*3.1. Topology of HBsAgS*

The correct folding of HBsAgS depends on two topogenic signal sequences, which determine the orientation of the S-domains in relation to the lipid layer. The insertion of the HBsAgS N-terminus into the ER membrane requires the presence of the topogenic N-terminal signal sequence 1 (transmembrane region 1, TM1, aa 8–22), which is not proteolytically cleaved by the host's peptidases, and allows the translocation of the N-terminus across the ER membrane. The second internal topogenic transmembrane sequence (TM2), which is located between aa 80 and 98, supports the translocation of flanking C-terminal sequences and serves as an anchor to hold the sequence in the membrane [17,55,56]. Both topogenic signal sequences are required for the correct folding of HBsAgS, resulting in the formation of a cytosolic loop (aa 23 to 79), and a loop reaching into the ER lumen (aa 99 to approximately 155), followed by a proposed amphipathic helix (aa 156 to 169) and the hydrophobic C-terminal region (aa 170 to 226) embedded in the ER membrane (Figures 1A and 3) [57]. The luminal loop region of the S-domain is located at the external surface of the mature SVPs and also infectious virions, and harbors the major HBsAg protein epitopes ("a"-determinant, and "d/y", "w/r" determinants) [13]. The hydrophobic C-terminal region (aa 170 to 226) possibly contains two additional transmembrane regions, as indicated by topological models [58,59]. The presence of transmembrane passages is supported by experimental data suggesting that the C-terminal sequences are exposed at internal and external surfaces. The sequence between residues 196 and 201 is important for packaging of hepatitis delta virus (HDV), a satellite of HBV, and therefore expected to be accessible to facilitate the interaction with the HDV ribonucleoprotein complex [60]. A second site between residue 178 and 186 is targeted by an anti-HBs monoclonal antibody, indicating surface exposure [61].

HBsAg proteins contain 14 cysteine (cys) residues located in the S-domain, which are highly conserved among different HBV genotypes; the preS1 and preS2 domains do not contain additional cysteine residues [12,17]. Eight cysteine residues are located in the external loop region, forming disulfide bonds which are important for the integrity of the major antigenic determinants [62,63], such as cys-107 in the external loop for retaining the "a"-determinant specific antigenicity [64]. The identification of HBsAgS oligomers and polymers by electrophoresis under non-reducing conditions suggests that the disulfide bonding is heterogeneous, consistent with the finding that only a fraction of HBsAgS subunits are exclusively linked by disulfide bonds formed between cys-121 and cys-147 (Figure 3) [62,63,65]. Reduction or absence of intermolecular disulfide bridges interferes with the native HBsAgS antigenicity but allows SVP formation [62,65,66].

The S-domain shared by the HBsAg proteins harbors an *N*-glycosylation site at position asparagine-146 (N146), which is partially utilized by the oligosaccharyltransferase [17,67], and hence, HBsAgS proteins are synthesized as unglycosylated p24 and *N*-glycosylated gp27 versions (Figure 1B). HBsAgS p24 and gp27 have identical transmembrane topologies and dimerize without preference for a specific pairing and form heterologous dimers with HBsAgM and HBsAgL [66]. Synthesis and secretion of HBsAgS SVPs do not depend on *N*-glycosylation at position N146 in the external loop region, in contrast to the formation and release of infectious HBV particles [67–70].

**Figure 3.** Proposed model for the formation of the HBsAgS homodimer based on Suffner et al. [57]. Dimerization of two HBsAgS monomers represented in purple and blue is facilitated by interactions of the transmembrane domains (TM2), cytosolic loops (CL) and intermolecular disulfide bridges (S-S) between cysteine residues (red circles) in the external loop region. The facultative *N*-glycosylation site at position N146 is indicated (dark blue). The orientation of the hydrophobic C-terminal region is illustrated in a simplified form.

#### *3.2. Topology of HBsAgM*

The middle HBsAg protein has the same transmembrane topology as HBsAgS, and hence is partly glycosylated at N146 in the S-domain. Translocation of the preS2 domain of HBsAgM is mediated by the topologic signal TM1 located in the S-domain [71]. The translocation event across the ER membrane into the lumen allows the *N*-glycosylation of the asparagine-4 (N4) residue in the preS2 domain, which is always glycosylated [72], resulting in glycoproteins with a molecular weight of 33 kDa and 36 kDa (Figure 1B). In addition to the *N*-glycosylation site in the preS2 domain, the preS2 domain can be partially *O*-glycosylated, depending on the genotype and the presence of the threonine (T37) (Figure 1B) [73]. Similar to HBsAgS, HBsAgM proteins assemble into SVPs and can be secreted independently from other viral proteins [74–77]. HBsAgM, however, is not essential for virion morphogenesis and infectivity [78].
