Hepatitis C Virus Glycoprotein Complex Localization in the Endoplasmic Reticulum Involves a Determinant for Retention and Not Retrieval

Sandrine Duvet,Laurence Cocquerel,André Pillez,René Cacan,André Verbert,Darius Moradpour,Czeslaw Wychowski,Jean Dubuisson

doi:10.1074/jbc.273.48.32088

Abstract

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2). These glycoproteins interact to form a noncovalent heterodimeric complex which in the cell accumulates in endoplasmic reticulum (ER)-like structures. The transmembrane domain of E2, at least, is involved in HCV glycoprotein complex localization in this compartment. In principle, ER localization of a protein can be the consequence of actual retention in this organelle or of retrieval from the Golgi. To determine which of these two mechanisms is responsible for HCV glycoprotein complex accumulation in the ER, the precise localization of these proteins was studied by immunofluorescence, and the processing of their glycans was analyzed. Immunolocalization of HCV glycoproteins after nocodazole treatment suggested an ER retention. In addition, HCV glycoprotein glycans were not modified by Golgi enzymes, indicating that the ER localization of these proteins is not because of their retrieval from the cis Golgi. Retention of HCV glycoprotein complexes in the ER without retrieval suggests that this compartment plays an important role for the acquisition of the envelope of HCV particles. A true retention in the ER was also observed for E2 expressed in the absence of E1 or for a chimeric protein containing the ectodomain of CD4 in fusion with the transmembrane domain of E2. These data indicate that, in HCV glycoprotein complex, the transmembrane domain of E2, at least, is responsible for true retention in the ER, without recycling through the Golgi.

Highlights

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2)
Immunolocalization of HCV Glycoprotein Complex—Recent data from our group indicate that native HCV glycoprotein complexes are localized in endoplasmic reticulum (ER)-like structures and are not transported beyond the cis or medial Golgi [15]
The formation of native E1E2 complexes in UHCV-11.4 cell line was slow, as shown by the kinetics of recognition of HCV glycoproteins with a conformation-sensitive E2reactive Monoclonal antibodies (mAbs) (H53), which reacts with the noncovalent E1E2 heterodimer (Fig. 1) [15, 19]. mAb H53 was used throughout this work to localize E2 or E1E2 complexes

Summary

Introduction

The hepatitis C virus (HCV) genome encodes two envelope glycoproteins (E1 and E2). These glycoproteins interact to form a noncovalent heterodimeric complex which in the cell accumulates in endoplasmic reticulum (ER)-like structures. A true retention in the ER was observed for E2 expressed in the absence of E1 or for a chimeric protein containing the ectodomain of CD4 in fusion with the transmembrane domain of E2 These data indicate that, in HCV glycoprotein complex, the transmembrane domain of E2, at least, is responsible for true retention in the ER, without recycling through the Golgi. More recent data indicate that E1E2 complexes in their native form do not interact any longer with calnexin and are still retained in a pre-medial Golgi compartment [15]. D, endo ␤-D-N-acetylglucosaminidase H and D, respectively; PNGase F, peptide N-glycanase F; HPLC, high performance liquid chromatography; PDI, protein disulfide isomerase; ERGIC, ER-to-Golgi intermediate compartment

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Conclusion