Assembly of hepatitis delta virus particles.

Hepatitis delta virus (HDV) is a subviral satellite of hepatitis B virus (HBV). Since the RNA genome of HDV can replicate in cultured cells in the absence of HBV, it has been suggested that the only helper function of HBV is to supply HBV coat proteins in the assembly process of HDV particles. To examine the factors involved in such virion assembly, we transiently cotransfected cells with various hepadnavirus constructs and cDNAs of HDV and analyzed the particles released into the medium. We report that the HDV genomic RNA and the delta antigen can be packaged by coat proteins of either HBV or the related hepadnavirus woodchuck hepatitis virus (WHV). Among the three co-carboxy-terminal coat proteins of WHV, the smallest form was sufficient to package the HDV genome; even in the absence of HDV RNA, the delta antigen could be packaged by this WHV coat protein. Also, of the two co-amino-terminal forms of the delta antigen, only the larger form was essential for packaging.     

Hepadnavirus infection requires interaction between the viral pre-S domain and a specific hepatocellular receptor.

To better define the molecules involved in the initial interaction between hepadnaviruses and hepatocytes, we performed binding and infectivity studies with the duck hepatitis B virus (DHBV) and cultured primary duck hepatocytes. In competition experiments with naturally occurring subviral particles containing DHBV surface proteins, these DNA-free particles were found to interfere with viral infectivity if used at sufficiently high concentrations. In direct binding saturation experiments with radiolabelled subviral particles, a biphasic titration curve containing a saturable component was obtained. Quantitative evaluation of both the binding and the infectivity data indicates that the duck hepatocyte presents about 10(4) high-affinity binding sites for viral and subviral particles. Binding to these productive sites may be preceded by reversible virus attachment to a large number of less specific, nonsaturable primary binding sites. To identify which of the viral envelope proteins is responsible for hepatocyte-specific attachment, subviral particles containing only one of the two DHBV surface proteins were produced in Saccharomyces cerevisiae. In infectivity competition experiments, only particles containing the large pre-S/S protein were found to markedly reduce the efficiency of DHBV infection, while particles containing the small S protein had only a minor effect. Similarly, physical binding of radiolabelled serum-derived subviral particles to primary duck hepatocytes was inhibited well only by the yeast-derived pre-S/S particles. Together, these results strongly support the notion that hepadnaviral infection is initiated by specific attachment of the pre-S domain of the large DHBV envelope protein to a limited number of hepatocellular binding sites.     

A metastable form of the large envelope protein of duck hepatitis B virus: low-pH release results in a transition to a hydrophobic, potentially fusogenic conformation

We have examined the structure and fusion potential of the duck hepatitis B virus (DHBV) envelope proteins by treating subviral particles with deforming agents known to release envelope proteins of viruses from a metastable to a fusion-active state. Exposure of DHBV particles to low pH triggered a major structural change in the large envelope protein (L), resulting in exposure of trypsin sites within its S domain but without affecting the same region in the small surface protein (S) subunits. This conformational change was associated with increased hydrophobicity of the particle surface, most likely arising from surface exposure of the hydrophobic first transmembrane domain (TM1). In the hydrophobic conformation, DHBV particles were able to bind to liposomes and intact cells, while in their absence these particles aggregated, resulting in viral inactivation. These results suggests that some L molecules are in a spring-loaded metastable state which, when released, exposes a previously hidden hydrophobic domain, a transition potentially representing the fusion-active state of the envelope.     

Role of N glycosylation of hepatitis B virus envelope proteins in morphogenesis and infectivity of hepatitis delta virus

Hepatitis delta virus (HDV) particles are coated with the large (L), middle (M), and small (S) hepatitis B virus envelope proteins. In the present study, we constructed glycosylation-defective envelope protein mutants and evaluated their capacity to assist in the maturation of infectious HDV in vitro. We observed that the removal of N-linked carbohydrates on the S, M, and L proteins was tolerated for the assembly of subviral hepatitis B virus (HBV) particles but was partially inhibitory for the formation of HDV virions. However, when assayed on primary cultures of human hepatocytes, virions coated with S, M, and L proteins lacking N-linked glycans were infectious. Furthermore, in the absence of M, HDV particles coated with nonglycosylated S and L proteins retained infectivity. These results indicate that carbohydrates on the HBV envelope proteins are not essential for the in vitro infectivity of HDV.     

Hepatitis B virus subviral envelope particle morphogenesis and intracellular trafficking

Hepatitis B virus (HBV) is unusual in that its surface proteins (small [S], medium, and large [L]) are not only incorporated into the virion envelope but they also bud into empty subviral particles in great excess over virions. The morphogenesis of these subviral envelope particles remains unclear, but the S protein is essential and sufficient for budding. We show here that, in contrast to the presumed model, the HBV subviral particle formed by the S protein self-assembles into branched filaments in the lumen of the endoplasmic reticulum (ER). These long filaments are then folded and bridged for packing into crystal-like structures, which are then transported by ER-derived vesicles to the ER-Golgi intermediate compartment (ERGIC). Within the ERGIC, they are unpacked and relaxed, and their size and shape probably limits further progression through the secretory pathway. Such progression requires their conversion into spherical particles, which occurred spontaneously during the purification of these filaments by affinity chromatography. Small branched filaments are also formed by the L protein in the ER lumen, but these filaments are not packed into transport vesicles. They are transported less efficiently to the ERGIC, potentially accounting for the retention of the L protein within cells. These findings shed light on an important step in the HBV infectious cycle, as the intracellular accumulation of HBV subviral filaments may be directly linked to viral pathogenesis.     

C-Terminal Substitution of HBV Core Proteins with Those from DHBV Reveals That Arginine-Rich (167)RRRSQSPRR(175) Domain Is Critical for HBV Replication

To investigate the contributions of carboxyl-terminal nucleic acid binding domain of HBV core (C) protein for hepatitis B virus (HBV) replication, chimeric HBV C proteins were generated by substituting varying lengths of the carboxyl-terminus of duck hepatitis B virus (DHBV) C protein for the corresponding regions of HBV C protein. All chimeric C proteins formed core particles. A chimeric C protein with 221-262 amino acids of DHBV C protein, in place of 146-185 amino acids of the HBV C protein, supported HBV pregenomic RNA (pgRNA) encapsidation and DNA synthesis: 40% amino acid sequence identity or 45% homology in the nucleic-acid binding domain of HBV C protein was sufficient for pgRNA encapsidation and DNA synthesis, although we predominantly detected spliced DNA. A chimeric C protein with 221-241 and 251-262 amino acids of DHBV C, in place of HBV C 146-166 and 176-185 amino acids, respectively, could rescue full-length DNA synthesis. However, a reciprocal C chimera with 242-250 of DHBV C ((242)RAGSPLPRS(250)) introduced in place of 167-175 of HBV C ((167)RRRSQSPRR(175)) significantly decreased pgRNA encapsidation and DNA synthesis, and full-length DNA was not detected, demonstrating that the arginine-rich (167)RRRSQSPRR(175) domain may be critical for efficient viral replication. Five amino acids differing between viral species (underlined above) were tested for replication rescue; R169 and R175 were found to be important.     

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.     

Structure and Glycosylation Patterns of Surface Proteins from Woodchuck Hepatitis Virus

Woodchucks chronically infected with woodchuck hepatitis virus (WHV) are a valuable model for human hepatitis B virus (HBV) in studies of pathogenesis, immunity, and antiviral therapy. For this reason, substantial efforts to characterize both the similarities and the differences between HBV and WHV are being made. The structure of the WHV surface proteins (WHs proteins) has not yet been adequately elucidated. The bands that would be expected for glycosylated and nonglycosylated small (S) WHs protein are found by sodium dodecyl sulfate gel electrophoresis of purified WHs protein, but the bands corresponding to the middle (M) and large (L) WHs proteins of HBV are not seen at the expected sizes, even though the sequences of the WHV and HBV surface protein genes are 60% homologous. By amino-terminal sequencing we have identified two bands at 41 and 45 kDa as the MWHs proteins, 8 kDa larger than expected. We have also confirmed that two bands at 24 and 27 kDa are SWHs proteins. A protein of 49 kDa was blocked at the N terminus, which using immunoblotting with an antiserum against WHV pre-S1 (positions 126 to 146) was identified, together with a part of the 45-kDa protein, as glycosylated and nonglycosylated LWHs protein of the expected size. Sialidase and O-glycosidase digestion showed that the larger size of MWHs protein results from the presence of O glycoside groups which are probably in the pre-S2 domain of MWHs protein. Since the pre-S2 domains of HBV and WHV have similar numbers of potential O glycosylation sites, it appears to be likely that the glycosyltransferases act differently on the viral proteins in woodchucks and humans.     

Topology of the large envelope protein of duck hepatitis B virus suggests a mechanism for membrane translocation during particle morphogenesis.

We have investigated the membrane topology of the large envelope protein of duck hepatitis B virus (DHBV) by protease protection and Western blot analysis, using monoclonal antibodies specific for the pre-S and S regions of the DHBV envelope to characterize protease-resistant polypeptides. These studies showed that DHBV L protein exhibits a mixed membrane topology similar to that of human hepatitis B virus L, with approximately half of the L molecules displaying pre-S on the surface of virus particles and the remainder with pre-S sequestered inside the virus envelope. The C-terminal region of DHBV pre-S was susceptible to protease digestion on all DHBV particle L protein, indicating that this region was externally disposed. DHBV L protein pre-S was entirely cytosolic immediately after synthesis. Our data, therefore, suggested that an intermediate form of the DHBV L molecule exists in mature envelope particles in which L is partially translocated or exists in a translocation-ready conformation. Incubation of virus particles at low pH and 37 degrees C triggered conversion of this intermediate into a fully translocated form. We have proposed a model for pre-S translocation based on our results that invokes the presence of an aqueous pore in the virus envelope, most likely created by oligomerization of transmembrane domains in the S region. The model predicts that pre-S is transported through this pore and that a loop structure is formed because the N terminus remains anchored to the inner face of the membrane. This translocation process occurs during particle morphogenesis and may also be a prerequisite to virus uncoating during infection.     

Analysis of host range phenotypes of primate hepadnaviruses by in vitro infections of hepatitis D virus pseudotypes

Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) have natural host ranges that are limited to closely related species. The barrier for infection of primates seems to be at the adsorption and/or entry steps of the viral replication cycle, since a human hepatoma cell line is permissive for HBV and WMHBV replication following transfection of cloned DNA. We hypothesized that the HBV and WMHBV envelope proteins contain the principal viral determinants of host range. As previously shown by using the hepatitis D virus (HDV) system, recombinant HBV-HDV particles were infectious in chimpanzee as well as human hepatocytes. We extended the HDV system to include HDV particles pseudotyped with the WMHBV envelope. In agreement with the natural host ranges of HBV and WMHBV, in vitro infections demonstrated that HBV-HDV and WM-HDV particles preferentially infected human and spider monkey cells, respectively. Previous studies have implicated the pre-S1 region of the large (L) envelope protein in receptor binding and host range; therefore, recombinant HDV particles were pseudotyped with the hepadnaviral envelopes containing chimeric L proteins with the first 40 amino acids from the pre-S1 domain exchanged between HBV and WMHBV. Surprisingly, addition of the human amino terminus to the WMHBV L protein increased infectivity on spider monkey hepatocytes but did not increase infectivity for human hepatocytes. Based upon these data, we discuss the possibility that the L protein may be comprised of two domains that affect infectivity and that sequences downstream of residue 40 may influence host range and receptor binding or entry.     

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.     

Primary human hepatocytes are susceptible to infection by hepatitis delta virus assembled with envelope proteins of woodchuck hepatitis virus

Hepatitis B virus (HBV) and hepatitis delta virus (HDV) share the HBV envelope proteins. When woodchucks chronically infected with woodchuck hepatitis virus (WHV) are superinfected with HDV, they produce HDV with a WHV envelope, wHDV. Several lines of evidence are provided that wHDV infects not only cultured primary woodchuck hepatocytes (PWH) but also primary human hepatocytes (PHH). Surprisingly, HBV-enveloped HDV (hHDV) and wHDV infected PHH with comparable efficiencies; however, hHDV did not infect PWH. The basis for these host range specificities was investigated using as inhibitors peptides bearing species-specific pre-S (where S is the small envelope protein) sequences. It was found that pre-S1 contributed to the ability of wHDV to infect both PHH and PWH. In addition, the inability of hHDV to infect PWH was not overcome using a chimeric form of hHDV containing WHV S protein, again supporting the essential role of pre-S1 in infection of target cells. One interpretation of these data is that host range specificity of HDV is determined entirely by pre-S1 and that the WHV and HBV pre-S1 proteins recognize different receptors on PHH.     

Antibody Responses during Hepatitis B Viral Infection

Hepatitis B is a DNA virus that infects liver cells and can cause both acute and chronic disease. It is believed that both viral and host factors are responsible for determining whether the infection is cleared or becomes chronic. Here we investigate the mechanism of protection by developing a mathematical model of the antibody response following hepatitis B virus (HBV) infection. We fitted the model to data from seven infected adults identified during acute infection and determined the ability of the virus to escape neutralization through overproduction of non-infectious subviral particles, which have HBs proteins on their surface, but do not contain nucleocapsid protein and viral nucleic acids. We showed that viral clearance can be achieved for high anti-HBV antibody levels, as in vaccinated individuals, when: (1) the rate of synthesis of hepatitis B subviral particles is slow; (2) the rate of synthesis of hepatitis B subviral particles is high but either anti-HBV antibody production is fast, the antibody affinity is high, or the levels of pre-existent HBV-specific antibody at the time of infection are high, as could be attained by vaccination. We further showed that viral clearance can be achieved for low equilibrium anti-HBV antibody levels, as in unvaccinated individuals, when a strong cellular immune response controls early infection.     

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.     

Properties of subviral particles of hepatitis B virus

In the sera of patients infected with hepatitis B virus (HBV), in addition to infectious particles, there is an excess (typically 1,000- to 100,000-fold) of empty subviral particles (SVP) composed solely of HBV envelope proteins in the form of relatively smaller spheres and filaments of variable length. Hepatitis delta virus (HDV) assembly also uses the envelope proteins of HBV to produce an infectious particle. Rate-zonal sedimentation was used to study the particles released from liver cell lines that produced SVP only, HDV plus SVP, and HBV plus SVP. The SVP made in the absence of HBV or HDV were further examined by electron microscopy. They bound efficiently to heparin columns, consistent with an ability to bind cell surface glycosaminoglycans. However, unlike soluble forms of HBV envelope protein that were potent inhibitors, the SVP did not inhibit the ability of HBV and HDV to infect primary human hepatocytes.     

Novel function of the endoplasmic reticulum degradation‐enhancing α‐mannosidase‐like proteins in the human hepatitis B virus life cycle,mediated by the middle envelope protein

Cells replicating the human hepatitis B virus (HBV) express high levels of degradation‐enhancing α‐mannosidase‐like proteins (EDEMs), a family of proteins involved in the endoplasmic reticulum associated degradation, one of the pathways activated during the unfolded protein response. Owing to their α‐1,2 mannosidase activity, the EDEM1–3 proteins are able to process the N‐linked glycans of misfolded or incompletely folded proteins, providing the recognition signal for their subsequent degradation. The HBV small (S), medium (M), and large (L) surface proteins bear an N‐linked glycosylation site in the common S domain that is partially occupied in all proteins. The M protein contains an additional site in its preS2 domain, which is always functional. Here, we report that these oligosaccharides are processed by EDEMs, more efficiently by EDEM3, which induces degradation of L and S proteins, accompanied by a reduction of subviral particles production. In striking contrast, M not only is spared from degradation but its trafficking is also accelerated leading to an improved secretion. This unusual behavior of the M protein requires strictly the mannose trimming of the preS2 N‐linked glycan. Furthermore, we show that HBV secretion is significantly inhibited under strong endoplasmic reticulum stress conditions when M expression is prevented by mutagenesis of the viral genome. These observations unfold unique properties of the M protein in the HBV life cycle during unfolded protein response and point to alternative mechanisms employed by EDEMs to alleviate this stress in case of necessity by promoting glycoprotein trafficking rather than degradation.     

Isolation and characterization of a hepatitis B virus endemic in herons.

A new hepadnavirus (designated heron hepatitis B virus [HHBV]) has been isolated; this virus is endemic in grey herons (Ardea cinerea) in Germany and closely related to duck hepatitis B virus (DHBV) by morphology of viral particles and size of the genome and of the major viral envelope and core proteins. Despite its striking similarities to DHBV, HHBV cannot be transmitted to ducks by infection or by transfection with cloned viral DNA. After the viral genome was cloned and sequenced, a comparative sequence analysis revealed an identical genome organization of HHBV and DHBV (pre-C/C-, pre-S/S-, and pol-ORFs). An open reading frame, designated X in mammalian hepadnaviruses, is not present in DHBV. DHBV and HHBV differ by 21.6% base exchanges, and thus they are less closely related than the two known rodent hepatitis B viruses (16.4%). The nucleocapsid protein and the 17-kilodalton envelope protein sequences of DHBV and HHBV are well conserved. In contrast, the pre-S part of the 34-kilodalton envelope protein which is believed to mediate virus attachment to the cell is highly divergent (less than 50% homology). The availability of two closely related avian hepadnaviruses will now allow us to test recombinant viruses in vivo and in vitro for host specificity-determining sequences.     

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.     

Sulfamoylbenzamide Derivatives Inhibit the Assembly of Hepatitis B Virus Nucleocapsids

Chronic hepatitis B virus (HBV) infection, a serious public health problem leading to cirrhosis and hepatocellular carcinoma, is currently treated with either pegylated alpha interferon (pegIFN-α) or one of the five nucleos(t)ide analogue viral DNA polymerase inhibitors. However, neither pegIFN-α nor nucleos(t)ide analogues are capable of reliably curing the viral infection. In order to develop novel antiviral drugs against HBV, we established a cell-based screening assay by using an immortalized mouse hepatocyte-derived stable cell line supporting a high level of HBV replication in a tetracycline-inducible manner. Screening of a library consisting of 26,900 small molecules led to the discovery of a series of sulfamoylbenzamide (SBA) derivatives that significantly reduced the amount of cytoplasmic HBV DNA. Structure-activity relationship studies have thus far identified a group of fluorine-substituted SBAs with submicromolar antiviral activity against HBV in human hepatoma cells. Mechanistic analyses reveal that the compounds dose dependently inhibit the formation of pregenomic RNA (pgRNA)-containing nucleocapsids of HBV but not other animal hepadnaviruses, such as woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV). Moreover, heterologous genetic complementation studies of capsid protein, DNA polymerase, and pgRNA between HBV and WHV suggest that HBV capsid protein confers sensitivity to the SBAs. In summary, SBAs represent a novel chemical entity with superior activity and a unique antiviral mechanism and are thus warranted for further development as novel antiviral therapeutics for the treatment of chronic hepatitis B.     

Persistence of the Recombinant Genomes of Woodchuck Hepatitis Virus in the Mouse Model

Hydrodynamic injection (HI) with a replication competent hepatitis B virus (HBV) genome may lead to transient or prolonged HBV replication in mice. However, the prolonged HBV persistence after HI depends on the specific backbone of the vector carrying HBV genome and the genetic background of the mouse strain. We asked whether a genetically closely related hepadnavirus, woodchuck hepatitis virus (WHV), may maintain the gene expression and replication in the mouse liver after HI. Interestingly, we found that HI of pBS-WHV1.3 containing a 1.3 fold overlength WHV genome in BALB/c mouse led to the long presence of WHV DNA and WHV proteins expression in the mouse liver. Thus, we asked whether WHV genome carrying foreign DNA sequences could maintain the long term gene expression and persistence. For this purpose, the coding region of HBV surface antigen (HBsAg) was inserted into the WHV genome to replace the corresponding region. Three recombinant WHV-HBV genomes were constructed with the replacement with HBsAg a-determinant, major HBsAg, and middle HBsAg. Serum HBsAg, viral DNA, hepatic WHV protein expression, and viral replication intermediates were detected in mice after HI with recombinant genomes. Similarly, the recombinant genomes could persist for a prolonged period of time up to 45 weeks in mice. WHV and recombinant WHV-HBV genomes did not trigger effective antibody and T-cell responses to viral proteins. The ability of recombinant WHV constructs to persist in mice is an interesting aspect for the future investigation and may be explored for in vivo gene transfer.