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.

bThe product of the human cytomegalovirus (HCMV) UL71 gene is conserved throughout the herpesvirus family. During HCMV infection, protein pUL71 is required for efficient virion egress and is involved in the final steps of secondary envelopment leading to infectious viral particles. We found strong indications for oligomerization of pUL71 under native conditions when recombinant pUL71 was negatively stained and analyzed by electron microscopy. Oligomerization of pUL71 during infection was further verified by native and reducing polyacrylamide gel electrophoresis (PAGE). By in silico analyses of the pUL71 sequence, we noticed a basic leucine zipper (bZIP)-like domain, which might serve as an oligomerization domain. We demonstrated the requirement of the bZIP-like domain for pUL71 oligomerization by coimmunoprecipitation and bimolecular fluorescence complementation using a panel of pUL71 mutants. These studies revealed that the mutation of two leucine residues is sufficient to abrogate oligomerization but that intracellular localization of pUL71 was unaffected. To investigate the relevance of the bZIP domain in the viral context, recombinant viruses carrying mutations identical to those in the panel of pUL71 mutants were generated. bZIP-defective viral mutants showed impaired viral growth, a small-plaque phenotype, and an ultrastructural phenotype similar to that of the previously described UL71 stop mutant virus. The majority of virus particles within the viral assembly compartment exhibited various stages of incomplete envelopment, which is consistent with the growth defect for the bZIP mutants. From these data we conclude that the bZIP-like domain is required for oligomerization of pUL71, which seems to be essential for correct envelopment of HCMV.