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. 2010 Oct;84(19):10159-68.
doi: 10.1128/JVI.01180-10. Epub 2010 Jul 28.

Characterization of the envelope glycoproteins associated with infectious hepatitis C virus

Gabrielle Vieyres  1 , Xavier Thomas, Véronique Descamps, Gilles Duverlie, Arvind H Patel, Jean Dubuisson
Affiliations

Characterization of the envelope glycoproteins associated with infectious hepatitis C virus

Gabrielle Vieyres et al. J Virol. 2010 Oct.

Abstract

Hepatitis C is caused by an enveloped virus whose entry is mediated by two glycoproteins, namely, E1 and E2, which have been shown to assemble as a noncovalent heterodimer. Despite extensive research in the field of such an important human pathogen, hepatitis C virus (HCV) glycoproteins have only been studied so far in heterologous expression systems, and their organization at the surfaces of infectious virions has not yet been described. Here, we characterized the envelope glycoproteins associated with cell-cultured infectious virions and compared them with their prebudding counterparts. Viral particles were analyzed by ultracentrifugation, and the envelope glycoproteins were characterized by coimmunoprecipitation and receptor pulldown assays. Furthermore, their oligomeric state was determined by sedimentation through sucrose gradients and by separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. In sucrose gradient analyses, HCV envelope glycoproteins were associated with fractions containing the most infectious viral particles. Importantly, besides maturation of some of their glycans, HCV envelope glycoproteins showed a dramatic change in their oligomeric state after incorporation into the viral particle. Indeed, virion-associated E1 and E2 envelope glycoproteins formed large covalent complexes stabilized by disulfide bridges, whereas the intracellular forms of these proteins assembled as noncovalent heterodimers. Furthermore, the virion-associated glycoprotein complexes were recognized by the large extracellular loop of CD81 as well as conformation-sensitive antibodies, indicating that these proteins are in a functional conformation. Overall, our study fills a gap in the description of HCV outer morphology and should guide further investigations into virus entry and assembly.

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Figures

FIG. 1.
FIG. 1.
Detection of HCV structural proteins in infected cell lysate and supernatant. (A) Lysates obtained from infected cells (lane 1), naïve cells (lane 2), or concentrated infectious supernatant (lane 3) were analyzed by reducing SDS-PAGE and Western blotting with specific anti-core, anti-E1, or anti-E2 antibodies. Molecular mass markers (in kDa) are indicated on the left. In supernatant, the asterisk corresponds to a disturbance in the E2 migration profile, likely due to the presence of the BSA protein, as seen by membrane staining with Ponceau solution (data not shown). The double asterisk indicates the slow-migrating band detected by the anti-E2 antibody in cell lysate which likely corresponds to the E2-p7-NS2 precursor, as previously reported (57). (B) GNA pulldown assay. Lysates obtained from infected cells (lane 1), naïve cells (lane 2), or concentrated infectious supernatant (lane 3) were incubated with GNA-conjugated beads. Proteins were eluted from the beads in reducing Laemmli buffer and analyzed by SDS-PAGE and Western blotting. #, the additional bands detected by the anti-E1 antibody might correspond to E1 dimers and trimers, as already observed in recombinant and HCVcc systems (7).
FIG. 2.
FIG. 2.
Secreted E1 and E2 glycoproteins are associated with infectious particles. (A) Separation of concentrated HCVcc in a sucrose gradient. (Top) Concentrated cell-cultured HCVs were separated by sedimentation through a 20-to-60% sucrose gradient. Fractions were collected from the top and analyzed for their infectivity by focus-forming assay (black diamonds) and for their viral RNA content by reverse transcription and quantitative PCR (white circles). Fraction density was measured in a control gradient spun in parallel (dotted line with white triangles). ffu, focus-forming units. (Bottom) Viral proteins were detected following total protein precipitation (core and NS5A proteins) or CD81 pulldown (E1 and E2 proteins), SDS-PAGE, and Western blotting with specific antibodies. The input virus preparation (inp.) was analyzed in parallel. The lower-molecular-weight band detected by the anti-E1 MAb is likely an artifact due to the comigration of one CD81 form (*) during SDS-PAGE (as observed by Ponceau staining) (data not shown); note that only one band is detectable after GNA pulldown (see panel B). (B) Analysis of cell contaminants in sucrose gradient-separated cell-cultured HCVs. In an experiment similar to that described in the legend to panel A, the cellular chaperone calnexin and the viral core protein were detected on the same membrane with specific antibodies. As a comparison, the E1 and E2 sedimentation profiles are also depicted; in that case, viral glycoproteins were precipitated from gradient fractions using the GNA lectin. Note that the distortion of the calnexin band in the first three lanes is likely due to the comigration of contaminant proteins such as BSA, as observed by membrane staining with Ponceau solution (data not shown).
FIG. 3.
FIG. 3.
Interaction of viral envelope glycoproteins with HCV entry factors. (A) Heparin pulldown assay. Infected cells or viral supernatants were lysed and incubated with heparin-conjugated beads or GNA-conjugated beads as a positive control. As a specificity control, E1E2-containing extracts were incubated beforehand with soluble heparin at various concentrations. *, the slow-migrating band detected by the anti-E2 antibody in cell lysate might correspond to the E2-p7-NS2 precursor. (B) CD81 pulldown assay. Infected cells or viral supernatants were lysed and incubated with GS4B beads or GS4B beads incubated beforehand with a recombinant form of mouse CD81 protein (mCD81) or human CD81 protein (hCD81). (A, B) Pulled-down proteins were eluted from the beads in Laemmli buffer and analyzed by SDS-PAGE and Western blotting.
FIG. 4.
FIG. 4.
Glycosylation pattern of HCV envelope glycoproteins. (Left) E1E2-containing extracts from HCVcc-infected cells or viral concentrated supernatant were denatured and divided into three equal aliquots. The first aliquot was kept untreated, the second one was digested with PNGase F, and the third one was digested with Endo H. The samples were analyzed by SDS-PAGE and Western blotting. The white band labeled with an asterisk corresponds to a disturbance in the E2 migration profile, likely due to the BSA protein, as seen by membrane staining with Ponceau solution (data not shown). (Right) A similar analysis was conducted with HCVpp-producing cells or secreted HCVpp after partial purification by ultracentrifugation over a sucrose cushion.
FIG. 5.
FIG. 5.
Oligomerization of envelope glycoproteins analyzed by SDS-PAGE. Glycoproteins from infected cell lysates or supernatants were separated by SDS-PAGE under reducing (R) or nonreducing (NR) conditions and analyzed by Western blotting with anti-E2 (A) or anti-E1 (B) antibodies. Molecular mass markers (in kDa) are indicated on the left. *, the slow-migrating band detected by the anti-E2 antibody in cell lysate might correspond to the E2-p7-NS2 precursor.
FIG. 6.
FIG. 6.
Separation of envelope glycoprotein complexes in sucrose density gradients. Glycoprotein complexes from infected cell lysates or supernatants were separated by sedimentation through a 5-to-20% sucrose gradient. Eleven 1-ml fractions and the gradient pellet (P) were harvested. After GNA pulldown (A) or CD81 pulldown (B), samples were analyzed for the presence of E1 and E2 glycoproteins by reducing SDS-PAGE and Western blotting. The sedimentation profiles of several standard proteins in a parallel gradient are indicated above. *, the slow-migrating band detected by the anti-E2 antibody in cell lysate might correspond to the E2-p7-NS2 precursor.
FIG. 7.
FIG. 7.
Covalent E1E2 complexes display a functional conformation. (A) HCV glycoproteins from concentrated viral supernatant were immunoprecipitated (IP) with CBH-5 or CBH-7 MAbs or precipitated with GNA or protein-A Sepharose beads alone as positive and negative controls, respectively. Samples were analyzed by SDS-PAGE and Western blotting under reducing (left) or nonreducing (right) conditions. The band indicated by an asterisk corresponds to the heavy chain of the immunoglobulin used for immunoprecipitation. (B) HCV glycoproteins from concentrated viral supernatant were precipitated with GS4B beads incubated beforehand with a recombinant form of mouse CD81 protein (mCD81) or human CD81 protein (hCD81) or with GNA beads as positive control. Samples were analyzed by SDS-PAGE and Western blotting under reducing (left) or nonreducing (right) conditions.
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