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. 1998 May;72(5):3851-8.
doi: 10.1128/JVI.72.5.3851-3858.1998.

Involvement of endoplasmic reticulum chaperones in the folding of hepatitis C virus glycoproteins

A Choukhi  1 , S Ung, C Wychowski, J Dubuisson
Affiliations

Involvement of endoplasmic reticulum chaperones in the folding of hepatitis C virus glycoproteins

A Choukhi et al. J Virol. 1998 May.

Abstract

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2) which interact noncovalently to form a heterodimer (E1-E2). During the folding and assembly of HCV glycoproteins, a large portion of these proteins are trapped in aggregates, reducing the efficiency of native E1-E2 complex assembly. To better understand this phenomenon and to try to increase the efficiency of HCV glycoprotein folding, endoplasmic reticulum chaperones potentially interacting with these proteins were studied. Calnexin, calreticulin, and BiP were shown to interact with E1 and E2, whereas no interaction was detected between GRP94 and HCV glycoproteins. The association of HCV glycoproteins with calnexin and calreticulin was faster than with BiP, and the kinetics of interaction with calnexin and calreticulin were very similar. However, calreticulin and BiP interacted preferentially with aggregates whereas calnexin preferentially associated with monomeric forms of HCV glycoproteins or noncovalent complexes. Tunicamycin treatment inhibited the binding of HCV glycoproteins to calnexin and calreticulin, indicating the importance of N-linked oligosaccharides for these interactions. The effect of the co-overexpression of each chaperone on the folding of HCV glycoproteins was also analyzed. However, the levels of native E1-E2 complexes were not increased. Together, our data suggest that calnexin plays a role in the productive folding of HCV glycoproteins whereas calreticulin and BiP are probably involved in a nonproductive pathway of folding.

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Figures

FIG. 1
FIG. 1
Coprecipitation of HCV glycoproteins with ER chaperones. BHK-21 cells were coinfected with vTF7-3 and vHCV1-1488 (lanes V) or vTF7-3 and vpTM1 (M). At 4.5 h postinfection, infected cells were labeled for 15 min with [35S]methionine and lysed with Triton X-100. Cell lysates were used for immunoprecipitation with anti-E1 (control A4), anti-calnexin (CNX), anti-calreticulin (CRT), anti-BiP, or anti-GRP94 antibodies. Immunoprecipitates were analyzed by SDS-PAGE (10% polyacrylamide). The sizes (in kilodaltons) of molecular mass markers are indicated on the left.
FIG. 2
FIG. 2
Western blotting analysis of the interaction between BiP and HCV glycoproteins. BHK-21 cells were coinfected with vTF7-3 and vHCV1-1488 (V) or vTF7-3 and pTM1 (M). At 5 h postinfection, infected cells were lysed with Triton X-100 in buffer containing or lacking 1 mM ATP. Cell lysates were used for immunoprecipitation with anti-E1 (MAb A4), anti-E2 (MAb A11 and H2) or anti-BiP antibodies. Immunoprecipitates were separated by SDS-PAGE (10% polyacrylamide) and revealed by Western blotting with an anti-BiP MAb.
FIG. 3
FIG. 3
Kinetics of HCV glycoprotein association with calnexin, calreticulin, and BiP. BHK-21 cells coinfected with vHCV170-809 and vTF7-3 were pulse-labeled for 5 min with [35S]methionine and chased for the indicated times. Cell lysates were used for immunoprecipitation with anti-BiP, anti-calnexin, or anti-calreticulin antibodies. Proteins were separated by SDS-PAGE, and quantifications of HCV glycoproteins coprecipitated with chaperones were performed with a PhosphorImager.
FIG. 4
FIG. 4
Analysis of HCV glycoproteins associated with BiP, calnexin (CNX), and calreticulin (CRT) under nonreducing conditions. BHK-21 cells coinfected with vTF7-3 and vHCV1-1488 were pulse-labeled for 15 min with [35S]methionine, chased for 10 min, and lysed with Triton X-100. Cell lysates were used for immunoprecipitation with anti-chaperone antibodies, and proteins associated with these chaperones were released by heating for 5 min at 37°C in 0.5% Nonidet P-40 and reprecipitated with MAb A4 (anti-E1). Immunoprecipitates were analyzed by SDS-PAGE (10% polyacrylamide) under reducing (R) or nonreducing (NR) conditions. Agg, aggregates.
FIG. 5
FIG. 5
Interaction of calnexin (CNX), BiP, or calreticulin (CRT) with HCV glycoprotein E1 (A) or E2 (B) expressed alone. BHK-21 cells were coinfected with vTF7-3 and vHCV1-383 (V, panel A) or vHCV371-809 (V, panel B) or vpTM1 (M). At 4 h postinfection, the cells were labeled for 15 min with [35S]methionine and lysed with Triton X-100. Cell lysates were used for immunoprecipitation with anti-chaperone (CNX, BiP, and CRT), anti-E1 (MAb A4), or anti-E2 (MAb A11) antibodies. The immunoprecipitates were analyzed by SDS-PAGE. The sizes (in kilodaltons) of molecular mass markers are indicated on the left.
FIG. 6
FIG. 6
Effect of CST and tunicamycin (Tun) on the interaction of HCV glycoproteins with calnexin and calreticulin. BHK-21 cells were coinfected with vTF7-3 and vHCV170-809 (V) or vpTM1 (M). Infected cells were incubated in the presence or absence of 1 mM CST or 5 μg of tunicamycin per ml, labeled for 30 min with [35S]methionine in the presence of the same concentrations of drugs, and lysed with Triton X-100. Cell lysates were used for immunoprecipitation with anti-E1 (MAb A4), anti-calnexin (CNX), or anti-calreticulin (CRT) antibodies. Immunoprecipitates were analyzed by SDS-PAGE (10% polyacrylamide). Deglycosylated proteins are indicated by asterisks. The sizes (in kilodaltons) of molecular mass markers are indicated on the right.
FIG. 7
FIG. 7
Formation of native E1-E2 complexes after castanospermine (CST) or tunicamycin treatment, analyzed with the conformation-sensitive MAb H2. BHK-21 cells were coinfected with vTF7-3 and vHCV170-809 (V) or vTF7-3 and vpTM1 (M). Infected cells were incubated in the presence or absence of 1 mM CST or 5 μg of tunicamycin per ml, labeled for 2 h with [35S]methionine in the presence of the same concentrations of drugs, and lysed with Triton X-100. Cell lysates were used for immunoprecipitation with a conformation-sensitive (H2) or -insensitive (A4) MAb and analyzed by SDS-PAGE (10% polyacrylamide). Agg, aggregates. The sizes (in kilodaltons) of molecular mass markers are indicated on the right.
FIG. 8
FIG. 8
Expression of the ER chaperones calnexin (CNX), BiP, or calreticulin (CRT) by vaccinia virus recombinants. (A) Analysis of chaperone expression by immunoprecipitation. BHK-21 cells were coinfected with vTF7-3 and the appropriate vaccinia virus-chaperone recombinant (V) or with vTF7-3 and vpTM1 (M). At 4.5 h postinfection, infected cells were labeled for 2 h with [35S]methionine and lysed with Triton X-100. Cell lysates were immunoprecipitated with anti-chaperone antibodies (CNX, BiP, or CRT) and analyzed by SDS-PAGE. Arrows correspond to the migration level of each chaperone. (B) Analysis of chaperone expression by Western blotting. BHK-21 cells were coinfected with vTF7-3 and the appropriate vaccinia virus-chaperone recombinant. At 0, 4, or 8 h postinfection, infected cells were lysed with Triton X-100. Cell lysates were separated by SDS-PAGE and revealed by Western blotting with specific anti-chaperone antibodies.
FIG. 9
FIG. 9
Effect of the co-overexpression of calnexin (CNX), BiP, or calreticulin (CRT) with HCV glycoproteins on the formation of native E1-E2 complexes. BHK-21 cells were coinfected with vTF7-3, vHCV170-809, and the appropriate vaccinia virus-chaperone recombinant or vpTM1 (control). At 4.5 h postinfection, infected cells were labeled for 2 h with [35S]methionine and lysed with Triton X-100. Cell lysates were used for immunoprecipitation with the conformation-sensitive MAb H2. Immunoprecipitates were analyzed by SDS-PAGE, and the intensity of coprecipitated E1 was quantified with a PhosphorImager.
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