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

Abstract

The hepatitis B virus (HBV) surface proteins not only are incorporated into the virion envelope but in addition form subviral particles (SVP) consisting solely of surface proteins and lipids. Heterologous expression of the small HBV envelope protein S produces secreted spherical SVP 20 nm in diameter, with approximately 100 S molecules per particle. The pathway leading from the initial S translation product as a multispanning transmembrane protein to the final SVP is largely unknown. To investigate the role of the four transmembrane domains (TM) of S in this process, we introduced mutations in these regions and characterized their effects on SVP formation in transfected Huh7 cells. We found that the insertion of one amino acid in the center of the ␣-helix of TM1 or the exchange of TM1 with a heterologous TM blocked SVP release and SVP formation by coexpressed wildtype S chains in a transdominant negative fashion. Surprisingly, this effect was partially neutralized when the mutations were expressed in the background of the HBV surface protein M, suggesting that mutations in TM1 could partially be complemented by the pre-S2 domain. The exchange of TM2 with heterologous TMs that form ␣-helices of the same lengths was also incompatible with SVP formation. However, these mutants no longer blocked SVP formation by coexpressed wild-type S. We conclude that TM2 is essential for the stable assembly of S chains by establishing intramembrane interactions. Hepatitis B virus (HBV) causes chronic infections of the liver in more than 350 million people worldwide and an increased risk for the development of hepatocellular carcinoma in these individuals (1). Today's options for the treatment of persistent HBV infections are unsatisfying. Therefore, the prevention of HBV infections is particularly important and can be achieved by active vaccination. The most common vaccine consists of the major HBV envelope protein S expressed in yeast. This protein is peculiar as it not only is incorporated into the envelope of virions which have a diameter of 42 nm but, together with host lipids, also assembles into spherical or filamentous lipoprotein particles of 20 nm in diameter (2). Such subviral particles (SVP) are secreted in great excess over virions from infected hepatocytes. Spherical SVP containing approximately 100 protein molecules are also formed by cells expressing the S protein in the absence of any other HBVencoded protein and represent the antigenic component of HBV vaccines.The gene encoding the S protein constitutes the 3= end of a longer continuous open reading frame that is divided into the regions pre-S1, pre-S2, and S. Two further HBV envelope proteins, M and L, are encoded by the pre-S2 plus S and by the pre-S1 plus pre-S2 plus S sequences, respectively. Therefore, all three proteins share the S domain at their C termini. Virion envelopes contain the three proteins S, M, and L at a ratio of approximately 4:1:1. Natural spherical SVP contain mainly S proteins and some M proteins but only small amounts of L, while filamen...

Specific Amino Acid Substitutions in the S Protein Prevent Its Excretion In Vitro and May Contribute to Occult Hepatitis B Virus Infection

“…Previous studies have shown that both virions and HBsAg secretion were affected by mutations within three of the putative transmembrane (TM) alpha-helix domains TM1, TM2, and TM4 of the S protein (18,22,23). Mutations in TM2 and TM4 may affect (i) possible intramolecular interactions between TM domains within the S protein, resulting in altered protein folding and defective insertion into the ER membrane, or (ii) intermolecular interactions with the peptide chains of other S proteins essential for HBsAg morphogenesis (11,23,24).…”

“…Mutations in TM2 and TM4 may affect (i) possible intramolecular interactions between TM domains within the S protein, resulting in altered protein folding and defective insertion into the ER membrane, or (ii) intermolecular interactions with the peptide chains of other S proteins essential for HBsAg morphogenesis (11,23,24). In the present study, the P178R substitution in the N-terminal part of the putative TM3 prevented HBsAg protein excretion in the two distinct HK3110-cl4 and HK3475-cl6 OBI clones and in the mutated M88-cl4 control.…”

Phylogeny of Host Response Proteins Activated in Silkworm Bombyx mori in Response to Infestation by Dipteran Endoparasitoid Revealed Functional Divergence and Temporal Molecular Adaptive Evolution

“…Notch showed eight TM regions, AIF with four, Toll with three and trehalase with two TM regions. The TM domains prevent proteins moving out of the locale [39] and are essential for transmembrane interactions and stability [40] thereby enhances immune reactions at specific infected locations. Divergence and mutations in these regions affect functional variability of the immune proteins [41].…”

“…All proteins of Cluster A were with TM regions indicating functional significance of TM region in the immune proteins by enhancing the transmembrane interactions [40,56,77]. An exception in this cluster was apoptotic enzyme Nedd2-like caspase, which possess TM region however without signal peptide.…”

Immunoprevalence and Immunodominance of HLA-Cw∗0801-Restricted T Cell Response Targeting the Hepatitis B Virus Envelope Transmembrane Region

“…Experimental analysis of the role of the transmembrane domains in the production of HBV surface protein has clearly shown that mutations introduced within the first transmembrane domain (at positions 172 and 178) completely blocked S protein synthesis and subviral particle production (17). Together with the demonstration that this domain is also crucial for infectivity (16), this means that it is unlikely that HBV will escape CTL pressure that targets this region due to the major inherent viral fitness cost, making the Env 171-180 epitope a T cell response with important antiviral potential.…”