Understanding and Targeting HBV Transcription and Post-Transcriptional Regulation.

Chronic hepatitis B (CHB) affects approximately 300 million people worldwide and current therapies rarely cure it [...

Hepatitis D virus interferes with hepatitis B virus RNA production via interferon-dependent and -independent mechanisms.

Background & Aims: Chronic coinfection with HBV and HDV leads to the most aggressive form of chronic viral hepatitis. Herein, we aimed to elucidate the molecular mechanisms underlying the widely reported observation that HDV interferes with HBV in most coinfected patients.

Methods: Patient liver tissues, primary human hepatocytes, HepaRG cells and human liver chimeric mice were used to analyze the effect of HDV on HBV using virological and RNA-sequencing analyses, as well as RNA synthesis, stability and association assays.

Characterization of a Novel Capsid Assembly Modulator for the Treatment of Chronic Hepatitis B Virus Infection.

The standard of care for the treatment of chronic hepatitis B (CHB) is typically lifelong treatment with nucleos(t)ide analogs (NAs), which suppress viral replication and provide long-term clinical benefits. However, infectious virus can still be detected in patients who are virally suppressed on NA therapy, which may contribute to the failure of these agents to cure most CHB patients. Accordingly, new antiviral treatment options are being developed to enhance the suppression of hepatitis B virus (HBV) replication in combination with NAs ("antiviral intensification").

Smc5/6 silences episomal transcription by a three-step function.

In addition to its role in chromosome maintenance, the six-membered Smc5/6 complex functions as a restriction factor that binds to and transcriptionally silences viral and other episomal DNA. However, the underlying mechanism is unknown. Here, we show that transcriptional silencing by the human Smc5/6 complex is a three-step process.

Therapeutic shutdown of HBV transcripts promotes reappearance of the SMC5/6 complex and silencing of the viral genome in vivo.

Objective: Therapeutic strategies silencing and reducing the hepatitis B virus (HBV) reservoir, the covalently closed circular DNA (cccDNA), have the potential to cure chronic HBV infection. We aimed to investigate the impact of small interferring RNA (siRNA) targeting all HBV transcripts or pegylated interferon-α (peg-IFNα) on the viral regulatory HBx protein and the structural maintenance of chromosome 5/6 complex (SMC5/6), a host factor suppressing cccDNA transcription. In particular, we assessed whether interventions lowering HBV transcripts can achieve and maintain silencing of cccDNA transcription in vivo.

Spatiotemporal Analysis of Hepatitis B Virus X Protein in Primary Human Hepatocytes.

The structural maintenance of chromosomes 5/6 complex (Smc5/6) is a host restriction factor that suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the host DNA damage-binding protein 1 (DDB1) E3 ubiquitin ligase to target Smc5/6 for degradation. HBx is an attractive therapeutic target for the treatment of chronic hepatitis B (CHB), but it is challenging to study this important viral protein in the context of natural infection due to the lack of a highly specific and sensitive HBx antibody.

Hepatitis B Virus X Protein Function Requires Zinc Binding.

The host structural maintenance of chromosomes 5/6 complex (Smc5/6) suppresses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing the X protein (HBx), which redirects the cellular DNA damage-binding protein 1 (DDB1)-containing E3 ubiquitin ligase to target Smc5/6 for degradation. However, the details of how HBx modulates the interaction between DDB1 and Smc5/6 remain to be determined.

In vitro resistance profile of hepatitis C virus NS5A inhibitor velpatasvir in genotypes 1 to 6.

Velpatasvir is a pan-genotypic hepatitis C virus (HCV) NS5A inhibitor, which is used with sofosbuvir for treatment of infection with HCV genotypes 1-6. In vitro resistance studies were performed to characterize NS5A changes that might confer reduced velpatasvir susceptibility in vivo. Resistance selection studies using HCV replicon cells for subtypes 1a, 1b, 2a, 2b, 3a, 4a, 5a and 6a identified NS5A resistance-associated substitutions (RASs) at nine positions, most often 28M/S/T, 31F/I/M/P/V and 93D/H/N/S.

Toll-like receptor 7 agonist GS-9620 induces prolonged inhibition of HBV via a type I interferon-dependent mechanism.

Background & Aims: GS-9620, an oral agonist of toll-like receptor 7 (TLR7), is in clinical development for the treatment of chronic hepatitis B (CHB). GS-9620 was previously shown to induce prolonged suppression of serum viral DNA and antigens in the woodchuck and chimpanzee models of CHB. Herein, we investigated the molecular mechanisms that contribute to the antiviral response to GS-9620 using in vitro models of hepatitis B virus (HBV) infection.

Identifying and Characterizing Interplay between Hepatitis B Virus X Protein and Smc5/6.

Hepatitis B X protein (HBx) plays an essential role in the hepatitis B virus (HBV) replication cycle, but the function of HBx has been elusive until recently. It was recently shown that transcription from the HBV genome (covalently-closed circular DNA, cccDNA) is inhibited by the structural maintenance of chromosome 5/6 complex (Smc5/6), and that a key function of HBx is to redirect the DNA-damage binding protein 1 (DDB1) E3 ubiquitin ligase to target this complex for degradation. By doing so, HBx alleviates transcriptional repression by Smc5/6 and stimulates HBV gene expression.

The Smc5/6 Complex Restricts HBV when Localized to ND10 without Inducing an Innate Immune Response and Is Counteracted by the HBV X Protein Shortly after Infection.

The structural maintenance of chromosome 5/6 complex (Smc5/6) is a restriction factor that represses hepatitis B virus (HBV) transcription. HBV counters this restriction by expressing HBV X protein (HBx), which targets Smc5/6 for degradation. However, the mechanism by which Smc5/6 suppresses HBV transcription and how HBx is initially expressed is not known.

Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor.

Chronic hepatitis B virus infection is a leading cause of cirrhosis and liver cancer. Hepatitis B virus encodes the regulatory HBx protein whose primary role is to promote transcription of the viral genome, which persists as an extrachromosomal DNA circle in infected cells. HBx accomplishes this task by an unusual mechanism, enhancing transcription only from extrachromosomal DNA templates.

A small-molecule inhibitor of hepatitis C virus infectivity.

One of the most challenging goals of hepatitis C virus (HCV) research is to develop well-tolerated regimens with high cure rates across a variety of patient populations. Such a regimen will likely require a combination of at least two distinct direct-acting antivirals (DAAs). Combining two or more DAAs with different resistance profiles increases the number of mutations required for viral breakthrough.

Hepatitis C viral entry inhibitors prolong viral suppression by replication inhibitors in persistently-infected Huh7 cultures.

Efforts to treat HCV patients are focused on developing antiviral combinations that lead to the eradication of infection. Thus, it is important to identify optimal combinations from the various viral inhibitor classes. Based on viral dynamic models, HCV entry inhibitors are predicted to reduce viral load in a monophasic manner reflecting the slow death rate of infected hepatocytes (t1/2 = 2-70 days) and the protection of naïve, un-infected cells from HCV infection.

Cellular growth kinetics distinguish a cyclophilin inhibitor from an HSP90 inhibitor as a selective inhibitor of hepatitis C virus.

During antiviral drug discovery, it is critical to distinguish molecules that selectively interrupt viral replication from those that reduce virus replication by adversely affecting host cell viability. In this report we investigate the selectivity of inhibitors of the host chaperone proteins cyclophilin A (CypA) and heat-shock protein 90 (HSP90) which have each been reported to inhibit replication of hepatitis C virus (HCV). By comparing the toxicity of the HSP90 inhibitor, 17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) to two known cytostatic compounds, colchicine and gemcitabine, we provide evidence that 17-AAG exerts its antiviral effects indirectly through slowing cell growth.

An adaptive mutation in NS2 is essential for efficient production of infectious 1b/2a chimeric hepatitis C virus in cell culture.

The development of JFH1 based intergenotypic recombinants which exploit the unique replication characteristics of JFH1 has made it possible to study infectious HCV encoding the structural genes of additional HCV genotypes including genotype 1b. Although, intergenotypic 1b/2a chimeric genomes replicate efficiently in transfected cells they produce very low viral titers, limiting the utility of this system. Here, intergenotypic 1b/2a variants were generated by serially passaging the virus in a novel highly permissive Huh-7 cell clone.

Novel mutations in a tissue culture-adapted hepatitis C virus strain improve infectious-virus stability and markedly enhance infection kinetics.

Hepatitis C virus (HCV) establishes persistent infections and leads to chronic liver disease. It only recently became possible to study the entire HCV life cycle due to the ability of a unique cloned patient isolate (JFH-1) to produce infectious particles in tissue culture. However, despite efficient RNA replication, yields of infectious virus particles remain modest.

Hepatitis C virus NS2 protein contributes to virus particle assembly via opposing epistatic interactions with the E1-E2 glycoprotein and NS3-NS4A enzyme complexes.

The hepatitis C virus NS2 protein has been recently implicated in virus particle assembly. To further understand the role of NS2 in this process, we conducted a reverse genetic analysis of NS2 in the context of a chimeric genotype 2a infectious cell culture system. Of 32 mutants tested, all were capable of RNA replication and 25 had moderate-to-severe defects in virus assembly.

The NS4A protein of hepatitis C virus promotes RNA-coupled ATP hydrolysis by the NS3 helicase.

Nonstructural protein 3 (NS3) is an essential replicative component of the hepatitis C virus (HCV) and a member of the DExH/D-box family of proteins. The C-terminal region of NS3 (NS3hel) exhibits RNA-stimulated NTPase and helicase activity, while the N-terminal serine protease domain of NS3 enhances RNA binding and unwinding by NS3hel. The nonstructural protein 4A (NS4A) binds to the NS3 protease domain and serves as an obligate cofactor for NS3 serine protease activity.

Establishing a mechanistic basis for the large kinetic steps of the NS3 helicase.

The NS3 helicase from hepatitis C virus is a prototypical DEx(H/D) RNA helicase. NS3 has been shown to unwind RNA in a discontinuous manner, pausing after long apparent steps of unwinding. We systematically examined the effects of duplex stability and ionic conditions on the periodicity of the NS3 unwinding cycle.

Hepatitis C viral NS3-4A protease activity is enhanced by the NS3 helicase.

Non-structural protein 3 (NS3) is a multifunctional enzyme possessing serine protease, NTPase, and RNA unwinding activities that are required for hepatitis C viral (HCV) replication. HCV non-structural protein 4A (NS4A) binds to the N-terminal NS3 protease domain to stimulate NS3 serine protease activity. In addition, the NS3 protease domain enhances the RNA binding, ATPase, and RNA unwinding activities of the C-terminal NS3 helicase domain (NS3hel).

The serine protease domain of hepatitis C viral NS3 activates RNA helicase activity by promoting the binding of RNA substrate.

Nonstructural (NS) protein 3 is a DEXH/D-box motor protein that is an essential component of the hepatitis C viral (HCV) replicative complex. The full-length NS3 protein contains two functional modules, both of which are essential in the life cycle of HCV: a serine protease domain at the N terminus and an ATPase/helicase domain (NS3hel) at the C terminus. Truncated NS3hel constructs have been studied extensively; the ATPase, nucleic acid binding, and helicase activities have been examined and NS3hel has been used as a target in the development of antivirals.

The C terminus of hepatitis C virus NS4A encodes an electrostatic switch that regulates NS5A hyperphosphorylation and viral replication.

Hepatitis C virus (HCV) nonstructural protein 4A (NS4A) is only 54 amino acids (aa) in length, yet it is a key regulator of the essential serine protease and RNA helicase activities of the NS3-4A complex, as well as a determinant of NS5A phosphorylation. Here we examine the structure and function of the C-terminal acidic region of NS4A through site-directed mutagenesis of a Con1 subgenomic replicon and through biophysical characterization of a synthetic peptide corresponding to this region. Our genetic studies revealed that in 8 of the 15 C-terminal residues of NS4A, individual Ala substitutions or charge reversal substitutions led to severe replication phenotypes, as well as decreased NS5A hyperphosphorylation.

Robust translocation along a molecular monorail: the NS3 helicase from hepatitis C virus traverses unusually large disruptions in its track.

The NS3 helicase is essential for replication of the hepatitis C virus. This multifunctional Superfamily 2 helicase protein unwinds nucleic acid duplexes in a stepwise, ATP-dependent manner. Although kinetic features of its mechanism are beginning to emerge, little is known about the physical determinants for NS3 translocation along a strand of nucleic acid.