Hepatitis B Virus Surface Antigen Assembly Function Persists when Entire Transmembrane Domains 1 and 3 Are Replaced by a Heterologous Transmembrane Sequence

Abstract

Native hepatitis B surface antigen (HBsAg) spontaneously assembles into 22-nm subviral particles. The particles are lipoprotein micelles, in which HBsAg is believed to span the lipid layer four times. The first two transmembrane domains, TM1 and TM2, are required for particle assembly. We have probed the requirements for particle assembly by replacing the entire first or third TM domain of HBsAg with the transmembrane domain of HIV gp41. We found that either TM domain of HBsAg could be replaced, resulting in HBsAg-gp41 chimeras that formed particles efficiently. HBsAg formed particles even when both TM1 and TM3 were replaced with the gp41 domain. The results indicate remarkable flexibility in HBsAg particle formation and provide a novel way to express heterologous membrane proteins that are anchored to a lipid surface by their own membrane-spanning domain. The membrane-proximal exposed region (MPER) of gp41 is an important target of broadly reactive neutralizing antibodies against HIV-1, and HBsAg-MPER particles may provide a good platform for future vaccine development.HBsAg is the surface antigen of hepatitis B virus. In the virus, translation of the same open reading frame can begin at one of three in-frame start codons, resulting in proteins of large (42 kDa), medium (33 kDa), and small size (27 kDa), which share the identical 226-amino-acid sequence at the carboxyl end. The 27-kDa form of HBsAg is fully capable of spontaneous assembly into virus-like particles, so we have focused on this protein to study the requirements for particle assembly. In nature, small HBsAg participates in forming at least four different kinds of particles: 42-nm infectious hepatitis B virions (Dane particles), 22-nm spherical subviral particles, 22-nm-diameter rods, or as the borrowed envelope protein in 36-nm hepatitis D virus (39, 45).Recombinant-derived small HBsAg forms subviral particles when expressed in a number of different cell types, including mammalian (39), yeast (25), and insect cells (19), but not Escherichia coli (25,26). The particles are lipoprotein micelles, which contain 25% lipid. In these particles, HBsAg is believed to span the lipid four times, with four transmembrane domains (TM1 to TM4). This conformation exposes three external domains on the lipid surface at the amino end, middle, and carboxyl end of the protein (7, 42). Prior studies, based on the deletion of membrane-spanning domains, showed that transmembrane domains TM1 and TM2 were required for particle formation (5, 6). However, subsequent studies have shown that, at least for TM1, the replacement of amino acids is permitted (21,33). In this paper, we have substituted an entire TM domain of another transmembrane protein (HIV gp41) for TM1 or TM3 of HBsAg and probed the requirements for particle assembly.When other proteins were expressed in tandem with HBsAg, the surface antigen acted as a carrier protein and incorporated proteins as large as gp120 into the particles (2). This method allowed us to express membrane proteins and transmembrane proteins...

Role of Transmembrane Domains of Hepatitis B Virus Small Surface Proteins in Subviral-Particle Biogenesis

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