor 4 (ATF4) element in the CHOP promoter. PERK is necessary and sufficient for CHOP induction by activating the canonical eIF2 phosphorylation-ATF4 pathway and a yet undefined pathway leading to transactivation of the ERSE. HCV E1 and E2 also cause engagement of the ER-associated degradation (ERAD) by inducing XBP1 splicing and UPRE transactivation, its product EDEM is directly involved in the recognition of malfolded proteins for degradation.

FOOTNOTES

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This Article Full Text (PDF) All Versions of this Article: 19/11/1510 04-3455fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Chan, S.-W. Articles by Egan, P. A. Search for Related Content PubMed PubMed Citation Articles by Chan, S.-W. Articles by Egan, P. A.