Expression of hepatitis B virus middle and large surface antigen genes in Saccharomyces cerevisiae.

The hepatitis B virus genome carries the surface antigen (SAg) gene and an open reading frame that encodes two SAg-related polypeptides: SAg with a 55-amino-acid N-terminal extension polypeptide and SAg with a 174-amino-acid N-terminal extension polypeptide. These are termed middle S and large S, respectively. These polypeptides or their glycosylated derivatives have been detected in Dane particles, but their chemical and biological properties have remained largely unknown because of their limited availability. We attempted to produce these proteins in Saccharomyces cerevisiae by placing the coding regions under the control of the promoter of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. Yeast cells carrying middle S and large S coding sequences produced 33,000- and 42,000-dalton products, respectively, each of which reacted with anti-S antibody and bound to polymerized human serum albumin, in accordance with the known properties of pre-S proteins from particles in human sera (K. H. Heermann, U. Goldmann, W. Schwartz, T. Seyffarth, H. Baumgarten, and W. H. Gerlich, J. Virol. 52:396-402, 1984; A. Machida, S. Kishimoto, H. Ohnuma, K. Baba, Y. Ito, H. Miyamoto, G. Funatsu, K. Oda, S. Usuda, S. Togami, T. Nakamura, Y. Miyakawa, and M. Mayumi, Gastroenterology 86:910-918, 1984). The middle S polypeptide is glycosylated and can be assembled into particles whose size and density are similar to those of SAg. However, this polypeptide was highly susceptible to proteolytic degradation into 29,000- and 26,000-dalton polypeptides, of which only the former retained the binding activity to polymerized albumin. The large S polypeptides are nonglycosylated, relatively stable, and do not seem to assemble into particles by themselves.     

Expression of human T-cell leukemia virus type I envelope protein in Saccharomyces cerevisiae

The entire envelope gene of human T-cell leukemia virus type I (HTLV-I) was inserted into an expression vector and expressed under the control of the repressible acid phosphatase promoter in yeast (Saccharomyces cerevisiae). The product in yeast cells was glycosylated into heterodisperse proteins.     

Post-translational alterations in transmembrane topology of the hepatitis B virus large envelope protein.

The preS domain at the N-terminus of the large envelope protein (LHBs) of the hepatitis B virus is involved in (i) envelopment of viral nucleocapsids and (ii) binding to the host cell. While the first function suggests a cytosolic location of the preS domain during virion assembly, the function as an attachment site requires its translocation across the lipid bilayer and final exposure on the virion surface. We compared the transmembrane topology of newly synthesized LHBs in the endoplasmic reticulum (ER) membrane with its topology in the envelope of secreted virions. Protease sensitivity and the absence of glycosylation suggest that the entire preS domain of newly synthesized LHBs remains at the cytosolic side of ER vesicles. However, virions secreted from transfected cell cultures or isolated from the blood of persistent virus carriers expose antibody binding sites and proteolytic cleavage sites of the preS domain at their surface in approximately half of the LHBs molecules. Thus, preS domains appear to be transported across the viral lipid barrier by a novel post-translational translocation mechanism to fulfil a dual function in virion assembly and attachment to the host cell.     

Role of the large hepatitis B virus envelope protein in infectivity of the hepatitis delta virion.

The hepatitis delta virus (HDV) is coated with large (L), middle (M), and small (S) envelope proteins encoded by coinfecting hepatitis B virus (HBV). To study the role of the HBV envelope proteins in the assembly and infectivity of HDV, we produced three types of recombinant particles in Huh7 cells by transfection with HBV DNA and HDV cDNA: (i) particles with an envelope containing the S HBV envelope protein only, (ii) particles with an envelope containing S and M proteins, and (iii) particles with an envelope containing S, M, and L proteins. Although the resulting S-, SM-, and SML-HDV particles contained both hepatitis delta antigen and HDV RNA, only particles coated with all three envelope proteins (SML) showed evidence of infectivity in an in vitro culture system susceptible to HDV infection. We concluded that the L HBV envelope protein, and more specifically the pre-S1 domain, is important for infectivity of HDV particles and that the M protein, which has been reported to bear a site for binding to polymerized albumin in the pre-S2 domain, is not sufficient for infectivity. Our data also show that the helper HBV is not required for initiation of HDV infection. The mechanism by which the L protein may affect HDV infectivity is discussed herein.     

Expression of hepatitis B virus large envelope polypeptide inhibits hepatitis B surface antigen secretion in transgenic mice.

The outer membrane of the hepatitis B virus consists of host lipid and the hepatitis B virus major (p25, gp28), middle (gp33, gp36), and large (p39, gp42) envelope polypeptides. These polypeptides are encoded by a large open reading frame that contains three in-phase translation start codons and a shared termination signal. The influence of the large envelope polypeptide on the secretion of hepatitis B surface antigen (HBsAg) subviral particles in transgenic mice was examined. The major polypeptide is the dominant structural component of the HBsAg particles, which are readily secreted into the blood. A relative increase in production of the large envelope polypeptide compared with that of the major envelope polypeptide led to profound reduction of the HBsAg concentration in serum as a result of accumulation of both envelope polypeptides in a relatively insoluble compartment within the cell. We conclude that inhibition of HBsAg secretion is related to a hitherto unknown property of the pre-S-containing domain of the large envelope polypeptide.     

Mammalian BiP controls posttranslational ER translocation of the hepatitis B virus large envelope protein

The hepatitis B virus L protein forms a dual topology in the endoplasmic reticulum (ER) via a process involving cotranslational membrane integration and subsequent posttranslational translocation of its preS subdomain. Here, we show that preS posttranslocation depends on the action of the ER chaperone BiP. To modulate the in vivo BiP activity, we designed an approach based on overexpressing its positive and negative regulators, ER-localized DnaJ-domain containing protein 4 (ERdj4) and BiP-associated protein (BAP), respectively. The feasibility of this approach was confirmed by demonstrating that BAP, but not ERdj4, destabilizes the L/BiP complex. Overexpressing BAP or ERdj4 inhibits preS posttranslocation as does the reduction of ATP levels. These results hint to a new role of BiP in guiding posttranslational polypeptide import into the mammalian ER.     

Dual topology of the hepatitis B virus large envelope protein: determinants influencing post-translational pre-S translocation

The large (L) envelope protein of the hepatitis B virus (HBV) has the peculiar capacity to form two transmembrane topologies via an as yet uncharacterized process of partial post-translational translocation of its pre-S domain across membranes. In view of a current model that predicts an HBV-specific channel generated during virion envelope assembly to enable pre-S translocation, we have examined parameters influencing L topogenesis by using protease protection analysis of wild-type and mutant L proteins synthesized in transfected cells. We demonstrate that contrary to expectation, all determinants, thought to be responsible for channel formation, are dispensable for pre-S reorientation. In particular, we observed that this process does not require (i) the helper function of the HBV S (small) and M (middle) envelope proteins, (ii) covalent dimer formation of envelope chains, or (iii) either of the three amphipathic transmembrane segments of L. Rather, the most hydrophobic transmembrane segment 2 of L was identified as a vital topogenic determinant, essential and sufficient for post-translational pre-S translocation. Cell fractionation studies revealed that pre-S refolding and thus the dual topology of L is established at the endoplasmic reticulum (ER) membrane rather than at a post-ER compartment as originally supposed. Together our data provide evidence to suggest that the topological reorientation of L is facilitated by a host cell transmembrane transport machinery such as the ER translocon.     

Immunoadhesins containing pre-S domains of hepatitis B virus large envelope protein are secreted and inhibit virus infection

Hepatitis B virus (HBV) replication produces three envelope proteins (L, M, and S) that have a common C terminus. L, the largest, contains a domain, pre-S1, not present on M. Similarly M contains a domain, pre-S2, not present on S. The pre-S1 region has important functions in the HBV life cycle. Thus, as an approach to studying these roles, the pre-S1 and/or pre-S2 sequences of HBV (serotype adw2, genotype A) were expressed as N-terminal fusions to the Fc domain of a rabbit immunoglobulin G chain. Such proteins, known as immunoadhesins (IA), were highly expressed following transfection of cultured cells and, when the pre-S1 region was present, >80% were secreted. The IA were myristoylated at a glycine penultimate to the N terminus, although mutation studies showed that this modification was not needed for secretion. As few as 30 amino acids from the N terminus of pre-S1 were both necessary and sufficient to drive secretion of IA. Even expression of pre-S1 plus pre-S2, in the absence of an immunoglobulin chain, led to efficient secretion. Overall, these studies demonstrate an unexpected ability of the N terminus of pre-S1 to promote protein secretion. In addition, some of these secreted IA, at nanomolar concentrations, inhibited infection of primary human hepatocytes either by hepatitis delta virus (HDV), a subviral agent that uses HBV envelope proteins, or HBV. These IA have potential to be part of antiviral therapies against chronic HDV and HBV, and may help understand the attachment and entry mechanisms used by these important human pathogens.     

Expression, purification and characterization of the Hepatitis B virus entire envelope large protein in Pichia pastoris

The current HBsAg vaccine has performed a vital role in preventing the transmission of HBV during the past 20 years. However, a number of individuals still show no response or a low response to the vaccine. In the present study, the HBV envelope large protein gene was cloned into the eukaryotic expression vector pPIC9k and was subsequently expressed in the yeast Pichia pastoris. The HBV large protein (L protein) was produced and secreted into the medium, where some of the L protein formed particles. The soluble L protein and particles were purified by column chromatography and sucrose density gradient centrifugation. Western blot analysis demonstrated that the particle was composed of both HBV L and S protein. To compare the antigenicity of the L protein and HBsAg, rabbits were immunized with the soluble L protein and the commercially available HBV vaccine and the increasing level of antibodies was determined by ELISA. The results showed that the anti-HBsAg antibody, from rabbits injected with the L protein at a dose of 2 and 10microg, was detected on day 14, whereas rabbits vaccinated with 10 and 2microg HBsAg did not develop antibodies until day 21 and 28, respectively. The antibody level in groups inoculated with the L protein was approximately 50% higher than in the group injected with HBsAg using the same dose. Furthermore, 2microg L protein induced a significant and rapid anti-HBsAg antibody response than 10microg HBsAg. Therefore, we suggest that the L protein is an ideal candidate for a new generation HB vaccine to protect people from HBV infection.     

Hepatitis B virus envelope L protein particles. Synthesis and assembly in Saccharomyces cerevisiae, purification and characterization.

The hepatitis B virus envelope gene encodes three transmembrane proteins in frame; S, the product of S gene; M, the product of M (pre-S2 + S) gene; and L, the product of L (pre-S1 + pre-S2 + S) gene. Unlike the S and M proteins, attempts to efficiently synthesize L proteins and assemble them into L protein particles in various eukaryotic cells have been unsuccessful, probably because of the presence of the pre-S1 peptide with an unknown function which appears to be inhibitory to the host secretory apparatus. To investigate the role of the pre-S1 peptide, we constructed an L gene fused with a synthetic gene for chicken-lysozyme signal peptide (C-SIG) at the 5'-terminal and placed the resultant gene under the control of the yeast glyceraldehyde-3-phosphate dehydrogenase gene promoter. After the fused-C-SIG peptide was correctly processed by the yeast secretory apparatus, a yeast transformant synthesized a protein with a molecular mass of approximately 52 kDa at a level of 42% of the total soluble protein. Electron micrographic observation showed that the gene products assembled into 23-nm spherical and filamentous particles. The pre-S peptide of the gene product was deposited into the endoplasmic reticulum (ER) lumen and well-glycosylated. It seemed that the gene products were accumulated as particles in certain specific membrane structures of the yeast secretory apparatus. Moreover, both the amount of mRNAs specific for the L gene and the in vivo stability of the synthesized L proteins did not change significantly by the addition of the C-SIG gene. These findings indicated that, if the pre-S1 peptide penetrates the ER membrane efficiently, the L proteins can be synthesized cotranslationally, translocate across the ER membrane with its S region, and then assemble by themselves into the particle form. Therefore, the pre-S1 peptide may involve weak or reduced signal peptide activity for recognition by the secretory apparatus and/or for the transport of the pre-S peptide into the ER lumen.     

Determination of the minimal distance between the matrix and transmembrane domains of the large hepatitis B virus envelope protein

The cytosolic matrix domain (MD) located between amino acids (aa) 103 and 124 of the large hepatitis B virus envelope protein L is essential for virion formation. We reduced the distance between MD and the transmembrane domain (TD; aa 254 to 272) by deletions starting at aa 132. Six mutants with deletions of up to aa 234 were wild type, and four mutants with slightly larger deletions were blocked with respect to virion morphogenesis. Thus, the minimal distance between MD and TD was around 26 aa. This spacer might be required by MD to reach contact sites on the capsid.     

Cholesterol depletion of hepatoma cells impairs hepatitis B virus envelopment by altering the topology of the large envelope protein

Previous reports have shown that cholesterol depletion of the membrane envelope of the hepatitis B virus (HBV) impairs viral infection of target cells. A potential function of this lipid in later steps of the viral life cycle remained controversial, with secretion of virions and subviral particles (SVP) being either inhibited or not affected, depending on the experimental approach employed to decrease the intracellular cholesterol level. This work addressed the role of host cell cholesterol on HBV replication, assembly, and secretion, using an alternative method to inhibition of the enzymes involved in the biosynthesis pathway. Growing HBV-producing cells with lipoprotein-depleted serum (LPDS) resulted in an important reduction of the amount of cholesterol within 24 h of treatment (about 40%). Cell exposure to chlorpromazine, an inhibitor of the clathrin-mediated pathway used by the low-density lipoprotein receptor for endocytosis, also impacted the cholesterol level; however, this level of inhibition was not achievable when the synthesis inhibitor lovastatin was used. HBV secretion was significantly inhibited in cholesterol-depleted cells (by ～80%), while SVP release remained unaffected. The viral DNA genome accumulated in LPDS-treated cells in a time-dependent manner. Specific immunoprecipitation of nucleocapsids and mature virions revealed an increased amount of naked nucleocapsids, while synthesis of the envelope proteins occurred as normally. Following analysis of the large envelope protein conformation in purified microsomes, we concluded that cholesterol is important in maintaining the dual topology of this polypeptide, which is critical for viral envelopment.     

Purification and characterization of the hepatitis B virus core antigen produced in the yeast Saccharomyces cerevisiae

Hepatitis B virus core antigen gene was expressed in Saccharomyces cerevisiae and the product (yHBcAg) was purified from a crude lysate of the yeast by three steps: sucrose step-gradient ultracentrifugation, hydroxyapatite chromatography and CsCl-isopycnic ultracentrifugation. yHBcAg was synthesized in yeast cells as a particle consisting of polypeptides which have a molecular weight of 21.5 kDa (p21.5). In the CsCl-density gradient, yHBcAg particles synthesized with the expression vector pYG701c (the GAP promoter) had two peaks, at 1.35 g cm⁻³ (HP; high-density particle) and 1.31 g cm⁻³ (LP; low-density particle). On the other hand, the particles synthesized with expression vector pAC701 (the PHO5 promoter) had only one peak at 1.32 g cm⁻³. The isoelectric points of HP and LP were estimated to be 4.05 and 4.07, respectively. Absorption spectrum analysis showed that the HP contains nucleic acids. yHBcAg particles possessed the immunogenicity of HBcAg and its component polypeptide (p21.5) possessed that of HBeAg in addition to HBcAg. Moreover, Western blotting analysis of p21.5 using a monoclonal antibody against yHBcAg or yHBeAg indicated that the hepatitis B virus C-gene-coded protein shares the antigenic sites responsible for both antibodies.     

St, a truncated envelope protein derived from the S protein of duck hepatitis B virus, acts as a chaperone for the folding of the large envelope protein

Envelope proteins of hepadnaviruses undergo a unique folding mechanism which results in the posttranslational translocation of 50% of the large envelope protein (L) chains across the endoplasmic reticulum. This mechanism is essential for the eventual positioning of the receptor-binding domain on the surface of the virus particle and in duck hepatitis B virus (DHBV) is dependent on the small (S) envelope protein as part of the assembly process. In this study, we report the identification of a third envelope protein, St, derived from the S protein and carrying functions previously attributed to S. Antibody mapping and mutagenesis studies indicated St to be C terminally truncated, spanning the N-terminal transmembrane domain (TM1) plus the adjacent cysteine loop. We have previously shown that the mutation of two conserved polar residues in TM1 of S (SAA) eliminates L translocation and assembly. A plasmid expressing a functional equivalent of St was able to rescue assembly, demonstrating that this assembly defect is due to mutations of the corresponding residues in St and not in S per se. Immunofluorescence analysis showed that St directly affects L protein cellular localization. These results indicate that St acts as a viral chaperone for L folding, remaining associated with the DHBV envelope upon secretion. The presence of St at a molar ratio of half that of L suggests that it is St which regulates L translocation to 50%.     

A hydrophobic domain in the large envelope protein is essential for fusion of duck hepatitis B virus at the late endosome

The duck hepatitis B virus (DHBV) envelope is comprised of two transmembrane (TM) proteins, the large (L) and the small (S), that assemble into virions and subviral particles. Secondary-structure predictions indicate that L and S have three alpha-helical, membrane-spanning domains, with TM1 predicted to act as the fusion peptide following endocytosis of DHBV into the hepatocyte. We used bafilomycin A1 during infection of primary duck hepatocytes to show that DHBV must be trafficked from the early to the late endosome for fusion to occur. Alanine substitution mutations in TM1 of L and S, which lowered TM1 hydrophobicity, were used to examine the role of TM1 in infectivity. The high hydrophobicity of the TM1 domain of L, but not of S, was shown to be essential for virus infection at a step downstream of receptor binding and virus internalization. Using wild-type and mutant synthetic peptides, we demonstrate that the hydrophobicity of this domain is required for the aggregation and the lipid mixing of phospholipid vesicles, supporting the role of TM1 as the fusion peptide. While lipid mixing occurred at pH 7, the kinetics of insertion of the fusion peptide was increased at pH 5, consistent with the location of DHBV in the late-endosome compartment and previous studies of the nonessential role of low pH for infectivity. Exchange of the TM1 of DHBV with that of hepatitis B virus yielded functional, infectious DHBV particles, suggesting that TM1 of all of the hepadnaviruses act similarly in the fusion mechanism.     

Mutations in the carboxyl-terminal domain of the small hepatitis B virus envelope protein impair the assembly of hepatitis delta virus particles

The carboxyl-terminal domain of the small (S) envelope protein of hepatitis B virus was subjected to mutagenesis to identify sequences important for the envelopment of the nucleocapsid during morphogenesis of hepatitis delta virus (HDV) virions. The mutations consisted of carboxyl-terminal truncations of 4 to 64 amino acid residues and small combined deletions and insertions spanning the entire hydrophobic domain between residues 163 and 224. Truncation of as few as 14 residues partially inhibited glycosylation and secretion of S and prevented assembly or stability of HDV virions. Short internal combined deletions and insertions were tolerated for secretion of subviral particles with the exceptions of those affecting residues 164 to 173 and 219 to 223. However, mutants competent for subviral particle secretion had a reduced capacity for HDV assembly compared to that of the wild type. One exception was a mutant carrying a deletion of residues 214 to 218, which exhibited a twofold increase in HDV assembly (or stability), whereas deletions of residues 179 to 183, 194 to 198, and 199 to 203 were the most inhibitory. Substitutions of single amino acids between residues 194 and 198 demonstrated that HDV assembly deficiency could be assigned to the replacement of the tryptophan residue at position 196. We concluded that assembly of stable HDV particles requires a specific function of the carboxyl terminus of S which is mediated at least in part by Trp-196.     

The middle hepatitis B virus envelope protein is not necessary for infectivity of hepatitis delta virus.

The hepatitis delta virus (HDV) envelope contains the large (L), middle (M), and small (S) surface proteins encoded by coinfecting hepatitis B virus. Although HDV-like particles can be assembled with only the S protein in the envelope, the L protein is essential for infectivity in vitro (C. Sureau, B. Guerra, and R. Lanford, J. Virol. 67:366-372, 1993). Here, we demonstrate that the M protein, previously described as carrying a site for binding to polymerized human albumin, is not necessary for infectivity. HDV-like particles coated with the S plus L or the S plus M plus L proteins are infectious in primary cultures of chimpanzee hepatocytes. We conclude that the S and L proteins serve two essential functions in the HDV replication cycle; the S protein ensures the export of the HDV genome from an infected cell by forming a particle, and the L protein ensures its import into a human hepatocyte.