Topological changes in the transmembrane domains of hepatitis C virus envelope glycoproteins

Abstract

L.Cocquerel and A.Op de Beeck contributed equally to this workHepatitis C virus proteins are synthesized as a polyprotein cleaved by a signal peptidase and viral proteases. The behaviour of internal signal sequences at the C-terminus of the transmembrane domains of hepatitis C virus envelope proteins E1 and E2 is essential for the topology of downstream polypeptides. We determined the topology of these transmembrane domains before and after signal sequence cleavage by tagging E1 and E2 with epitopes and by analysing their accessibility in selectively permeabilized cells. We showed that, after cleavage by signal peptidase in the endoplasmic reticulum, the C-terminal orientation of these transmembrane domains changed from luminal to cytosolic. The dynamic behaviour of these transmembrane domains is unique and it is linked to their multifunctionality. By reorienting their C-terminus toward the cytosol and being part of a transmembrane domain, the signal sequences at the C-terminus of E1 and E2 contribute to new functions: (i) membrane anchoring; (ii) E1E2 heterodimerization; and (iii) endoplasmic reticulum retention.

Characterization of GB Virus B Polyprotein Processing Reveals the Existence of a Novel 13-kDa Protein with Partial Homology to Hepatitis C Virus p7 Protein

“…1 and 4), the HCV E2 is considerably more heavily glycosylated with 11 putative N-glycosylation sites (HCV genotype 1a). Despite these differences, structure predictions indicate the presence of a putative transmembrane domain at the actual C terminus of GBV-B E2 which is similar to that observed in HCV (50).…”

Regulation of Hepatitis C Virus Polyprotein Processing by Signal Peptidase Involves Structural Determinants at the p7 Sequence Junctions

“…HCV glycoprotein E1 was used as a reporter protein because it is a glycosylated protein, and we can easily discriminate between glycosylated and nonglycosylated forms. E1 has indeed been shown to be a good reporter protein in some of our previous works (24,28).…”

“…The pTM1 plasmid contains a polycloning region immediately downstream of the T7 promoter and the encephalomyocarditis virus internal ribosome entry site cap-independent translation initiation (26). Plasmids pTM1/E2E1, pTM1/Spp7E1, and pTM1/SpNS2E1 have been described previously (24,27,28). Plasmid pTM1/ CE1E2p7NS2Myc contains the sequence of a truncated HCV polyprotein including C, E1, E2, p7, and NS2.…”

“…1). Indeed, the C termini of E2 and p7 contain a sequence for reinitiation of translocation, and when fused to a reporter protein, these sequences function as signal peptides (24,28). For this reason, the translocation reinitiation sequence, which is present at the C terminus of E2, has been named the signal peptide of p7 (Spp7) (Fig.…”

“…2) have been shown to function as signal sequences (24,28). To determine whether structural features in these sequences potentially lead to partial cleavages at the E2/p7 and p7/NS2 sites, we analyzed the efficiency of cleavage of a reporter protein fused to them.…”