Conserved use of the sodium/bile acid cotransporter (NTCP) as an entry receptor by hepatitis B virus and domestic cat hepadnavirus.

The Orthohepadnavirus genus includes hepatitis B virus (HBV) that can cause chronic hepatitis and hepatocarcinoma in humans. Recently, a novel hepadnavirus in cats, domestic cat hepadnavirus (DCH), was identified that is genetically close to HBV. DCH infection is associated with chronic hepatitis in cats, suggesting a similarity with HBV pathogenesis and the potential to use DCH as a novel animal model for HBV research. HBV is shown to use the sodium/bile acid cotransporter (NTCP) as a major cell entry receptor, but the equivalent receptor for DCH remains unknown. Here we sought to identify the entry receptor for DCH. HBV- and DCH-derived preS1 peptides efficiently bound to both human and cat NTCPs, and residue 158 of NTCP proteins determined the species-specific binding of the DCH preS1 peptide. Myrcludex B, an HBV entry inhibitor, blocked the binding of the DCH preS1 peptide. Thus, DCH and HBV may share cell entry molecules, suggesting a possibility of inter-species transmission. Furthermore, our study suggests that DCH can be useful as a novel model for HBV research.

Structural conservation of HBV-like capsid proteins over hundreds of millions of years despite the shift from non-enveloped to enveloped life-style.

The discovery of nackednaviruses provided new insight into the evolutionary history of the hepatitis B virus (HBV): The common ancestor of HBV and nackednaviruses was non-enveloped and while HBV acquired an envelope during evolution, nackednaviruses remained non-enveloped. We report the capsid structure of the African cichlid nackednavirus (ACNDV), determined by cryo-EM at 3.7 Å resolution. This enables direct comparison with the known capsid structures of HBV and duck HBV, prototypic representatives of the mammalian and avian lineages of the enveloped Hepadnaviridae, respectively. The sequence identity with HBV is 24% and both the ACNDV capsid protein fold and the capsid architecture are very similar to those of the Hepadnaviridae and HBV in particular. Acquisition of the hepadnaviral envelope was thus not accompanied by a major change in capsid structure. Dynamic residues at the spike tip are tentatively assigned by solid-state NMR, while the C-terminal domain is invisible due to dynamics. Solid-state NMR characterization of the capsid structure reveals few conformational differences between the quasi-equivalent subunits of the ACNDV capsid and an overall higher capsid structural disorder compared to HBV. Despite these differences, the capsids of ACNDV and HBV are structurally highly similar despite the 400 million years since their separation.

Codon usage pattern and its influencing factors in different genomes of hepadnaviruses.

Codon usage bias (CUB) arises from the preference for a codon over codons for the same amino acid. The major factors contributing to CUB are evolutionary forces, compositional properties, gene expression, and protein properties. The present analysis was performed to investigate the compositional properties and the extent of CUB across the genomes of members of the family Hepadnaviridae, as previously no work using bioinformatic tools has been reported. The viral genes were found to be AT rich with low CUB. Analysis of relative synonymous codon usage (RSCU) was used to identify overrepresented and underrepresented codons for each amino acid. Correlation analysis of overall nucleotide composition and its composition at the third codon position suggested that mutation pressure might influence the CUB. A highly significant correlation was observed between GC12 and GC3 (r = 0.910, p < 0.01), indicating that directional mutation affected all three codon positions across the genome. Translational selection (P2) and mutational responsive index (MRI) values of genes suggested that mutation plays a more important role than translational selection in members of the family Hepadnaviridae.

The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation.

Hepadnavirus covalently closed circular (ccc) DNA is the bona fide viral transcription template, which plays a pivotal role in viral infection and persistence. Upon infection, the non-replicative cccDNA is converted from the incoming and de novo synthesized viral genomic relaxed circular (rc) DNA, presumably through employment of the host cell's DNA repair mechanisms in the nucleus. The conversion of rcDNA into cccDNA requires preparation of the extremities at the nick/gap regions of rcDNA for strand ligation. After screening 107 cellular DNA repair genes, we herein report that the cellular DNA ligase (LIG) 1 and 3 play a critical role in cccDNA formation. Ligase inhibitors or functional knock down/out of LIG1/3 significantly reduced cccDNA production in an in vitro cccDNA formation assay, and in cccDNA-producing cells without direct effect on viral core DNA replication. In addition, transcomplementation of LIG1/3 in the corresponding knock-out or knock-down cells was able to restore cccDNA formation. Furthermore, LIG4, a component in non-homologous end joining DNA repair apparatus, was found to be responsible for cccDNA formation from the viral double stranded linear (dsl) DNA, but not rcDNA. In conclusion, we demonstrate that hepadnaviruses utilize the whole spectrum of host DNA ligases for cccDNA formation, which sheds light on a coherent molecular pathway of cccDNA biosynthesis, as well as the development of novel antiviral strategies for treatment of hepatitis B.

Nuclear factor Y regulates ancient budgerigar hepadnavirus core promoter activity.

Endogenous viral elements (EVE) in animal genomes are the fossil records of ancient viruses and provide invaluable information on the origin and evolution of extant viruses. Extant hepadnaviruses include avihepadnaviruses of birds and orthohepadnaviruses of mammals. The core promoter (Cp) of hepadnaviruses is vital for viral gene expression and replication. We previously identified in the budgerigar genome two EVEs that contain the full-length genome of an ancient budgerigar hepadnavirus (eBHBV1 and eBHBV2). Here, we found eBHBV1 Cp and eBHBV2 Cp were active in several human and chicken cell lines. A region from nt -85 to -11 in eBHBV1 Cp was critical for the promoter activity. Bioinformatic analysis revealed a putative binding site of nuclear factor Y (NF-Y), a ubiquitous transcription factor, at nt -64 to -50 in eBHBV1 Cp. The NF-Y core binding site (ATTGG, nt -58 to -54) was essential for eBHBV1 Cp activity. The same results were obtained with eBHBV2 Cp and duck hepatitis B virus Cp. The subunit A of NF-Y (NF-YA) was recruited via the NF-Y core binding site to eBHBV1 Cp and upregulated the promoter activity. Finally, the NF-Y core binding site is conserved in the Cps of all the extant avihepadnaviruses but not of orthohepadnaviruses. Interestingly, a putative and functionally important NF-Y core binding site is located at nt -21 to -17 in the Cp of human hepatitis B virus. In conclusion, our findings have pinpointed an evolutionary conserved and functionally critical NF-Y binding element in the Cps of avihepadnaviruses.

Sodium taurocholate cotransporting polypeptide mediates woolly monkey hepatitis B virus infection of Tupaia hepatocytes.

Primary Tupaia hepatocytes (PTHs) are susceptible to woolly monkey hepatitis B virus (WMHBV) infection, but the identity of the cellular receptor(s) mediating WMHBV infection of PTHs remains unclear. Recently, sodium taurocholate cotransporting polypeptide (NTCP) was identified as a functional receptor for human hepatitis B virus (HBV) infection of primary human and Tupaia hepatocytes. In this study, a synthetic pre-S1 peptide from WMHBV was found to bind specifically to cells expressing Tupaia NTCP (tsNTCP) and it efficiently blocked WMHBV entry into PTHs; silencing of tsNTCP in PTHs significantly inhibited WMHBV infection. Ectopic expression of tsNTCP rendered HepG2 cells susceptible to WMHBV infection. These data demonstrate that tsNTCP is a functional receptor for WMHBV infection of PTHs. The result also indicates that NTCP's orthologs likely act as a common cellular receptor for all known primate hepadnaviruses.

Cyclin-dependent kinase 2 phosphorylates s/t-p sites in the hepadnavirus core protein C-terminal domain and is incorporated into viral capsids.

Phosphorylation of the hepadnavirus core protein C-terminal domain (CTD) is important for viral RNA packaging, reverse transcription, and subcellular localization. Hepadnavirus capsids also package a cellular kinase. The identity of the host kinase that phosphorylates the core CTD or gets packaged remains to be resolved. In particular, both the human hepatitis B virus (HBV) and duck hepatitis B virus (DHBV) core CTDs harbor several conserved serine/threonine-proline (S/T-P) sites whose phosphorylation state is known to regulate CTD functions. We report here that the endogenous kinase in the HBV capsids was blocked by chemical inhibitors of the cyclin-dependent kinases (CDKs), in particular, CDK2 inhibitors. The kinase phosphorylated the HBV CTD at the serine-proline (S-P) sites. Furthermore, we were able to detect CDK2 in purified HBV capsids by immunoblotting. Purified CDK2 phosphorylated the S/T-P sites of the HBV and DHBV CTD in vitro. Inhibitors of CDKs, of CDK2 in particular, decreased both HBV and DHBV CTD phosphorylation in vivo. Moreover, CDK2 inhibitors blocked DHBV CTD phosphorylation, specifically at the S/T-P sites, in a mammalian cell lysate. These results indicate that cellular CDK2 phosphorylates the functionally critical S/T-P sites of the hepadnavirus core CTD and is incorporated into viral capsids.

Carbonyl J acid derivatives block protein priming of hepadnaviral P protein and DNA-dependent DNA synthesis activity of hepadnaviral nucleocapsids.

Current treatments for chronic hepatitis B are effective in only a fraction of patients. All approved directly antiviral agents are nucleos(t)ide analogs (NAs) that target the DNA polymerase activity of the hepatitis B virus (HBV) P protein; resistance and cross-resistance may limit their long-term applicability. P protein is an unusual reverse transcriptase that initiates reverse transcription by protein priming, by which a Tyr residue in the unique terminal protein domain acts as an acceptor of the first DNA nucleotide. Priming requires P protein binding to the ε stem-loop on the pregenomic RNA (pgRNA) template. This interaction also mediates pgRNA encapsidation and thus provides a particularly attractive target for intervention. Exploiting in vitro priming systems available for duck HBV (DHBV) but not HBV, we demonstrate that naphthylureas of the carbonyl J acid family, in particular KM-1, potently suppress protein priming by targeting P protein and interfering with the formation of P-DHBV ε initiation complexes. Quantitative evaluation revealed a significant increase in complex stability during maturation, yet even primed complexes remained sensitive to KM-1 concentrations below 10 µM. Furthermore, KM-1 inhibited the DNA-dependent DNA polymerase activity of both DHBV and HBV nucleocapsids, including from a lamivudine-resistant variant, directly demonstrating the sensitivity of human HBV to the compound. Activity against viral replication in cells was low, likely due to low intracellular availability. KM-1 is thus not yet a drug candidate, but its distinct mechanism of action suggests that it is a highly useful lead for developing improved, therapeutically applicable derivatives.

Hepatitis delta virus infects the cells of hepadnavirus-induced hepatocellular carcinoma in woodchucks.

UNLABELLED: Hepatitis delta virus (HDV) is a natural subviral agent of human hepatitis B virus (HBV). HDV enhances liver damage during concomitant infection with HBV. The molecular pathogenesis of HDV infection remains poorly understood. To advance our understanding of the relationship between HDV infection and liver cancer, it was determined whether HDV could infect in vivo the cells of hepadnavirus-induced hepatocellular carcinoma (HCC). Woodchucks (Marmota monax) that were chronically infected with HBV-related woodchuck hepatitis virus (WHV) and already developed HCCs were used as an experimental model. The locations of HCCs within the livers were determined using ultrasound imaging followed by open surgery. One week after surgery the WHV carrier woodchucks were superinfected with WHV-enveloped HDV (wHDV). Six weeks later the animals were sacrificed and HDV replication in normal liver tissues and in center masses of HCCs was evidenced by Northern analysis, real-time polymerase chain reaction assay, and immunohistochemistry. Based on accumulation levels of HDV RNAs and numbers of infected cells, the efficiency of wHDV infection appears to be comparable in most HCCs and normal liver tissues. CONCLUSION: Cells of WHV-induced HCCs are susceptible to HDV infection in vivo, and therefore express functional putative WHV receptors and support the steps of the attachment/entry governed by the hepadnavirus envelope proteins. Because others previously hypothesized that hepadnavirus-induced HCCs are resistant to reinfection with a hepadnavirus in vivo, our data suggest that if such a resistance exists it likely occurs via a block at the post-entry step. The demonstrated ability of HDV to infect already formed HCCs may facilitate development of novel strategies further dissecting the mechanism of liver pathogenesis associated with HDV infection.

Somatic hypermutation of human mitochondrial and nuclear DNA by APOBEC3 cytidine deaminases, a pathway for DNA catabolism.

The human APOBEC3 (A3A-A3H) locus encodes six cytidine deaminases that edit single-stranded DNA, the result being DNA peppered with uridine. Although several cytidine deaminases are clearly restriction factors for retroviruses and hepadnaviruses, it is not known if APOBEC3 enzymes have roles outside of these settings. It is shown here that both human mitochondrial and nuclear DNA are vulnerable to somatic hypermutation by A3 deaminases, with APOBEC3A standing out among them. The degree of editing is much greater in patients lacking the uracil DNA-glycolyase gene, indicating that the observed levels of editing reflect a dynamic composed of A3 editing and DNA catabolism involving uracil DNA-glycolyase. Nonetheless, hyper- and lightly mutated sequences went hand in hand, raising the hypothesis that recurrent low-level mutation by APOBEC3A could catalyze the transition from a healthy to a cancer genome.

Phosphorylation state-dependent interactions of hepadnavirus core protein with host factors.

Dynamic phosphorylation and dephosphorylation of the hepadnavirus core protein C-terminal domain (CTD) are required for multiple steps of the viral life cycle. It remains unknown how the CTD phosphorylation state may modulate core protein functions but phosphorylation state-dependent viral or host interactions may play a role. In an attempt to identify host factors that may interact differentially with the core protein depending on its CTD phosphorylation state, pulldown assays were performed using the CTD of the duck hepatitis B virus (DHBV) and human hepatitis B virus (HBV) core protein, either with wild type (WT) sequences or with alanine or aspartic acid substitutions at the phosphorylation sites. Two host proteins, B23 and I2PP2A, were found to interact preferentially with the alanine-substituted CTD. Furthermore, the WT CTD became competent to interact with the host proteins upon dephosphorylation. Intriguingly, the binding site on the DHBV CTD for both B23 and I2PP2A was mapped to a region upstream of the phosphorylation sites even though B23 or I2PP2A binding to this site was clearly modulated by the phosphorylation state of the downstream and non-overlapping sequences. Together, these results demonstrate a novel mode of phosphorylation-regulated protein-protein interaction and provide new insights into virus-host interactions.

An interdomain RNA binding site on the hepadnaviral polymerase that is essential for reverse transcription.

The T3 motif on the duck hepatitis B virus reverse transcriptase (P) is proposed to be a binding site essential for viral replication, but its ligand and roles in DNA synthesis are unknown. Here, we found that T3 is needed for P to bind the viral RNA, the first step in DNA synthesis. A second motif, RT-1, was predicted to assist T3. T3 and RT-1 appear to form a composite RNA binding site because mutating T3 and RT-1 had similar effects on RNA binding, exposure of antibody epitopes on P, and DNA synthesis. The T3 and RT-1 motifs bound RNA non-specifically, yet they were essential for specific interactions between P and the viral RNA. This implies that specificity for the viral RNA is provided by a post-binding step. The T3:RT-1 motifs are conserved with the human hepatitis B virus and may be an attractive target for novel antiviral drug development.

Chaperones activate hepadnavirus reverse transcriptase by transiently exposing a C-proximal region in the terminal protein domain that contributes to epsilon RNA binding.

All hepatitis B viruses replicate by protein-primed reverse transcription, employing a specialized reverse transcriptase, P protein, that carries a unique terminal protein (TP) domain. To initiate reverse transcription, P protein must bind to a stem-loop, epsilon, on the pregenomic RNA template. TP then provides a Y residue for covalent attachment of the first nucleotide of an epsilon-templated DNA oligonucleotide (priming reaction) that serves to initiate full-length minus-strand DNA synthesis. epsilon binding requires the chaperone-dependent conversion of inactive P protein into an activated, metastable form designated P*. However, how P* differs structurally from P protein is not known. Here we used an in vitro reconstitution system for active duck hepatitis B virus P combined with limited proteolysis, site-specific antibodies, and defined P mutants to structurally compare nonactivated versus chaperone-activated versus primed P protein. The data show that Hsp70 action, under conditions identical to those required for functional activation, transiently exposes the C proximal TP region which is, probably directly, involved in epsilon RNA binding. Notably, after priming and epsilon RNA removal, a very similar new conformation appears stable without further chaperone activity; hence, the activation of P protein is triggered by energy-consuming chaperone action but may be completed by template RNA binding.

Translation of stable hepadnaviral mRNA cleavage fragments induced by the action of phosphorothioate-modified antisense oligodeoxynucleotides.

Phosphorothioate-modified antisense oligodeoxynucleotides (ASOs) are used to suppress gene expression by inducing RNase H-mediated cleavage with subsequent degradation of the target mRNA. However, previous observations suggest that ASO/RNase H can also result in the generation of stable mRNA cleavage fragments and expression of truncated proteins. Here, we addressed the underlying translational mechanisms in more detail using hepadnavirus-transfected hepatoma cells as a model system of antisense therapy. Generation of stable mRNA cleavage fragments was restricted to the ASO/RNase H pathway and not observed upon cotransfection of isosequential small interfering RNA or RNase H-incompetent oligonucleotides. Furthermore, direct evidence for translation of mRNA fragments was established by polysome analysis. Polysome-associated RNA contained cleavage fragments devoid of a 5' cap structure indicating that translation was, at least in part, cap-independent. Further analysis of the uncapped cleavage fragments revealed that their 5' terminus and initiation codon were only separated by a few nucleotides suggesting a 5' end-dependent mode of translation, whereas internal initiation could be ruled out. However, the efficiency of translation was moderate compared to uncleaved mRNA and amounted to 13-24% depending on the ASO used. These findings provide a rationale for understanding the translation of mRNA fragments generated by ASO/RNase H mechanistically.

In vitro reconstitution of functional hepadnavirus reverse transcriptase with cellular chaperone proteins.

Initiation of reverse transcription in hepadnaviruses (hepatitis B viruses) depends on the specific binding of an RNA signal (the packaging signal, epsilon) on the pregenomic RNA template by the viral reverse transcriptase (RT) and is primed by the RT itself (protein priming). We have previously shown that the RT-epsilon interaction and protein priming require the cellular heat shock protein, Hsp90. However, additional host factors required for these reactions remained to be identified. We now report that five cellular chaperone proteins, all known cofactors of Hsp90, were sufficient to reconstitute a duck hepatitis B virus RT active in epsilon binding and protein priming in vitro. Four proteins, Hsp90, Hsp70, Hsp40, and Hop, were required for reconstitution of RT activity, and the fifth protein, p23, further enhanced the kinetics of reconstitution. RT activation by the chaperone proteins is a dynamic process dependent on ATP hydrolysis and the Hsp90 ATPase activity. Thus, our results have defined a minimal complement of host factors necessary and sufficient for RT activation. Furthermore, this defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT activation and chaperone functions.

Impact of viral oncogenesis on responses to anti-cancer drugs and irradiation.

The view that chemical or physical oncogenesis and tumor therapy resistance represent different parts of common cellular alterations gained considerable attractiveness, because it explains the inherent unreponsiveness of many tumors. Viruses are potent oncogenes and are causally linked to approximately one-fifth of all human malignancies. Whether viral oncogenesis exerts comparable effects was less clear. Recent progress in experimental research provided ample evidence that viruses affect response of tumor cells toward anti-cancer drugs and irradiation. Resistance to cytostatic drugs and radiation develops by alterations at the drug-target sites (i.e., DNA or specific target proteins), upstream (i.e., detoxification mechanisms), or downstream of them (i.e., programmed cell death). Viruses interfere with specific cellular genes at these three levels. Viral proteins induce the expression and expression of drug resistance genes, that is, MDR1, DHFR, or CAD. Viral interactions with the tumor suppressor genes (p53, pRB) abrogate cell cycle arrests and disturb DNA repair of drug- and radiation-induced DNA lesions. The readiness to commit cellular suicide (apoptosis) is also affected by viral genes. The connection between viral oncogenesis and the response of tumor cells to treatment adds a new dimension to tumor biology and may have important consequences for oncological treatment modalities in the future.

Why are hepadnaviruses DNA and not RNA viruses?

Virion assembly in hepadnaviruses is a two-step process leading to (1) the packaging of viral pregenomic RNA and reverse transcriptase into nucleocapsids and (2) the assembly of nucleocapsids with envelope components, which results in the formation of mature virus particles. Characteristically, both steps are intimately coupled to viral DNA synthesis. While assembly of nucleocapsids is coupled to the protein priming of reverse transcription, virion formation is linked to genome maturation.

Hepadnavirus enhancer and its binding proteins.

In the hepadnavirus enhancer region, a 33 bp DNA sequence is strongly conserved among mammalian hepadnavirus genomes. To elucidate the role of the sequence, we tested enhancer activities and capability to form DNA-protein complex of several synthetic DNAs. Not only two tandem copies of a 46 bp DNA covering the sequence but also two tandem copies of a 23 bp in the sequence exhibit enhancer activity. Also the activity was augmented by treatment of a tumor promoter, TPA. DNA binding proteins complexes with the 23 bp DNA were augmented in extracts of HepG2 or HeLa cells stimulated with TPA. These results imply that the conserved sequence of hepadnavirus enhancer is a TPA-inducible enhancer which is transactivated by ubiquitous DNA-binding proteins. We presented results showing that DNA-protein complexes with a 23 bp DNA are similar to but distinct from those with a TPA-responsive element DNA, the recognition site for c-jun/fos products. We also presented results suggesting that hepadnavirus X protein may not directly or indirectly affect DNA-protein complex formation with the conserved sequence in the hepadnavirus enhancer.

Sequences 5' to the polyadenylation signal mediate differential poly(A) site use in hepatitis B viruses.

Most genetic elements that employ reverse transcription generate a terminally redundant genomic RNA that serves as the template for this reaction. Because the identical polyadenylation signal is present in each terminally redundant segment, synthesis of this RNA requires that this signal be ignored on the first pass of the transcription machinery, then recognized and used on the second pass. We have studied the mechanism of this differential poly(A) site use in one family of retroid elements, the hepatitis B viruses (hepadnaviruses). Our results indicate that two features are involved: the presence of a variant poly(A) signal (TATAAA) and the participation of multiple sequences 5' to this signal that act to increase the efficiency of its use. Deletion of these upstream elements abolishes proper poly(A) site use, despite the presence of the poly(A) signal and downstream GT- and T-rich motifs known to be required for polyadenylation. Sequences from the corresponding regions of retroviral genomes can restore proper processing to these hepadnaviral deletion mutants. Thus, functionally analogous upstream elements exist in other classes of retroid elements, including those employing the canonical AATAAA hexanucleotide signal.