Organization of the TMDs of HCV glycoproteins. (A) Processing of the N-terminal region of HCV polyprotein generating the envelope proteins E1 and E2. The positions of the C-terminal residues of C, E1, and E2 are indicated at the top. (B) Amino acid variability of the putative TMDs of HCV glycoproteins E1 and E2. The consensus patterns of 340 and 133 natural variants of E1 and E2, respectively, are presented. The observed residues at each position are indicated in decreasing order of frequency from top to bottom. The less frequently observed residues (<5%) are not presented. The positions of the amino acid residues indicated above the consensus sequences correspond to their position in the polyprotein. Fully conserved, conserved, and similar residues at each position are symbolized by an asterisk, colon, and dot, respectively. The fully conserved Lys₃₇₀ residue in E1 and Asp₇₂₈ and Arg₇₃₀ residues in E2 which have been mutated in this study are shown in boldface. #, prediction of minimal membrane segments deduced from dense alignment surface (DAS) analysis (12).

The first hydrophobic stretch of the TMDs of HCV glycoproteins is not sufficient for an efficient arrest of translocation. HepG2 cells were coinfected with vTF7-3 and the appropriate vaccinia virus recombinant at a multiplicity of infection of 5 PFU/cell. At 4.5 h postinfection, cells were pulse-labeled for 10 min and chased for 4 h. Cell lysates and supernatants (sup) were immunoprecipitated with MAb H53 (E2 and E2₇₃₀) (A) or OKT4 (CD4-E1 and CD4-E1₃₇₀) (B). Proteins were separated by SDS-PAGE (10% polyacrylamide). The sizes (in kilodaltons) of protein molecular mass markers are indicated on the left. The slow-migrating form of E2 is indicated by an asterisk.

Analysis of the endo H sensitivity of E2, E2_DR, and E2-CD4 glycoproteins. HepG2 cells were coinfected with vTF7-3 and the appropriate vaccinia virus recombinant at a multiplicity of infection of 5 PFU/cell. At 4.5 h postinfection, cells were pulse-labeled for 10 min and chased for the indicated times (in hours). Cell lysates were immunoprecipitated with MAb H53 and then treated (+) or not (−) with endo H. Proteins were separated by SDS-PAGE (10% polyacrylamide) under reducing conditions. E2*, slow-migrating form of E2; E2^R, endo H-resistant form of E2; E2^S, deglycosylated form of E2.

Role of charged residues in ER retention mediated by the TMD of HCV glycoproteins. Chimeras made of the ectodomain of CD4 in fusion with wild-type or mutated TMD of HCV glycoproteins were expressed by vaccinia virus recombinants. HepG2 cells were coinfected with vTF7-3 and the appropriate vaccinia virus recombinant at a multiplicity of infection of 5 PFU/cell. At 4.5 h postinfection, cells were pulse-labeled for 10 min and chased for the indicated times (in hours). Cell lysates were immunoprecipitated with MAb OKT4 and then treated (+) or not (−) with endo H. Proteins were separated by SDS-PAGE (10% polyacrylamide) under reducing conditions. Deglycosylated proteins are indicated by an asterisk.

Cell surface expression of mutant proteins analyzed by indirect immunofluorescence. HepG2 cells were coinfected with vTF7-3 and the appropriate vaccinia virus recombinant at a multiplicity of infection of 3 PFU/cell. At 8 h postinfection, cells were fixed with paraformaldehyde, permeabilized or not with Triton X-100, and immunostained with anti-E2 (E2, E2_DR, and E2-CD4) or anti-CD4 (CD4, CD4-E1_NK, and CD4-E1) MAbs.

Role of charged residues on the signal sequence function of the TMD of HCV glycoprotein E1. HepG2 cells were coinfected with vTF7-3 and a vaccinia virus recombinant expressing E1E2 or E1_NKE2 at a multiplicity of infection of 5 PFU/cell. (A) At 4.5 h postinfection, cells were pulse-labeled for 10 min and chased for the indicated times (in hours). Cell lysates were immunoprecipitated with MAb H53 (anti-E2) or A4 (anti-E1). Proteins were separated by SDS-PAGE (10% polyacrylamide) under reducing conditions. (B) Infected cells were lysed at 4.5 h postinfection. After separation by SDS-PAGE, the proteins of interest were revealed by Western blotting with an anti-E1 (A4) or anti-E2 (A11) MAb. The uncleaved E1_NKE2 polyprotein is indicated by an asterisk.

Role of charged residues in the signal sequence function of the TMD of HCV glycoprotein E2. HepG2 cells were coinfected with vTF7-3 and a vaccinia virus recombinant expressing E2E1 or E2_DRE1 at a multiplicity of infection of 5 PFU/cell. Infected cells were lysed at 4.5 h postinfection. After separation by SDS-PAGE (10% polyacrylamide) under reducing conditions, proteins of interest were revealed by Western blotting with an anti-E1 (A4) or anti-E2 (A11) MAb. The uncleaved E2_DRE1 polyprotein is indicated by an asterisk.

Role of charged residues in the assembly of the E1E2 polyprotein complex. HepG2 cells were coinfected with vTF7-3 and the appropriate vaccinia virus recombinants at a multiplicity of infection of 5 PFU/cell. At 4.5 h postinfection, cells were pulse-labeled for 10 min and chased for 4 h. Cell lysates were immunoprecipitated with MAb H53 (anti-E2) or A4 (anti-E1), and proteins were separated by SDS-PAGE (10% polyacrylamide) under reducing conditions. The sizes (in kilodaltons) of protein molecular mass markers are indicated on the right. The slow-migrating form of E2 is indicated by an asterisk.

Comparison and conservation of amino acid sequences of the putative TMD of envelope proteins in the Flaviviridae family. The consensus sequence of most represented amino acid was deduced from the sequence analysis of natural variants available in the EMBL database as described in Materials and Methods. The number of variants analyzed is indicated in parentheses, and the amino acid homology at each position is symbolized as follows: asterisk, fully conserved; colon, conserved; dot, similar. The consensus of putative minimal TM segments is shown at the top (#). Conserved charge residues located between the two stretches of hydrophobic residues are in boldface. The amino acid numbering refers to the EMBL database sequence accession number when indicated. For consensus sequences, numbering is according to the sequences for which the EMBL accession number is indicated (in parentheses) in the following references: GBV-A and GBV-B viruses (49, 67); GBV-C/HGV virus (U44402) (34, 37); BVDV, bovine viral diarrhea virus (M96687) (15); CSFV, classical swine fever virus (J04358) (61); BDV, border disease virus (AF037405) (1); DEN1, dengue virus type 1 (M23027) (45); DEN2 (M20558) (16); DEN3 (M93130) (51); DEN4 (M14931) (77); YF, yellow fever virus (X03700) (60); JE, Japanese encephalitis virus (M55506) (50); WN, West Nile virus (5); KUN, Kunjin virus (9); MVE, Murray Valley encephalitis virus (13); SLE, St. Louis encephalitis virus (72); TBE, tick-borne encephalitis virus (M27157) (39); SSEV, Spanish sheep encephalitis virus (43); OHFV, Omsk hemorrhagic fever virus (28); LIV, Louping ill virus (M59376) (65); LANV, Langat virus (41); KFDV, Kyasanur Forest disease virus (73); POWV, tick-borne Powassan virus (40). Note that no prM sequence is available for OHFV or SSEV.