A short linear sequence in the pre-S domain of the large hepatitis B virus envelope protein required for virion formation.

Envelopment of the hepatitis B virus (HBV) nucleocapsid depends on the large envelope protein L, which is expressed as a transmembrane polypeptide at the endoplasmic reticulum membrane. Previous studies demonstrated that the cytosolic exposure of the N-terminal pre-S domain (174 amino acids) of L was required for virion formation. N-terminal truncations of L up to Arg 103 were tolerated. To map sites in the remaining C-terminal part of pre-S important for virion morphogenesis, a series of 11 L mutants with linker substitutions between Asn 98 and Pro 171 was generated. The mutants formed stable proteins and were secreted in transfected cell cultures, probably as components of subviral hepatitis B surface antigen particles. All four constructs with mutations between Asn 98 and Thr 125 were unable to complement in trans the block in virion formation of an L-negative HBV genome in cotransfected HuH7 cells. These mutants had a transdominant negative effect on virus yield in cotransfections with the wild-type HBV genome. In contrast, all seven mutants with substitutions downstream of Ser 124 were able to envelop the nucleocapsid and to secrete HBV. The sequence between Arg 103 and Ser 124 is highly conserved among different HBV isolates and also between HBV and the woodchuck hepatitis virus. Point mutations in this region introducing alanine residues at conserved positions blocked virion formation, in contrast to mutations at nonconserved residues. These results demonstrate that the pre-S sequence between Arg 103 and Ser 124 has an important function in HBV morphogenesis.

Functions of the internal pre-S domain of the large surface protein in hepatitis B virus particle morphogenesis.

The large hepatitis B virus (HBV) surface protein (L) forms two isomers which display their N-terminal pre-S domain at the internal and external side of the viral envelope, respectively. The external pre-S domain has been implicated in binding to a virus receptor. To investigate functions of the internal pre-S domain, a secretion signal sequence was fused to the N terminus of L (sigL), causing exclusive expression of external pre-S domains. A fusion construct with a nonfunctional signal (s25L), which corresponds in its primary sequence to sigL cleaved by signal peptidase, was used as a control. SigL was N glycosylated in transfected COS cells at both potential sites in pre-S in contrast to s25L or wild-type L, confirming the expected transmembrane topologies of sigL and s25L. Phenotypic characterization revealed the following points. (i) SigL lost the inhibitory effect of L or s25L on secretion of subviral hepatitis B surface antigen particles, suggesting that the retention signal mapped to the N terminus of L is recognized in the cytosol and not in the lumen of the endoplasmic reticulum. (ii) SigL was secreted into the culture medium even in the absence of the major HBV surface protein (S), while release of an L mutant lacking the retention signal was still dependent on S coexpression. (iii) s25L but not sigL could complement an L-negative HBV genome defective for virion secretion in cotransfections. This suggests that the cytosolic pre-S domain, like a matrix protein, is involved in the interaction of the viral envelope with preformed cytosolic nucleocapsids during virion assembly.

Mutational analysis of hepatitis B surface antigen particle assembly and secretion.

Cells infected with hepatitis B virus produce both virions and 20-nm subviral (surface antigen or HBsAg) particles; the latter are composed of viral envelope proteins and host-derived lipid. Although hepatitis B virus encodes three envelope proteins (L, M, and S), all of the information required to produce an HBsAg particle resides within the S protein. This polypeptide spans the bilayer at least twice and contains three hydrophobic regions, two of which are known to harbor topogenic signal sequences that direct this transmembrane orientation. We have examined the effects of mutations in these and other regions of the S protein on particle assembly and export. Lesions in the N terminal signal sequence (signal I) can still insert into the endoplasmic reticulum bilayer but do not participate in any of the subsequent steps in assembly. Deletion of the major internal signal (signal II) completely destabilizes the chain. Deletion of the C-terminal hydrophobic domain results in a stable, glycosylated, but nonsecreted chain. However, when coexpressed with wild-type S protein this mutant polypeptide can be incorporated into particles and secreted, indicating that the chain is still competent for some of the distal steps in particle assembly. The correct transmembrane disposition of the N terminus of the molecule is important for particle formation: addition of a heterologous (globin) domain to this region impairs secretion, but the defect can be corrected by provision of an N-terminal signal sequence that restores the proper topology of this region. The resulting chimeric chain is assembled into subviral particles that are secreted with normal efficiency.

The role of envelope proteins in hepatitis B virus assembly.

Hepatitis B virus (HBV) particles are generated by budding of preformed cytoplasmic nucleocapsids into endoplasmic reticulum (ER) membranes containing the three viral envelope proteins (L, M, and S). We have examined the contributions of the envelope proteins to virion assembly by using cultured hepatoma cells transfected with mutant HBV genomes bearing lesions in the envelope coding regions. We show here that HBV nucleocapsids are not released from cells without expression of envelope proteins, implying an active role for these proteins in viral morphogenesis. S and L but not M proteins are necessary for virion production. L protein over-expression inhibits virion release, just as it inhibits the release of subviral hepatitis B surface antigen (HBsAg) particles. Mutant L proteins that are no longer capable of retaining HBsAg particles in the ER still allow virion formation, indicating that this ER retention reaction is not required for viral budding. Myristoylation of L protein is also dispensable for virion formation. A chimeric protein bearing foreign epitopes fused to the S protein can be incorporated into virions when coexpressed with the wild-type envelope proteins. Models for the dependence of virion formation on both L and S proteins are discussed.

Mapping a region of the large envelope protein required for hepatitis B virion maturation.

The hepatitis B virion is a spherical double-shelled particle carrying three surface proteins (large [L], middle [M], and small [S]) in its envelope. All three proteins are translated from a single open reading frame by means of three different in-frame start codons from unspliced mRNAs. This organization defines three protein domains (pre-S1, pre-S2, and S). All three domains together form the L protein, whereas the M protein consists of domains pre-S2 plus S. The L and S proteins are both necessary for virion production, whereas the M protein is dispensable, suggesting an important function of the pre-S1 domain in virion morphogenesis. To investigate this point, we created a series of N-terminal-truncated L mutants and tested their ability to substitute for the wild-type L protein in virion formation. We found that the constructs fell into two classes, (i) N-terminal deletion mutants lacking up to 102 of the 119 amino acids of the pre-S1 domain still allowed virion maturation, showing that the N-terminal 5/6 of the pre-S1 sequence is dispensable for this process. (ii) Mutants lacking 110 or more N-terminal amino acids were unable to substitute for the L protein in virion assembly, although they were stably expressed and secreted as components of subviral 20-nm hepatitis B surface antigen particles. This suggests that a short C-terminal region of pre-S1 is important for virion formation. Like the wild-type L protein, the mutants of the first class were not glycosylated in their pre-S2 domains; however, this site was used for glycosylation in mutants of the second class, similar to that in the M protein. These findings can be related to a model for the function of the L protein in virion maturation.

Characterization of early hepatitis B virus surface protein oligomers.

The small surface protein (S) of the hepatitis B virus (HBV) is synthesized as unglycosylated p24 and N-glycosylated gp27 and forms disulfide linked dimers. Former models proposed that these complexes consist preferentially of p24-gp27 heterodimers. Furthermore, cell free in vitro experiments suggested that p24 has a transmembrane topology different from gp27. We tested these models by expressing the HBV surface proteins in transfected cell cultures and characterizing early maturation products after short pulse labelings. Two dimensional unreduced-reduced polyacrylamide gel electrophoresis demonstrated that p24 and gp27 dimerized without preference for a specific pairing. Protease protection experiments showed that both, p24 and gp27, had identical transmembrane topologies in cell culture. The middle sized (M) and large HBV surface proteins formed mixed dimers with the S protein. Mutant M and S protein in which all 10 cysteine residues in the ectodomain and transmembrane regions were replaced by serine residues formed no intermolecular S-S bridges but were secreted like wild type M and S protein.

Phenotypic mixing of rodent but not avian hepadnavirus surface proteins into human hepatitis B virus particles.

The virus family Hepadnaviridae comprises two genera: orthohepadnaviruses isolated from humans (hepatitis B virus [HBV]) and rodents (e.g., woodchuck hepatitis virus [WHV]) and avihepadnaviruses isolated from birds (e.g., duck hepatitis B virus [DHBV]). They carry in their envelopes two (DHBV) or three (HBV and WHV) coterminal proteins referred to as small (S), middle (M), or large (L) surface protein. These proteins are also secreted from infected cells as subviral particles consisting of surface protein and lipid (e.g., 20-nm hepatitis B surface antigen for HBV). To investigate the assembly of these proteins, we asked whether surface proteins from different hepadnaviruses are able to mix phenotypically with each other. By coexpression and coimmunoprecipitation with species-specific antibodies, we could show the formation of mixed subviral particles and disulfide-linked heterodimers between the WHV S and HBV M proteins whereas the DHBV and HBV surface proteins did not coassemble. Complementation of HBV genomes defective in expressing the S or L protein and therefore incompetent to form virions was possible with the closely related WHV S protein or a WHV pre-S-HBV S chimera, respectively, but not with the less related DHBV S or L protein or with a DHBV L-HBV S chimera. The results suggest that the assembly of HBV subviral particles and virion envelopes requires relatively precise molecular interactions of their surface proteins, which are not conserved between the two hepadnavirus genera. This contrasts with the ability of, e.g., rhabdoviruses or retroviruses, to incorporate envelope proteins even from unrelated viruses.

Formation of transmembraneous hepatitis B e-antigen by cotranslational in vitro processing of the viral precore protein.

The gene encoding the major core protein P22c of hepatitis B virus is preceded by a precore sequence. Expression of the core gene with the precore in Escherichia coli results in a membrane protein of HBe antigenicity. Expression in mammalian cells generates secreted HBeAg. To study the biosynthetic pathway of HBeAg and the function of precore in this process, we translated mRNAs for core proteins with and without precore using reticulocyte lysates and microsomal vesicles. The precore sequence was cleaved cotranslationally as a signal peptide, probably at alanine 19. The processed product P23e was partially translocated to the lumen of the microsomes. The arginine-rich carboxy-terminal domain of P23e was however not translocated and susceptible to trypsin. Clusters of positive-charged amino acids seem to act as a novel type of translocation stop signal. Trypsin generated a P16e which no longer had a transmembraneous configuration. The findings may explain the biosynthesis and potential function of HBeAg in hepatitis B virus-infected hepatocytes.

Extensive mutagenesis of the hepatitis B virus core gene and mapping of mutations that allow capsid formation.

We generated a large number of mutations in the hepatitis B virus (HBV) core gene inserted into a bacterial expression vector. The new mutagenesis procedure generated deletions and insertions (as sequence repeats) of various lengths at random positions between M1 and E145 but not substitutions. The R-rich 30-amino-acid C-terminal domain was not analyzed. A total of 50,000 colonies were tested with a polyclonal human serum for the expression of hepatitis B core or e antigen. A total of 110 mutants randomly chosen from 1,500 positive colonies were genotyped. Deletions and insertions were clustered in four regions: D2 to E14, corresponding to the N-terminal loop in a model for the core protein fold (B. Bottcher, S. A. Wynne, and R. A. Crowther, Nature 386:88-91, 1997); V27 to P50 (second loop); L60 to V86 (upper half of the alpha helix forming the N-terminal part of the spike and the tip of the spike); and V124 to L140 (C-terminal part of the C-terminal helix and downstream loop). Deletions or insertions in the remaining parts of the molecule forming the compact center of the fold seemed to destabilize the protein. Of the 110 mutations, 38 allowed capsid formation in Escherichia coli. They mapped exclusively to nonhelical regions of the proposed fold. The mutations form a basis for subsequent analysis of further functions of the HBV core protein in the viral life cycle.

Formation of intracellular particles by hepatitis B virus large surface protein.

Hepatitis B virus small surface protein is synthesized as a transmembrane protein of the rough endoplasmic reticulum (RER) and then buds into the lumen in the form of subviral particles that are secreted. The closely related large surface protein is also targeted to the RER but is retained in a pre-Golgi compartment and cannot be secreted. It has been assumed that the large surface protein remains as a transmembrane RER protein and hence cannot form particles, possibly because of binding to a host factor on the cytosolic face of the RER membranes. We have reexamined this question and found the following results. (i) The retained large surface protein is associated not with RER but, rather, with a more distal compartment. (ii) Electron microscopy reveals intravesicular 20-nm particles, similar to those formed by the small surface protein. (iii) The large surface protein colocalizes with and binds to calnexin, an ER chaperone protein. Therefore, our results indicate that the large surface protein is capable of budding and forming particles, and hence its intracellular retention cannot be attributed to a cytosolic factor. We interpret the data as evidence that the large surface protein is retained by virtue of interacting with calnexin, a component of what is considered the quality control mechanism of the ER.

Functions of the large hepatitis B virus surface protein in viral particle morphogenesis.

The hepatitis B virus (HBV) envelope and the subviral lipoprotein particles contain three viral surface proteins (L, M, and S) which are expressed from one open reading frame by the usage of three start codons and a common stop codon. The largest surface protein L has some unusual properties. It adopts two different transmembrane topologies due to a posttranslational switch of the folding in approximately half of the L proteins. L molecules which expose their N-terminal preS1 domain on the viral particle surface are probably ligands for a putative virus receptor and determine the species specificity and liver tropism of this virus. L chains with internal preS1 domains are required in virion morphogenesis and mediate the contact to the nucleocapsid like a matrix protein. Overexpression of this form of the L protein is also responsible for the inhibition of viral particle release. This short review summarizes our knowledge on the biosynthesis and maturation of the HBV surface proteins and their functions in viral particle morphogenesis with special emphasis on the L protein.

The hepatitis B virus large surface protein (LHBs) is a transcriptional activator.

It has been shown that a C-terminally truncated form of the middle-sized hepatitis B virus (HBV) surface protein (MHBst) functions as a transcriptional activator. This function is dependent on the cytosolic orientation of the N-terminal PreS2 domain of MHBst, but in the case of wild-type MHBs, the PreS2 domain is contranslationally translocated into the ER lumen. Recent reports demonstrated that the PreS2 domain of the large HBV surface protein (LHBs) initially remains on the cytosolic side of the ER membrane after translation. Therefore, the question arose as to whether the LHBs protein exhibits the same transcriptional activator function as MHBst. We show that LHBs, like MHBst, is indeed able to activate a variety of promoter elements. There is evidence for a PKC-dependent activation of AP-1 and NF-kappa B by LHBs. Downstream of the PKC the functionality of c-Raf-1 kinase is a prerequisite for LHBs-dependent activation of AP-1 and NF-kappa B since inhibition of c-Raf-1 kinase abolishes LHBs-dependent transcriptional activation of AP-1 and NF-kappa B.

Hepatitis B virus nucleocapsid envelopment does not occur without genomic DNA synthesis.

Assembly of the enveloped hepatitis B virus (HBV) is initiated by packaging of the RNA pregenome and the viral reverse transcriptase-DNA polymerase into a nucleocapsid. The pregenome is then reverse transcribed into single-stranded minus-polarity DNA, which is subsequently replicated to double-stranded DNA. All replicative intermediates are observable in capsids within infected liver, but only relatively mature nucleocapsids containing partially double stranded DNA are found in secreted virions. This observation suggests that maturation of the genome within the capsid is required for envelopment and secretion. We show that the differential distribution of replicative intermediates between intracellular nucleocapsids and secreted virions is also observable in human hepatoma cells transfected with wild-type HBV genomes. However, nucleocapsids were not enveloped or secreted when they were produced by an HBV genome carrying a missense mutation in the DNA polymerase that eliminates all DNA synthesis. An HBV missense mutant defective in the RNase H activity of the polymerase which allowed minus-strand DNA synthesis but not formation of double-stranded DNA was able to form virion-like particles. These experiments demonstrate that immature nucleocapsids containing pregenomic RNA are incompetent for envelopment and that minus-strand DNA synthesis in the interior lumen of the capsid is coupled to the appearance of a signal on the exterior of the nucleocapsid that is essential for its envelopment.

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.

Myristylation of a duck hepatitis B virus envelope protein is essential for infectivity but not for virus assembly.

In addition to the major surface (S) protein, the envelope of the duck hepatitis B virus (DHBV) contains a related presurface (preS) protein whose N-terminus bears a covalently attached myristate group. We have explored the functional significance of this modification by examining the replicative potential of a mutant viral genome whose myristylation signal has been inactivated. Following transfection into permissive hepatoma cells, the mutant expresses an unmyristylated preS protein of normal size, immunoreactivity and stability. Cytoplasmic cores containing viral DNA are synthesized, and Dane particles are assembled and exported into the medium. However, the mutant is noninfectious when inoculated into susceptible ducklings. We conclude that myristylation of preS proteins is essential for hepadnaviral infectivity but not for viral assembly; myristylation is most likely required for an early step of the life cycle involving the entry or uncoating of virus particles.

Myristylation of the large surface protein is required for hepatitis B virus in vitro infectivity.

The large surface protein (L) of the enveloped hepatitis B virus (HBV) is myristylated at glycine 2. To investigate whether this fatty acid moiety is required for HBV infectivity we made use of a point mutant in which the myristyl acceptor was mutated to a nonfunctional alanine. The mutant virus and a wild-type control were expressed in a human hepatoma cell line by transfection of genomic DNA constructs. Comparable amounts of virions were secreted by both strains as measured by the endogenous polymerase activity of immunoprecipitated virions. The presentation of an N-terminal epitope of L on the virion surface was not influenced by the mutation. To test the infectivity of this mutant virus primary human hepatocytes were incubated with the media of transfected cells. The covalently circular closed HBV DNA molecules generated after infection were discriminated from the open circular DNA genomes of inoculated virions by a sensitive PCR-based technique. The experiments demonstrated that the wild type was infectious but not the myristate negative mutant. This reflects the phenotype of an homologous duck hepatitis B virus mutant although the N-terminal L protein domains of this virus and of HBV show no primary sequence homology.

Precore sequence of hepatitis B virus inducing e antigen and membrane association of the viral core protein.

Hepatitis B virus (HBV) DNA contains a precore (pre-c) sequence of 29 codons with unknown function upstream of its gene for the major core protein. Its significance was studied by expression of core proteins with and without pre-c in Escherichia coli. Core protein without pre-c, P22c, assembled spontaneously to core particles and formed core antigen. It had the same size and antigenicity as core particles from infected liver. Core protein with pre-c, P25e, instead formed membrane-associated e antigen (HBeAg). The data suggest that pre-c functions as a signal peptide for the attachment of core protein P25e to cellular membranes. This hypothesis can explain the not yet understood relation between viremia and HbeAg and the protective role of anti-HBe antibody.

Hepatitis B virus core gene mutations which block nucleocapsid envelopment.

Recently we generated a panel of hepatitis B virus core gene mutants carrying single insertions or deletions which allowed efficient expression of the core protein in bacteria and self-assembly of capsids. Eleven of these mutations were introduced into a eukaryotic core gene expression vector and characterized by trans complementation of a core-negative HBV genome in cotransfected human hepatoma HuH7 cells. Surprisingly, four mutants (two insertions [EFGA downstream of A11 and LDTASALYR downstream of R39] and two deletions [Y38-R39-E40 and L42]) produced no detectable capsids. The other seven mutants supported capsid formation and pregenome packaging/viral minus- and plus-strand-DNA synthesis but to different levels. Four of these seven mutants (two insertions [GA downstream of A11 and EHCSP downstream of P50] and two deletions [S44 and A80]) allowed virion morphogenesis and secretion. The mutant carrying a deletion of A80 at the tip of the spike protruding from the capsid was hepatitis B virus core antigen negative but wild type with respect to virion formation, indicating that this site might not be crucial for capsid-surface protein interactions during morphogenesis. The other three nucleocapsid-forming mutants (one insertion [LS downstream of S141] and two deletions [T12 and P134]) were strongly blocked in virion formation. The corresponding sites are located in the part of the protein forming the body of the capsid and not in the spike. These mutations may alter sites on the particle which contact surface proteins during envelopment, or they may block the appearance of a signal for the transport or the maturation of the capsid which is linked to viral DNA synthesis and required for envelopment.

Intracellular retention of hepatitis B virus surface proteins reduces interleukin-2 augmentation after genetic immunizations.

We have previously shown that hepatitis B virus (HBV) surface antigens (HBsAgs) are highly immunogenic after genetic immunization. Compared to the secreted middle HBV surface proteins (MHBs) or small HBV surface proteins (SHBs), the nonsecreted large HBV surface protein (LHBs), however, induced significantly weaker humoral and cellular immune responses that could not be augmented by genetic coimmunizations with cytokine expression plasmids. In order to understand the mechanisms underlying this phenomenon, we examined the effect of coimmunizations with an interleukin-2 (IL-2) DNA expression plasmid on the immunogenicity at the B- and T-cell level of nonsecreted wild-type LHBs, a secreted mutant LHBs, wild-type SHBs, and a nonsecreted mutant SHBs. Coimmunizations of mice with plasmids encoding wild-type SHBs or the secreted mutant LHBs and IL-2 increased anti-HBs responses, helper T-cell proliferative activity and cytotoxic T-lymphocyte killing. By contrast, coimmunizations of plasmids encoding wild-type LHBs or nonsecreted mutant SHBs and IL-2 had no significant effects on immune responses. Interestingly, mice immunized with cytokine expression plasmids 14 days after the injection of the wild-type LHBs plasmid showed augmented immune responses compared to animals simultaneously injected with both expression constructs. Anti-HBs responses in mice injected with plasmids encoding secreted forms of HBsAgs were detectable about 10 days earlier than those in mice immunized with plasmids encoding nonsecreted forms of HBsAgs. Based on these observations, we conclude that cytokines produced by DNA plasmids at the initial site of antigen presentation cannot augment LHBs specific immune responses because LHBs is not produced at high enough levels or is not accessible for uptake by antigen-presenting cells.