Analysis of intermolecular disulfide bonds and free sulfhydryl groups in hepatitis B surface antigen particles

Abstract

The envelope proteins of hepadnaviruses are highly cross-linked by disulfide bonds in complete virions and 20 nm subviral envelope particles. We have previously shown which of the cysteines in the envelope proteins of the human hepatitis B virus (HBV) are essential for assembly and secretion of 20 nm particles and for the structure of the major antigenic determinants (HBsAg). Now we have analyzed the intermolecular disulfide bonds between S proteins. We have constructed mutants lacking cysteines and have analyzed their capacity for oligomerization in COS-7 cells. We demonstrate that C121 and C147 located in the second hydrophilic region carrying the major antigenic determinants of the HBV S protein participate in intermolecular disulfide bonding. A disulfide bond involving C124 blocks the accessibility of arginine/lysine at position 122, as shown by trypsin digestion of cysteine mutants. Alkylation studies using N-ethyl-maleimide indicate that C76, C90, and/or C221 carry the only free sulfhydryl group(s) present in 20 nm particles secreted from cell lines.

Entry of Hepatitis Delta Virus Requires the Conserved Cysteine Residues of the Hepatitis B Virus Envelope Protein Antigenic Loop and Is Blocked by Inhibitors of Thiol-Disulfide Exchange

“…As shown previously (25)(26)(27)42), the AGL disulfide network that crosslinks the HBV envelope proteins is essential to structure the surface-exposed "a" determinant. However, the latter is not an absolute requirement for particle morphogenesis and secretion, which suggests that its strict conservation among all HBV genotypes is linked to a function at viral entry and, eventually, to a binding event.…”

“…1) (25)(26)(27)42). To study the importance of AGL cysteines at viral entry, we used the HDV/HepaRG model (19,21), in which envelope proteins carrying cysteine substitutions were assayed for function at viral entry at the surface of HDV virions.…”

Maturation of Recombinant Hepatitis B Virus Surface Antigen Particles

“…7,14 Although there are no definitive assignments on Figure 1, the chromatographically purified Sample A is a process intermediate and the most upstream sample in the study. The subsequent treatment steps (KSCN treatment followed by dialysis back into PBS, followed by heat treatment and prolonged storage) are intended to demonstrate the different degrees of rHBsAg maturation or aging.…”

“…b Wampler et al, 1985. the exact disulfide bond pairings in rHBsAg, C121 and C147 are proposed to be involved in intermolecular disulfides. 14 Multiple intermolecular disulfides are formed in the lipid-containing particles to cross-link the adjacent monomers together to form dimers and high-order oligomers, 13 coupled with structural changes within the subunits or at the interface. Higher degrees of cross-linking correlate with higher conformational stability and lower conformational flexibility in the mature or aged antigen preparations.…”

Toward the development of monoclonal antibody-based assays to probe virion-like epitopes in hepatitis B vaccine antigen

“…As previously reported, cross-linking, likely coupled with conformational consolidation which can be probed with conformation-sensitive mAbs, is important for both antigenicity and immunogenicity of theses in vitro assembled VLPs. 12,[28][29][30] In early years of vaccine development, HBsAg VLP characterization was performed mainly with SDS-PAGE gel to show the degree of cross-linking, lacking information on 3-dimensional features of key epitopes. 19 Now with different anti-HBsAg mAbs against various epitopes available, the proposed assays with mAbs of choice can be performed on process intermediates or final products, yielding quantitative data and orthogonal information on antigen content ("mass ELISA") or integrity of clinically relevant epitopes (e.g., RF-1, or A1.2-like mAb binding activity, a surrogate marker for vaccine clinical efficacy).…”