Contribution of redox status to hepatitis C virus E2 envelope protein function and antigenicity.
Disulfide bonding contributes to the function and antigenicity of many viral envelope glycoproteins. We assessed here its significance for the hepatitis C virus E2 envelope protein and a counterpart deleted for hypervariable region-1 (HVR1). All 18 cysteine residues of the antigens were involved in disulfides. Chemical reduction of up to half of these disulfides was compatible with anti-E2 monoclonal antibody reaction, CD81 receptor binding, and viral entry, whereas complete reduction abrogated these properties. The addition of 5,5'-dithiobis-2-nitrobenzoic acid had no effect on viral entry. Thus, E2 function is only weakly dependent on its redox status, and cell entry does not require redox catalysts, in contrast to a number of enveloped viruses. Because E2 is a major neutralizing antibody target, we examined the effect of disulfide bonding on E2 antigenicity. We show that reduction of three disulfides, as well as deletion of HVR1, improved antibody binding for half of the patient sera tested, whereas it had no effect on the remainder. Small scale immunization of mice with reduced E2 antigens greatly improved serum reactivity with reduced forms of E2 when compared with immunization using native E2, whereas deletion of HVR1 only marginally affected the ability of the serum to bind the redox intermediates. Immunization with reduced E2 also showed an improved neutralizing antibody response, suggesting that potential epitopes are masked on the disulfide-bonded antigen and that mild reduction may increase the breadth of the antibody response. Although E2 function is surprisingly independent of its redox status, its disulfide bonds mask antigenic domains. E2 redox manipulation may contribute to improved vaccine design.
Fenouillet
Emmanuel
E
UMR 6184, CNRS, Université Aix-Marseille II, F-13015, France. emmanuel.fenouillet@univmed.fr
Lavillette
Dimitri
D
Loureiro
Silvia
S
Krashias
George
G
Maurin
Guillemette
G
Cosset
François-Loïc
FL
Jones
Ian M
IM
Barbouche
Rym
R
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D000917:Antibody Formation / Q000235:genetics / Q000276:immunology; D015703:Antigens, CD / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D004220:Disulfides / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D016174:Hepacivirus / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D018936:Hepatitis C Antigens / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007114:Immunization; D051379:Mice; D008807:Mice, Inbred BALB C; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary / Q000235:genetics; D017384:Sequence Deletion; D013439:Sulfhydryl Reagents / Q000737:chemistry; D060225:Tetraspanin 28; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism / Q000494:pharmacology; D014761:Viral Hepatitis Vaccines / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism / Q000494:pharmacology; D053586:Virus Internalization
D000914:Antibodies, Viral; D015703:Antigens, CD; C554798:CD81 protein, human; C554799:Cd81 protein, mouse; D004220:Disulfides; D018936:Hepatitis C Antigens; D013439:Sulfhydryl Reagents; D060225:Tetraspanin 28; D014759:Viral Envelope Proteins; D014761:Viral Hepatitis Vaccines; D004228:Dithionitrobenzoic Acid