Insights From Deep Sequencing of the HBV Genome-Unique, Tiny, and Misunderstood.
ABSTRACT: Hepatitis B virus (HBV) is a unique, tiny, partially double-stranded, reverse-transcribing DNA virus with proteins encoded by multiple overlapping reading frames. The substitution rate is surprisingly high for a DNA virus, but lower than that of other reverse transcribing organisms. More than 260 million people worldwide have chronic HBV infection, which causes 0.8 million deaths a year. Because of the high burden of disease, international health agencies have set the goal of eliminating HBV infection by 2030. Nonetheless, the intriguing HBV genome has not been well characterized. We summarize data on the HBV genome structure and replication cycle, explain and quantify diversity within and among infected individuals, and discuss advances that can be offered by application of next-generation sequencing technology. In-depth HBV genome analyses could increase our understanding of disease pathogenesis and allow us to better predict patient outcomes, optimize treatment, and develop new therapeutics.
Project description:The burden of chronic hepatitis B virus (HBV) infections is increasingly detected nowadays. Herein, we report a complete genome of HBV subgenotype C2 (HBV/C2) from a HBV infected patient. Complete genome analysis revealed that the isolated strain was a non-recombinant wild type and had several regular substitutions in the reverse transcriptase domain and small surface proteins of HBV. This study may help clinicians and scientists gain in-depth knowledge on the current substitutions of HBV/C2 genome and to identify potential therapies against HBV infections.
Project description:Hepatitis B Virus (HBV) is a small virus whose genome has only four open reading frames. We argue that the simplicity of the virion correlates with a complexity of functions for viral proteins. We focus on the HBV core protein (Cp), a small (183 residue) protein that self-assembles to form the viral capsid. However, its functions are a little more complicated than that. In an infected cell Cp modulates almost every step of the viral lifecycle. Cp is bound to nuclear viral DNA and affects its epigenetics. Cp correlates with RNA specificity. Cp assembles specifically on a reverse transcriptase-viral RNA complex or, apparently, nothing at all. Indeed Cp has been one of the model systems for investigation of virus self-assembly. Cp participates in regulation of reverse transcription. Cp signals completion of reverse transcription to support virus secretion. Cp carries both nuclear localization signals and HBV surface antigen (HBsAg) binding sites; both of these functions appear to be regulated by contents of the capsid. Cp can be targeted by antivirals - while self-assembly is the most accessible of Cp activities, we argue that it makes sense to engage the broader spectrum of Cp function. This article forms part of a symposium in Antiviral Research on "From the discovery of the Australia antigen to the development of new curative therapies for hepatitis B: an unfinished story."
Project description:Infection with HBV is common worldwide, with over 350 million chronic carriers. Chronic HBV infection is associated with cirrhosis and hepatocellular carcinoma. All currently available oral antivirals are directed against the HBV polymerase enzyme, a reverse transcriptase. HBV polymerase contains several important domains and motifs which define its functions and reveal ways to further target it. This enzyme executes many functions required for the HBV replication cycle, including viral RNA binding, RNA packaging, protein priming, template switching, DNA synthesis and RNA degradation. In addition, HBV polymerase must interact with host proteins for its functions. Future therapeutics may inhibit not only the DNA synthesis steps which are carried out by the reverse transcriptase domain (as all current antivirals do) but other domains, functions and interactions which are essential to the HBV replication cycle.
Project description:Hepadnaviruses, including human hepatitis B virus (HBV), replicate their tiny DNA genomes by protein-primed reverse transcription of a pregenomic (pg) RNA. Replication initiation as well as pgRNA encapsidation depend on the interaction of the viral polymerase, P protein, with the ? RNA element, featuring a lower and an upper stem, a central bulge, and an apical loop. The bulge, somehow assisted by the loop, acts as template for a P protein-linked DNA oligo that primes full-length minus-strand DNA synthesis. Phylogenetic conservation and earlier mutational studies suggested the highly based-paired ? structure as crucial for productive interaction with P protein. Using the tractable duck HBV (DHBV) model we here interrogated the entire apical DHBV ? (D?) half for sequence- and structure-dependent determinants of in vitro priming activity, replication, and, in part, in vivo infectivity. This revealed single-strandedness of the bulge, a following G residue plus the loop subsequence GUUGU as the few key determinants for priming and initiation site selection; unexpectedly, they functioned independently of a specific structure context. These data provide new mechanistic insights into avihepadnaviral replication initiation, and they imply a new concept towards a feasible in vitro priming system for human HBV.
Project description:OBJECTIVE:As the leading risk factor for the development of liver cancer, chronic infection with hepatitis B virus (HBV) represents a significant global health concern. Although an effective HBV vaccine exists, at least 240 million people are chronically infected with HBV worldwide. Therapeutic options for the treatment of chronic HBV remain limited, and none achieve an absolute cure. To develop novel therapeutic targets, a better understanding of the complex network of virus-host interactions is needed. Because of the central metabolic role of the liver, we assessed the metabolic impact of HBV infection as a means to identify viral dependency factors and metabolic pathways that could serve as novel points of therapeutic intervention. METHODS:Primary rat hepatocytes were infected with a control adenovirus, an adenovirus expressing a greater-than-unit-length copy of the HBV genome, or an adenovirus expressing the HBV X protein (HBx). A panel of 369 metabolites was analyzed for HBV- or HBx-induced changes 24 and 48?h post infection. Pathway analysis was used to identify key metabolic pathways altered in the presence of HBV or HBx expression, and these findings were further supported through integration of publically available gene expression data. RESULTS:We observed distinct changes to multiple metabolites in the context of HBV replication or HBx expression. Interestingly, a panel of 7 metabolites (maltotriose, maltose, myristate [14:0], arachidate [20:0], 3-hydroxybutyrate [BHBA], myo-inositol, and 2-palmitoylglycerol [16,0]) were altered by both HBV and HBx at both time points. In addition, incorporation of data from a transcriptome-based dataset allowed us to identify metabolic pathways, including long chain fatty acid metabolism, glycolysis, and glycogen metabolism, that were significantly altered by HBV and HBx. CONCLUSIONS:Because the liver is a central regulator of metabolic processes, it is important to understand how HBV replication and HBV protein expression affects the metabolic function of hepatocytes. Through analysis of a broad panel of metabolites we investigated this metabolic impact. The results of these studies have defined metabolic consequences of an HBV infection of hepatocytes and will help to lay the groundwork for novel research directions and, potentially, development of novel anti-HBV therapeutics.
Project description:Viral vectors are engineered virus variants able to deliver nonviral genetic information into cells, usually by the same routes as the parental viruses. For several virus families, replication-competent vectors carrying reporter genes have become invaluable tools for easy and quantitative monitoring of replication and infection, and thus also for identifying antivirals and virus susceptible cells. For hepatitis B virus (HBV), a small enveloped DNA virus causing B-type hepatitis, such vectors are not available because insertions into its tiny 3.2 kb genome almost inevitably affect essential replication elements. HBV replicates by reverse transcription of the pregenomic (pg) RNA which is also required as bicistronic mRNA for the capsid (core) protein and the reverse transcriptase (Pol); their open reading frames (ORFs) overlap by some 150 basepairs. Translation of the downstream Pol ORF does not involve a conventional internal ribosome entry site (IRES). We reasoned that duplicating the overlap region and providing artificial IRES control for translation of both Pol and an in-between inserted transgene might yield a functional tricistronic pgRNA, without interfering with envelope protein expression. As IRESs we used a 22 nucleotide element termed Rbm3 IRES to minimize genome size increase. Model plasmids confirmed its activity even in tricistronic arrangements. Analogous plasmids for complete HBV genomes carrying 399 bp and 720 bp transgenes for blasticidin resistance (BsdR) and humanized Renilla green fluorescent protein (hrGFP) produced core and envelope proteins like wild-type HBV; while the hrGFP vector replicated poorly, the BsdR vector generated around 40% as much replicative DNA as wild-type HBV. Both vectors, however, formed enveloped virions which were infectious for HBV-susceptible HepaRG cells. Because numerous reporter and effector genes with sizes of around 500 bp or less are available, the new HBV vectors should become highly useful tools to better understand, and combat, this important pathogen.
Project description:Chronic HBV infection is a major public health concern affecting over 240 million people worldwide. Although suppression of HBV replication is achieved in the majority of patients with currently available newer antivirals, discontinuation of therapy prior to hepatitis B surface antigen loss or seroconversion is associated with relapse of HBV in the majority of cases. Thus, new therapeutic modalities are needed to achieve eradication of the virus from chronically infected patients in the absence of therapy. The basis of HBV persistence includes viral and host factors. Here, we review novel strategies to achieve sustained cure or elimination of HBV. The novel approaches include targeting the viral and or host factors required for viral persistence, and novel immune-based therapies, including therapeutic vaccines.
Project description:Hepatitis B virus (HBV) infection is a leading cause of liver cirrhosis, liver cancer, and liver failure, affecting 350 million people worldwide. Currently available anti-HBV drugs include (PEGylated-) interferon-? and nucleos(t)ide analogs, which can cause significant side effects and drug-resistance in many cases of long-term treatment. The lack of a reliable and robust in vitro infection system is a major barrier for understanding the HBV life cycle and discovering novel therapeutic targets. In the present study, we demonstrate that overexpression of the hepatitis B surface antigen binding protein (SBP) in HepG2 cells (HepG2-SBP) resulted in their susceptibility to HBV infection. HepG2-SBP cells supported the uptake of the viral surface protein (HBsAg-preS), HBV-pseudotyped virus, and live HBV in patient sera. Moreover, SBP-mediated HBsAg-preS uptake, and HBV pseudotyped virus infections were efficiently blocked by preS1- and SBP-specific antibodies. These observations suggest that SBP is involved in HBV entry and that HepG2-SBP cells can serve as a cellular model to study the post-binding steps of HBV infection.
Project description:Hepatitis B virus (HBV) is a partially double-stranded DNA virus that replicates by reverse transcription. We previously demonstrated that the host restriction factor-APOBEC3B (A3B) inhibited HBV replication which was dependent on its deaminase activity during reverse transcription. However, the host factors involved in the process of regulating the anti-HBV function of A3B are less known. In this research, to obtain a comprehensive understanding of the interaction networks of A3B, we conducted coimmunoprecipitation and mass spectrometry to identify A3B-interacting proteins in the presence of HBV. By this approach, we determined that DExD/H-box helicase 9 (DHX9) suppressed the anti-HBV effect of A3B, and this suppression was dependent on their interaction. Although DHX9 did not affect the deamination activity of A3B in vitro assay or the viral DNA editing of A3B in HepG2-NTCP cells that support HBV infection, it inhibited the binding of A3B with pgRNA. These data suggest that DHX9 can interact with A3B and attenuate the anti-HBV efficacy of A3B.Abbreviations: 3D-PCR: differential DNA denaturation PCR; APOBEC3: apolipoprotein B mRNA-editing catalytic polypeptide 3; cccDNA: covalently closed circular DNA; co-IP: coimmunoprecipitation; DDX: DExD-box RNA helicases; HBc: HBV core protein; HBV: hepatitis B virus; HepAD38: HepG2 cell line stably transfected with HBV DNA; HepG2-NTCP: HepG2 cell line stably transfected with Na+/taurocholate cotransporter polypeptide; Huh7: human hepatoma cell line; pgRNA: pregenomic RNA; PPI: protein-protein interactions; RC DNA: relaxed circular DNA.
Project description:Chronic hepatitis B virus (HBV) infection represents a worldwide public health concern with approximately 250 million people chronically infected and at risk of developing liver cirrhosis and hepatocellular carcinoma. Nucleos(t)ide analogues (NUC) are the most widely used therapies for HBV infection, but they often require long-lasting administration to avoid the risk of HBV reactivation at withdrawal. Therefore, there is an urgent need to develop novel treatments to shorten the duration of NUC therapy by accelerating virus control, and to complement the effect of available anti-viral therapies. In chronic HBV infection, virus-specific T cells are functionally defective, and this exhaustion state is a key determinant of virus persistence. Reconstitution of an efficient anti-viral T cell response may thus represent a rational strategy to treat chronic HBV patients. In this perspective, the enhancement of adaptive immune responses by a checkpoint inhibitor blockade, specific T cell vaccines, lymphocyte metabolism targeting, and autologous T cell engineering, including chimeric antigen receptor (CAR) and TCR-redirected T cells, constitutes a promising immune modulatory approach for a therapeutic restoration of protective immunity. The advances of the emerging immune-based therapies in the setting of the HBV research field will be outlined.