Abstract
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.
Highlights
The primary function of disulfides is in folding of nascent proteins [1]
Because the bovine viral diarrhea virus envelope is functionally similar to the related flavivirus HCV3 [9], yet the redox dependence of viral envelopes is quite diverse [7], we have studied the role of the disulfides of mature HCV envelope on its function during virus entry
E2 was the focus of the present study because: (i) E2 is the larger of the two envelope proteins, a Ϸ370 residue polypeptide including 18 Cys in its outer membrane region; (ii) E2 mediates virus attachment to cell receptor(s) such as CD81 (10 –12), and by analogy with other viral subunits responsible for receptor binding that exhibit a high Cys content [7], E2 is a candidate-substrate for redox reactions during entry; and (iii) E2 is the target of a neutralizing immune response, anti-E2 antibodies protecting from infection and making the protein a candidate vaccine component [13,14,15,16]
Summary
Materials—Expression of the proteins using the baculovirus system is described in Ref. 19. 22 and 23, the samples were treated with reducing agents (15 min; 25 °C) before dot-blotting and processing to assess the thiol content, as described above. The cells were treated using Bafilomycin A1 (15 nM) prior to incubation with HCVpp (2 h; 4 °C) followed by washing and DTT reduction together with treatment using fusion buffer (130 mM NaCl, 15 mM sodium citrate, 10 mM 2-(N-morpholino)ethanesulfonic acid, 5 mM Hepes) adjusted to various acidic pH levels. Viral supernatants were incubated with Huh7.5 cells for 4 h prior to washing and overlay with medium. The antigens were added (1 g for 90 min at 21 °C) before blocking the protein G-Fc binding domain with calf serum (10% in buffer). Anti-E2 antibody dilution in calf serum (5% in buffer) was used for labeling
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