Domains of the Hepatitis B Virus Small Surface Protein S Mediating Oligomerization

Abstract

During hepatitis B virus (HBV) infections, subviral particles (SVP) consisting only of viral envelope proteins and lipids are secreted. Heterologous expression of the small envelope protein S in mammalian cells is sufficient for SVP generation. S is synthesized as a transmembrane protein with N-terminal (TM1), central (TM2), and hydrophobic C-terminal (HCR) transmembrane domains. The loops between TM1 and TM2 (the cytosolic loop [CL]) and between TM2 and the HCR (the luminal loop [LL]) are located in the cytosol and the endoplasmic reticulum (ER) lumen, respectively. To define the domains of S mediating oligomerization during SVP morphogenesis, S mutants were characterized by expression in transiently transfected cells. Mutation of 12 out of 15 amino acids of TM1 to alanines, as well as the deletion of HCR, still allowed SVP formation, demonstrating that these two domains are not essential for contacts between S proteins. Furthermore, the oligomerization of S was measured with a fluorescence-activated cell sorter (FACS)-based Förster resonance energy transfer (FRET) assay. This approach demonstrated that the CL, TM2, and the LL independently contributed to S oligomerization, while TM1 and the HCR played minor roles. Apparently, intermolecular homo-oligomerization of the CL, TM2, and the LL drives S protein aggregation. Detailed analyses revealed that the point mutation C65S in the CL, the mutation of 13 out of 19 amino acids of TM2 to alanine residues, and the simultaneous replacement of all 8 cysteine residues in the LL by serine residues blocked the abilities of these domains to support S protein interactions. Altogether, specific domains and residues in the HBV S protein that are required for oligomerization and SVP generation were defined. The small hepatitis B virus envelope protein S has the intrinsic ability to direct the morphogenesis of spherical 20-nm subviral lipoprotein particles. Such particles expressed in yeast or mammalian cells represent the antigenic component of current hepatitis B vaccines. Our knowledge about the steps leading from the initial, monomeric, transmembrane translation product of S to SVP is very limited, as is our information on the structure of the complex main epitope of SVP that induces the formation of protective antibodies after vaccination. This study contributes to our understanding of the oligomerization process of S chains during SVP formation and shows that the cytoplasmic loop, one membrane-embedded domain, and the luminal loop of S independently drive S-S oligomerization.

Hepatitis B subviral envelope particles use the COPII machinery for intracellular transport via selective exploitation of Sec24A and Sec23B

“…In principal, the four Sec24 paralogs recognize ER export motifs of cargo proteins on the cytosolic side of the ER membrane (Barlowe & Helenius, ; Bethune & Wieland, ; Miller & Schekman, ; Wendeler et al, ). According to the model for the transmembrane topology of the S monomer at the ER membrane (Prange, ; Stirk, Thornton, & Howard, ; Suffner et al, ), we suspected its two cytosolic loops as candidate binding regions for Sec24A (Figure b). Because previous deletion analyses had shown that residues including the second cytosolic loop (i.e., aa 172 to 226) are dispensable for S assembly and secretion (Suffner et al, ), we focused on the first cytosolic loop connecting TM segments I and II.…”

“…According to the model for the transmembrane topology of the S monomer at the ER membrane (Prange, ; Stirk, Thornton, & Howard, ; Suffner et al, ), we suspected its two cytosolic loops as candidate binding regions for Sec24A (Figure b). Because previous deletion analyses had shown that residues including the second cytosolic loop (i.e., aa 172 to 226) are dispensable for S assembly and secretion (Suffner et al, ), we focused on the first cytosolic loop connecting TM segments I and II. Intriguingly, this loop is terminated by two consecutive arginine residues (R78 and R79) that partly resemble known dibasic ER export motifs (Dong et al, ).…”

Hepatitis B Virus (HBV) Subviral Particles as Protective Vaccines and Vaccine Platforms

“…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].…”

“…Approximately 100 HBsAgS proteins assemble with lipids into lipoprotein particles, fifty HBsAgS dimers were identified in SVPs purified from sera of transgenic mice [49,89,90]. Three different regions of the S-domain contribute to the oligomerization of the HBsAg proteins, the cytosolic loop, TM2, and the luminal loop ( Figure 3) [57]. The SVPs are compact particles with a reported density of 1.21 g/mL in caesium chloride (CsCl) compared to a density of infectious virions between 1.24 and 1.26 g/mL [23,91,92].…”

“…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.…”