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. 2010 Jun;84(11):5751-63.
doi: 10.1128/JVI.02200-09. Epub 2010 Mar 31.

Hepatitis C virus hypervariable region 1 modulates receptor interactions, conceals the CD81 binding site, and protects conserved neutralizing epitopes

Dorothea Bankwitz  1 , Eike Steinmann, Julia Bitzegeio, Sandra Ciesek, Martina Friesland, Eva Herrmann, Mirjam B Zeisel, Thomas F Baumert, Zhen-yong Keck, Steven K H Foung, Eve-Isabelle Pécheur, Thomas Pietschmann
Affiliations

Hepatitis C virus hypervariable region 1 modulates receptor interactions, conceals the CD81 binding site, and protects conserved neutralizing epitopes

Dorothea Bankwitz et al. J Virol. 2010 Jun.

Abstract

The variability of the hepatitis C virus (HCV), which likely contributes to immune escape, is most pronounced in hypervariable region 1 (HVR1) of viral envelope protein 2. This domain is the target for neutralizing antibodies, and its deletion attenuates replication in vivo. Here we characterized the relevance of HVR1 for virus replication in vitro using cell culture-derived HCV. We show that HVR1 is dispensable for RNA replication. However, viruses lacking HVR1 (Delta HVR1) are less infectious, and separation by density gradients revealed that the population of Delta HVR1 virions comprises fewer particles with low density. Strikingly, Delta HVR1 particles with intermediate density (1.12 g/ml) are as infectious as wild-type virions, while those with low density (1.02 to 1.08 g/ml) are poorly infectious, despite quantities of RNA and core similar to those in wild-type particles. Moreover, Delta HVR1 particles exhibited impaired fusion, a defect that was partially restored by an E1 mutation (I347L), which also rescues infectivity and which was selected during long-term culture. Finally, Delta HVR1 particles were no longer neutralized by SR-B1-specific immunoglobulins but were more prone to neutralization and precipitation by soluble CD81, E2-specific monoclonal antibodies, and patient sera. These results suggest that HVR1 influences the biophysical properties of released viruses and that this domain is particularly important for infectivity of low-density particles. Moreover, they indicate that HVR1 obstructs the viral CD81 binding site and conserved neutralizing epitopes. These functions likely optimize virus replication, facilitate immune escape, and thus foster establishment and maintenance of a chronic infection.

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Figures

FIG. 1.
FIG. 1.
HCV particles devoid of HVR1 have reduced infectivity. RNA transcripts of Jc1 and a Jc1 derivative lacking HVR1 (ΔHVR1) were transfected into Huh-7.5 cells. (A) Forty-eight hours later, intra- and extracellular core protein levels were determined using a core-specific ELISA as an indicator for efficiency of RNA replication and virus production. (B) In parallel, virus infectivity normalized for equal quantity of core was determined by inoculation of naïve Huh-7.5 cells using a limiting dilution assay. Error bars show standard errors. (C) Representative microscopic views of Huh-7.5 cells inoculated with Jc1 and ΔHVR1 particles. Cells were stained with NS5A-specific antibodies by immunohistochemistry. Results of one representative experiment out of three independent repetitions are shown.
FIG. 2.
FIG. 2.
Serial cell culture passage of an ΔHVR1 mutant yields viruses with increased fitness. (A) Huh-7.5 cells were transfected with the ΔHVR1 mutant RNA. Forty-eight hours later, the cell culture fluid of these cells was harvested and used to infect naïve Huh-7.5 cells. Subsequently, cell-free culture fluids were passaged in naive Huh-7.5 cells every two to three days in total for 10 serial rounds of passaging. Infectivity of the respective culture fluid at the time of passaging was determined using the limiting dilution assay. (B) Huh-7.5 cells were inoculated with ΔHVR1 virus harvested 72 h posttransfection or with ΔHVR1 viruses collected after 10 consecutive passages at a multiplicity of infection of 0.1. Inoculated cells were fixed at the indicated time points and stained for NS5A expression by indirect immunofluorescence (green). Nuclear DNA was stained with DAPI (blue). (C) The percentage of infected cells at the respective time points was calculated by counting total cells (DAPI stain) and infected cells (NS5A expression) in five arbitrarily chosen microscopic views, respectively (left). Data are means and standard errors of the means. Production of viral progeny was quantified by inoculation of naïve Huh-7.5 cells (right).
FIG. 3.
FIG. 3.
The suppressor mutation I347L restores infectivity of ΔHVR1 viruses. (A) RNA transcripts of Jc1, Jc1/ΔHVR1, and Jc1/ΔHVR1/I347L were transfected into Huh-7.5 cells. (A) Forty-eight hours later, intra- and extracellular core protein levels were determined using a core-specific ELISA. Results from a representative of two independent repetitions are shown. (B) Cell supernatants were harvested at the indicated time points and used for infection of naïve Huh-7.5 cells. The 50% tissue culture infectious dose of the samples was determined by a limiting dilution assay. Data are means from five independent repetitions. (C) RNA transcripts of JFH1, JFH1/ΔHVR1, and JFH1/ΔHVR1/I347L were transfected as for panel A, and infectivity released was quantified as for panel B. Data are means from three independent experiments.
FIG. 4.
FIG. 4.
Deletion of HVR1 and the I347L mutation does not affect glycosylation of HCV E1-E2 complexes. Huh-7.5 cells were transfected with the indicated viral RNAs and metabolically labeled. Cells were then lysed under nondenaturing conditions, and E1/E2 complexes were precipitated using the E2-specific mouse monoclonal antibody AP33 or an E1-specific polyclonal serum. After extensive washing, proteins were deglycosylated with Endo-H or left untreated. Finally, immune complexes were resolved by denaturing SDS-PAGE and detected by autoradiography. Deglycosylated protein species are marked with asterisks.
FIG. 5.
FIG. 5.
The suppressor mutation I347L does not restore infectivity of HCVpp with the ΔHVR1 mutation. Murine leukemia virus (MLV)-based pseudotypes bearing vesicular stomatitis virus glycoproteins (VSV-G), HCV J6 wild-type (J6), J6/ΔHVR1 (ΔHVR1), or J6/ΔHVR1/I347L (ΔHVR1/I347L) glycoproteins were prepared by transfection of 293T cells. Preparations created in the absence of a viral envelope protein expression construct (pcDNA) served as controls. Infectivity of the pseudoparticles transducing a luciferase transgene was determined by inoculation of Huh-7.5 cells. Data are the means of quadruple measurements and are from a representative of three independent repetitions.
FIG. 6.
FIG. 6.
Buoyant density and specific infectivity of Jc1, Jc1/ΔHVR1, and Jc1/ΔHVR1/I347L viruses. Individual viruses were harvested 48 h after transfection of Huh-7.5 cells and were resolved using an iodixanol step gradient. Ten fractions were harvested from the bottom, and HCV core protein (A), HCV RNA (B), and infectivity (C) were determined for each fraction. Values are plotted against the density of the respective fraction measured by refractometry. (D) Specific infectivity of viruses contained in individual fractions is expressed as TCID50 per fmol core protein. The results are from a representative of five independent experiments.
FIG. 7.
FIG. 7.
HCV particles lacking HVR1 are more resistant to neutralization by SR-BI-specific sera. Huh7-Lunet N hCD81 cells were infected with luciferase reporter viruses normalized to equal quantities of HCV core and mixed with increasing doses of SR-BI-specific immunoglobulins (left) or antibodies purified from a control rat (right) for 72 h at 37°C. Luciferase reporter activity was determined and is expressed relative to that in infections performed in the absence of rat serum. Data are means and standard deviations of triplicate measurements. Two independent experiments were conducted.
FIG. 8.
FIG. 8.
Deletion of HVR1 exposes the viral CD81 binding site. (A) Huh7-Lunet N hCD81 cells were inoculated with indicated luciferase reporter viruses normalized to equal quantities of HCV core in the presence of increasing doses of CD81-specific monoclonal antibodies or control antibodies directed against CD13. The efficiency of infection was determined 72 h later by luciferase reporter assay and is expressed relative to infections performed in the absence of antibodies. Data are means and standard deviations of triplicate measurements and are from a representative of three independent experiments. (B) Infections (as described for panel A) were conducted in the presence of increasing doses of recombinant human CD81 large extracellular loop fused to GST (hCD81-LEL) or of GST alone. Two independent experiments were conducted. (C) Equal numbers of viruses were incubated with 10 μg hCD81-LEL or GST. Subsequently, complexes were precipitated using glutathione-coated beads, and associated HCV RNA was quantified by qRT-PCR. Data are means of duplicate measurements. Two independent experiments were performed.
FIG. 9.
FIG. 9.
Impaired fusion of viruses lacking HVR1 is partially compensated for by the I347L suppressor mutation in E1. Equal quantities of cell culture-produced viruses (normalized for core protein content) were used for fusion assays with fluorescently labeled liposomes. Dequenching of R18 was used as indicator of fusion between liposomes and viral membrane. Maximal dequenching of R18 was determined after 0.1% Triton X-100 was added to the liposomes. Data are from a representative of five independent experiments.
FIG. 10.
FIG. 10.
Deletion of HVR1 enhances neutralization by monoclonal antibodies and patient sera and causes increased exposure of conserved epitopes. Huh7-Lunet N hCD81 cells were inoculated with viruses normalized to equal quantities of HCV core in the presence of increasing doses of human or murine HCV E2-specific monoclonal antibodies (A) or sera from HCV infected patients or a healthy donor (B). The RO4 monoclonal antibody, recognizing a cytomegalovirus protein, served as a control. Infection efficiency was determined by luciferase assays 72 h postinoculation and is expressed relative to that of infections performed in the absence of antibodies and sera. Data are means and standard deviations of triplicate measurements and are from one representative of at least two independent experiments.
FIG. 11.
FIG. 11.
Deletion of HVR1 causes increased exposure of conserved epitopes. Equal numbers of viruses (normalized to HCV RNA) were incubated with 1 μg of the specified antibodies. Subsequently, immune complexes were precipitated using protein G-coated beads, and associated HCV RNA was quantified by qRT-PCR. Data are representative of the results of two independent experiments.
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References

    1. Alter, M. J. 2007. Epidemiology of hepatitis C virus infection. World J. Gastroenterol. 13:2436-2441. - PMC - PubMed
    1. Andre, P., F. Komurian-Pradel, S. Deforges, M. Perret, J. L. Berland, M. Sodoyer, S. Pol, C. Brechot, G. Paranhos-Baccala, and V. Lotteau. 2002. Characterization of low- and very-low-density hepatitis C virus RNA-containing particles. J. Virol. 76:6919-6928. - PMC - PubMed
    1. Bartosch, B., J. Dubuisson, and F. L. Cosset. 2003. Infectious hepatitis C virus pseudo-particles containing functional E1-E2 envelope protein complexes. J. Exp. Med. 197:633-642. - PMC - PubMed
    1. Bartosch, B., G. Verney, M. Dreux, P. Donot, Y. Morice, F. Penin, J. M. Pawlotsky, D. Lavillette, and F. L. Cosset. 2005. An interplay between hypervariable region 1 of the hepatitis C virus E2 glycoprotein, the scavenger receptor BI, and high-density lipoprotein promotes both enhancement of infection and protection against neutralizing antibodies. J. Virol. 79:8217-8229. - PMC - PubMed
    1. Bartosch, B., A. Vitelli, C. Granier, C. Goujon, J. Dubuisson, S. Pascale, E. Scarselli, R. Cortese, A. Nicosia, and F. L. Cosset. 2003. Cell entry of hepatitis C virus requires a set of co-receptors that include the CD81 tetraspanin and the SR-B1 scavenger receptor. J. Biol. Chem. 278:41624-41630. - PubMed

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