Monitoring of intracellular localization of Hepatitis B virus P22 protein using Laser Scanning Confocal Microscopy and Airyscan.

The aim of this study was to assess nucleo-cytoplasmic protein localization to better understand the exact intracellular localization of viral proteins involved with infections. Having determined the general protein localization of hepatitis B virus P22 precore protein, the aim was to more specifically resolve its intracellular organization. This was done using both laser scanning microscopy and Airyscan techniques. Using a 63× objective, the resolution obtained with Airyscan was increased by 1.5-fold as compared to confocal microscopy (p value <.00001).

Hepatitis B core protein promotes liver cancer metastasis through miR-382-5p/DLC-1 axis.

The hepatitis B virus core protein (HBc), also named core antigen, is well-known for its key role in viral capsid formation and virus replication. Recently, studies showed that HBc has the potential to control cell biology activity by regulating host gene expression. Here, we utilized miRNA microarray to identify 24 upregulated miRNAs and 21 downregulated miRNAs in HBc-expressed HCC cells, which were involved in multiple biological processes, including cell motility. Consistently, the in vitro transwell assay and the in vivo tail-vein injection model showed HBc promotion on HCC metastasis. Further, the miRNA-target gene network analysis displayed that the deleted in liver cancer (DLC-1) gene, an important negative regulator for cell motility, was potentially targeted by several differentially expressed miRNAs in HBc-introduced cells. Introduction of miRNAs mimics or inhibitors and 3'UTR luciferase activity assay proved that miR-382-5p efficiently suppressed DLC-1 expression and its 3'-UTR luciferase reporter activity. Importantly, cotransfection of miR-382-5p mimics/inhibitors and the DLC-1 expression vector almost abrogated HBc promotion on cell motility, indicating that the miR-382-5p/DLC-1 axis is important for mediating HBc-enhanced HCC motility. Clinical HCC samples also showed a negative correlation between miR-382-5p and DLC-1 expression level. Furthermore, HBc-positive HCC tissues showed high miR-382-5p level and reduced DLC-1 expression. In conclusion, our findings revealed that HBc promoted HCC motility by regulating the miR-382-5p/DLC-1 axis, which might provide a novel target for clinical diagnosis and treatment.

MxA inhibits hepatitis B virus replication by interaction with hepatitis B core antigen.

UNLABELLED: Human MxA, an interferon-inducible cytoplasmic dynamin-like GTPase, possesses antiviral activity against multiple RNA viruses. Recently, MxA has also been demonstrated to have activity against the hepatitis B virus (HBV), a well-known DNA virus responsible for acute and chronic liver disease in humans. We investigated the molecular mechanism for the anti-HBV activity of MxA. Our results demonstrated that in HepG2.2.15 cells, MxA GTPase independently suppressed the production of hepatitis B surface antigen and HBV DNA without changing the level of hepatitis B core antigen (HBcAg) and the distribution of HBV mRNA. MxA significantly reduced the level of the encapsidated pregenomic RNA. Through its central interactive domain, MxA interacted with HBcAg, causing accumulation of the proteins in perinuclear compartments. MxA-HBcAg interaction significantly affected the dynamics of HBcAg by immobilizing HBcAg in the perinuclear structures. CONCLUSION: MxA displays antiviral activity against HBV involving a mechanism of MxA-HBcAg interaction that may interfere with core particle formation.

HBcAg induces PD-1 upregulation on CD4+T cells through activation of JNK, ERK and PI3K/AKT pathways in chronic hepatitis-B-infected patients.

Hyper-expression of programmed death-1 (PD-1) is a hallmark of exhausted T cells. In chronic hepatitis-B virus (HBV)-infected patients, PD-1 upregulation on T cells was often observed. The mechanism of it has not been fully understood. In this study, we examined the dynamic changes of PD-1 expression on T cells during the natural history of chronic HBV infection and explored the signaling pathway of PD-1 upregulation by the hepatitis-B core antigen (HBcAg). Sixty-seven chronic HBV-infected patients were categorized into an immune tolerance group, an immune clearance group and an inactive virus carrier group, and 20 healthy volunteers were chosen as normal control group. Peripheral blood mononuclear cells from patients and healthy volunteers, and T lymphocytes from healthy volunteers were separated. Results showed that the PD-1 expression level on CD4(+)T cells in every phase of chronic HBV infection was significantly higher than that in healthy volunteers, whereas such effects were not observed on CD8(+)T cells. In the immune clearance phase, a positive correlation was found between serum HBV DNA level and the PD-1 expression level on CD4(+)T cells. In all phases, no correlation was shown between serum alanine amino transferase (ALT) level and PD-1 expression level. Phosphorylation of JNK, ERK and AKT was induced by HBcAg, and inhibitors of JNK, ERK and PI3K/AKT significantly decreased the HBcAg-induced PD-1 upregulation on CD4(+)T cells. In conclusion, the PD-1 expression level on CD4(+)T cells was upregulated in every phase of chronic HBV infection, which was induced by HBcAg through JNK, ERK and PI3K/AKT signaling pathways.

The arginine clusters of the carboxy-terminal domain of the core protein of hepatitis B virus make pleiotropic contributions to genome replication.

The carboxy-terminal domain (CTD) of the core protein of hepatitis B virus is not necessary for capsid assembly. However, the CTD does contribute to encapsidation of pregenomic RNA (pgRNA). The contribution of the CTD to DNA synthesis is less clear. This is the case because some mutations within the CTD increase the proportion of spliced RNA to pgRNA that are encapsidated and reverse transcribed. The CTD contains four clusters of consecutive arginine residues. The contributions of the individual arginine clusters to genome replication are unknown. We analyzed core protein variants in which the individual arginine clusters were substituted with either alanine or lysine residues. We developed assays to analyze these variants at specific steps throughout genome replication. We used a replication template that was not spliced in order to study the replication of only pgRNA. We found that alanine substitutions caused defects at both early and late steps in genome replication. Lysine substitutions also caused defects, but primarily during later steps. These findings demonstrate that the CTD contributes to DNA synthesis pleiotropically and that preserving the charge within the CTD is not sufficient to preserve function.

Low replication and variability of HBV pre-core in concomitant infection with hepatitis B and hepatitis C viruses.

In an attempt to define the virological profile of HBV in HCV co-infection, we analysed the viral load, the infecting genotype, and the mutational pattern of the HBV pre-core region (pre-C), which is involved in viral encapsidation and DNA replication. Eighty-six patients were studied: 32 with serological HBV/HCV-1b co-infection (group BC), 32 infected by HBV alone (group B), and 22 by HCV-1b alone (group C). Sequence analysis of the HBV pre-S and pre-C regions identified genotypes and mutational patterns. The HBV viral load was significantly lower in group BC than in group B (p < 0.001), and the distribution of HBV pre-C mutations showed a higher prevalence of wild type in concomitant infection than in the control group (p < 0.006). The predominant HBV infecting strain was genotype D in both the BC (96%) and B (87%) groups. No difference was observed in HCV viremia levels between the two groups, whereas in HBV/HCV infection, the low levels of circulating HBV were closely associated with the low degree of variability of pre-C domain (p = 0.005). In conclusion, in HBV/HCV infection, the virological pattern was characterised by the dominance of HCV associated with lower HBV replication capacity and decreased emergence of HBV pre-C variants.

Regulation of Toll-like receptor-2 expression in chronic hepatitis B by the precore protein.

UNLABELLED: Toll-like receptors (TLRs) play a key role in the innate immune response. The aim of this study was to examine the expression of TLR2 and TLR4 in chronic hepatitis B (CHB). The TLR2 and TLR4 expression on hepatocytes and Kupffer cells from fresh liver biopsies was measured from 21 patients with untreated hepatitis B e antigen (HBeAg)-positive and HBeAg-negative CHB. Parallel studies were also undertaken on monocytes from their peripheral blood. Expression of TLR2 on hepatocytes, Kupffer cells, and peripheral monocytes was significantly reduced in patients with HBeAg-positive CHB in comparison with HBeAg-negative CHB and controls, whereas it was significantly increased in HBeAg-negative CHB compared with controls. The level of TLR4 expression did not differ significantly between the groups. These results were confirmed in vitro using hepatic cell lines transduced with recombinant HBV baculovirus expressing wild-type HBV (HBeAg-positive), precore stop codon (G1896A) mutant HBV (HBeAg-negative). The functional relevance of these findings was established by the demonstration of significantly reduced cytokine production (TNF-alpha) and phospho-p38 kinase expression in the presence of the HBeAg. In the absence of HBeAg, HBV replication was associated with up-regulation of the TLR2 pathway leading to increased TNF-alpha production. CONCLUSION: This study demonstrates a potentially important interaction between HBeAg, HBV, and the innate immune response.

Mechanism of T cell hyporesponsiveness to HBcAg is associated with regulatory T cells in chronic hepatitis B.

AIM: To study the mechanisms of hyporesponsiveness of HBV-specific CD4+ T cells by testing TH1 and TH2 commitment and regulatory T cells. METHODS: Nine patients with chronic hepatitis B were enrolled. Peripheral blood mononuclear cells were stimulated with HBcAg or HBsAg to evaluate their potential to commit to TH1 and TH2 differentiation. HBcAg-specific activity of regulatory T cells was evaluated by staining with antibodies to CD4, CD25, CTLA-4 and interleukin-10. The role of regulatory T cells was further assessed by treatment with anti-interleukin-10 antibody and depletion of CD4+CD25+ cells. RESULTS: Level of mRNAs for T-bet, IL-12R beta2 and IL-4 was significantly lower in the patients than in healthy subjects with HBcAg stimulation. Although populations of CD4+CD25highCTLA-4+ T cells were not different between the patients and healthy subjects, IL-10 secreting cells were found in CD4+ cells and CD4+CD25+ cells in the patients in response to HBcAg, and they were not found in cells which were stimulated with HBsAg. Addition of anti-IL-10 antibody recovered the amount of HBcAg-specific TH1 antibody compared with control antibody (P < 0.01, 0.34% +/- 0.12% vs 0.15% +/- 0.04%). Deletion of CD4+CD25+ T cells increased the amount of HBcAg-specific TH1 antibody when compared with lymphocytes reconstituted using regulatory T cells (P < 0.01, 0.03% +/- 0.02% vs 0.18% +/- 0.05%). CONCLUSION: The results indicate that the mechanism of T cell hyporesponsiveness to HBcAg includes activation of HBcAg-induced regulatory T cells in contrast to an increase in TH2-committed cells in response to HBsAg.

The arginine-rich carboxy-terminal domain of the hepatitis B virus core protein mediates attachment of nucleocapsids to cell-surface-expressed heparan sulfate.

Binding of hepatitis B virus nucleocapsids to mouse B cells leads to production of nucleocapsid-specific antibodies, class II presentation of peptides and the generation of T helper-1 immunity. This T-cell-independent activation of B cells is thought to result from cross-linking of cell-surface immunoglobulin molecules, if these contain a specific motif in the framework region 1-complementarity determining region 1 junction. In the present study, it was observed that nucleocapsids bound to different B-cell lines, an interaction that was not dependent on cell-surface-expressed immunoglobulins. Furthermore, binding to several non-B-cell lines was observed. Capsids that lacked the carboxy-terminal protamine-like domains did not bind to cells. Treatment of nucleocapsids with ribonucleases enhanced the attachment of nucleocapsids to cells. Various soluble glycosaminoglycans inhibited attachment of nucleocapsids, while treatment of cells with heparinase I also reduced binding. These observations demonstrated that the arginine-rich protamine-like regions of the core proteins are responsible for the attachment of nucleocapsids to glycosaminoglycans expressed on the plasma membranes of cells.

[Study on anti-HBV effects of genetically engineered replication-defective hepatitis B virus expressing dominant negative mutants of core protein].

BACKGROUND: To explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular expression of dominant negative mutants of core protein. METHODS: Two kinds of full length mutant HBV genome, which express Core-partial P and Core-S fusion protein, were transfected into HepG 2.2.15 cell lines. Positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA and intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR. RESULTS: The mean inhibitory rates of HBsAg were 2.74+/-3.83%, 40.08+/-2.05% (P less than 0.01) and 52.94+/-1.93% (P less than 0.01) in group 2.2.15-pMEP4, 2.2.15-CP and 2.2.15-CS, respectively. The mean inhibitory rates of HBeAg were 4.46+/-4.25%, 52.86+/-1.32% (P less than 0.01) and 41.60+/-1.65% (P less than 0.01), respectively. The inhibitory rates of HBc related HBV DNA were 15.3%, 82.0% and 67.2%, respectively. Recombinant HBV virion was detectable in the culture medium of only group 2.2.15-CP. CONCLUSION: Dominant negative mutants of core protein can efficiently suppress wt-HBV replication and the expressions of HBV antigens. With the help of wild-type HBV, the recombinant HBV genome can form and secret HBV like particles, which provides evidence that the antiviral gene will be hepatotropic expression and the antiviral effects will be amplified.

Hepatitis B virus core protein stimulates the proteasome-mediated degradation of viral X protein.

Hepatitis B virus (HBV) X protein (HBx) plays an essential role in viral replication and in the development of hepatocellular carcinoma. HBx has the ability to transactivate the expression of all HBV proteins, including the viral core protein HBc. Consistent with its regulatory role, HBx is relatively unstable and is present at low levels in the cell. We report here that the level of HBx was significantly reduced by the coexpression of HBc in cultured human hepatoma cells, whereas the level of HBx mRNA was unaffected. The repression of HBx by HBc was relieved by treating cells with the proteasome inhibitor MG132, indicating that HBc acts by stimulating the proteasome-mediated degradation of HBx. Moreover, the inhibitory effect of HBc was specific to HBx and did not affect other proteins, including p53, a known target of the proteasome. Although no direct physical interaction between HBc and HBx could be demonstrated, mutational analysis indicated that the C-terminal half of HBc is responsible for its inhibitory effect. These results suggest that HBc functions as a novel regulator of the HBV life cycle and of hepatocellular carcinogenesis through control of the HBx level via an inhibitory feedback type of mechanism.

Transcomplementation of core and polymerase functions of the woolly monkey and human hepatitis B viruses.

Woolly monkey hepatitis B virus (WMHBV) is a new member of Hepadnaviridae that was isolated from a New World monkey and is phylogenetically distinct from the HBV family. In this study, we explored the functional significance of sequence divergence in the HBV and WMHBV genomes. Independently expressed TP and RT domains of the WMHBV reverse transcriptase (Pol) formed a complex functional for in vitro nucleotide priming, consistent with previous results from priming reactions conducted with HBV. Transcomplementation assays between HBV and WMHBV TP and RT components for in vitro priming demonstrated functional compatibility, although priming with the combination of WMHBV RT and HBV TP was reduced. Examination of cross-species protein-protein interactions revealed that WMHBV core coprecipitated with HBV TP and RT, as well as with WMHBV TP and RT. Analysis in Huh7 cells revealed that WMHBV core and Pol complemented core-negative and Pol-negative HBV mutant genomes for replication. These results highlight the conservation of function despite significant sequence divergence in these viruses.

Hepatitis B viral polymerase fusion proteins are biologically active and can interact with the hepatitis C virus core protein in vivo.

Hepadnaviruses and retroviruses are evolutionarily related families because they both require a process of reverse transcription for genome replication. However, hepadnaviruses produce polymerase (pol) and core proteins separately, while retroviruses synthesize a gag-pol fusion protein that is subsequently cleaved by a virally encoded protease to release a functional polymerase. To test whether an additional sequence at the N-terminus of pol in hepatitis B virus (HBV) interferes with its function, we created two plasmids expressing core-pol fusion proteins, core144-pol and core31-pol. Secreted particles obtained from HuH-7 cells, which were cotransfected with a core-pol fusion protein-expressing plasmid and a core-expressing plasmid, showed a positive signal of HBV DNA by the endogenous polymerase assay, indicating that the core-pol fusion proteins retain DNA priming, polymerization and RNase H activities. The fusion protein was detected in the cytoplasm of transfected cells and in secreted virions by immunoprecipitation. Furthermore, we found by immunofluorescence staining that the HBV core-pol fusion protein colocalized with the hepatitis C virus (HCV) core protein in cytoplasm and in lipid droplets. Immunoprecipitation studies showed that the anti-HCV core complex contained the HBV core-pol fusion protein while the anti-HBV pol complex contained the HCV core protein, which supports the hypothesis that the HCV core protein can form a complex with the HBV core-pol fusion protein.

Hepatitis B virus maturation is affected by the incorporation of core proteins having a C-terminal substitution of arginine or lysine stretches.

Assembly of replication-competent hepadnavirus nucleocapsids requires interaction of core protein, polymerase and encapsidation signal (epsilon) with viral pregenomic RNA. The N-terminal portion (aa 1-149) of the core protein is able to self-assemble into nucleocapsids, whereas the C-terminal portion (aa 150-183) is known to interact with pregenomic RNA. In this study, two hepatitis B virus (HBV) core mutants (C144Arg and C144Lys) in which the C-terminal SPRRR (Ser-Pro-Arg-Arg-Arg) motif was replaced by a stretch of arginine or lysine residues were generated to test their role in pregenome encapsidation and virus maturation. Mutant or wild-type core-expression plasmids were co-transfected with a core-negative plasmid into human hepatoma HuH-7 cells to compare trans-complementation efficiency for virus replication. Both low- and high-density capsids were present in -the cytoplasm and culture medium of HuH-7 cells in all transfections. Nucleocapsids formed by C144Arg and C144Lys, however, lost the endogenous polymerase activity to repair HBV DNA. Furthermore, in co-transfection of pHBVC144Arg or pHBVC144Lys with a plasmid which produces replication-competent nucleocapsids, the HBV DNA repairing signal was reduced 40- to 80-fold. This is probably due to formation of mosaic particles of wild-type and mutant cores. Results indicated that the SPRRR motif at the core protein C terminus is important for HBV DNA replication and maturation. Additionally, triple-plasmid transfection experiments showed that nucleocapsids containing various amounts of C144Arg and wild-type core proteins exhibited a bias in selecting a shorter pregenome for encapsidation and DNA replication. It is therefore suggested that unknown factors are also involved in HBV pregenome packaging.

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.

Hepatitis B core particles as a universal display model: a structure-function basis for development.

Because it exhibits a remarkable capability to accept mutational intervention and undergo correct folding and self-assembly in all viable prokaryotic and eukaryotic expression systems, hepatitis B core (HBc) protein has been favored over other proposed particulate carriers. Structurally, the unusual alpha-helical organization of HBc dimeric units allows introduction of foreign peptide sequences into several areas of HBc shells, including their most protruding spikes. Progress toward full resolution of the spatial structure as well as accumulation of chimeric HBc-based structures has brought closer the knowledge-based design of future vaccines, gene therapy tools and other artificial particulate objects.

Core particles of hepatitis B virus as carrier for foreign epitopes.

To be effective as vaccines, most monomeric proteins and peptides either require chemical coupling to high molecular weight carriers or application together with adjuvants. More recently, recombinant DNA techniques have been used to insert foreign epitopes into proteins with inherent multimerization capacity, such as particle-forming viral capsid or envelope proteins. The core protein of hepatitis B virus (HBcAg), because of its unique structural and immunological properties, has gained widespread interest as a potential antigen carrier. Foreign sequences of up to approximately 40 amino acid residues at the N terminus, 50 or 100 amino acids in the central immunodominant c/e 1 epitope region of HBcAg, and up to 100 or even more residues at the C terminus, did not interfere with particle formation. The humoral immunogenicity of inserted epitopes is determined by the immunogenicity of the peptide itself and its surface exposure, and is influenced by the route of application. The probably flexible and surface-exposed c/e1 region emerged as the most promising insertion site. When applied together with adjuvants approved for human and veterinary use, or even without adjuvants, such chimeric particles induced B and T cell immune responses against the inserted epitopes. In some cases neutralizing antibodies, cytotoxic T cells and protection against challenge with the intact pathogen were demonstrated. Major factors for the potentiated immune response against the foreign epitopes are the multimeric structure of chimeric HBcAg that results in a high epitope density per particle, and the provision of T cell help by the carrier moiety. Beyond its use as subunit vaccine, chimeric HBcAg produced in attenuated Salmonella strains may be applicable as live vaccine.

Modulation of the trans-suppression activity of hepatitis C virus core protein by phosphorylation.

We previously demonstrated that the core protein of hepatitis C virus (HCV) can suppress gene expression and replication of hepatitis B virus (HBV) in a human hepatoma cell line (HuH-7). In this study, we have characterized the phosphorylation property of HCV core protein and examined the effect of phosphorylation on its suppressive activity of HBV. Our results indicated that both the full-length HCV core protein (22 kDa) and its processed or degraded forms (14 to 18 kDa) were phosphorylated in insect cells. As demonstrated by using the glutathione S-transferase fusion protein expression system and in vitro transcription and translation system, the phosphorylation of HCV core protein was carried out by protein kinase A (PKA) and protein kinase C (PKC) in vitro. In both kinase reactions, it was determined that the phosphorylated amino acid was a serine residue. The potential phosphorylated sites in core protein were identified as residues Ser-53 and Ser-116 for PKA and Ser-53 and Ser-99 for PKC. Comparison of the phosphorylation intensities of the wild type and Ser mutants suggested that Ser-99 and Ser-116 were the major phosphorylation sites for PKC and PKA, respectively. The phosphorylation of Ser-99 and Ser-116, but not Ser-53, in HCV core protein was essential for the suppressive activity of HCV core protein on HBV gene expression and replication in HuH-7 cells. Mutation of the former two serine residues to alanine or aspartate residues led to a drastic loss of the inhibitory effects of HCV core protein on HBV gene expression (both transcription and antigen production) and pregenomic RNA encapsidation, as well as the release of HBV virus particles. In contrast, the Ser-53 mutant conferred the same level of suppressive activity as the wild type did. This property is in accordance with the observation that Ser-99 and Ser-116 are the predominant phosphorylation sites in the HCV core construct. All serine mutants (including those with mutations in PKA, PKC, and both kinase recognition sites) of HCV core protein retained the ability to translocate into the nucleus. Furthermore, wild-type HCV core protein diminished its suppressive activity when cells were treated with PKA or PKC inhibitor. In conclusion, HCV core protein is a phospho-protein and in HuH-7 cells, its trans suppression of HBV gene expression and replication is positively regulated by PKA and PKC. The role of phosphorylation in the control of trans-suppressive activity cannot be reproduced by introducing an acidic residue.(ABSTRACT TRUNCATED AT 250 WORDS)

Human non-hepatocytes support hepadnaviral replication and virion production.

The competence of non-hepatocytes to support hepatitis B virus (HBV) gene expression and replication was studied by transient transfection of various human cell lines with a head-to-tail dimer of HBV DNA. Independent of their neuroectodermal, mesenchymal or epithelial origin, all non-hepatocyte cell lines tested synthesized and secreted hepatitis B surface antigen (HBsAg) and hepatitis B core/e antigen (HBc/eAg). Further analyses of two of these cell lines (LS 180 and COLO 320) identified the two major HBV transcripts of 3.6 and 2.2/2.4 kb length, respectively. LS 180 cells were permissive for HBV and duck hepatitis B virus (DHBV) DNA replication and secretion of infectious virions. COLO 320 cells also supported HBV DNA replication, but did not appear to export complete viral particles. These findings provide direct evidence that both HBV and DHBV can replicate in non-hepatic tumour cell lines, one of which is shown also to produce infectious virions.