Johnson Y.N. Lau

Interleukin 10 treatment reduces fibrosis in patients with chronic hepatitis C: A pilot trial of interferon nonresponders ☆ ☆☆

BACKGROUND & AIMS Interleukin (IL)-10 is a cytokine that down-regulates the proinflammatory response and has a modulatory effect on hepatic fibrogenesis. The aim of this study was to determine the effect of IL-10 on hepatic injury in patients with chronic hepatitis C. METHODS Twenty-four patients with chronic hepatitis C who had not previously responded to interferon-based therapy were enrolled in a randomized, double-blinded 2-dose trial in which they received either 4 or 8 microgram/kg IL-10 subcutaneously daily for 90 days. Liver biopsies were performed before and at the end of therapy. RESULTS IL-10 was well tolerated with 22 patients completing the study. Serum ALT levels normalized in 19 of 22 patients by the end of therapy and were sustained in 5 of 22. Hepatic inflammation decreased in 19 of 22 patients, with 11 having a decrease by >/=2. Fibrosis decreased in 14 of 22 patients (mean change, 3.6-2.6; P = 0.001). There was no change in serum HCV RNA levels. IL-10 therapy was associated with changes in serological markers, suggesting a reduction of immune response and fibrogenesis. CONCLUSIONS IL-10 therapy is safe and well tolerated in patients with chronic hepatitis C. Although it has no apparent antiviral activity, IL-10 normalizes serum ALT levels, improves liver histology, and reduces liver fibrosis in a large proportion of patients receiving treatment. Therefore, IL-10 may have therapeutic potential in patients with chronic hepatitis C patients who do not respond to interferon-based therapy.

De Novo Initiation of RNA Synthesis by Hepatitis C Virus Nonstructural Protein 5B Polymerase

ABSTRACT RNA-dependent RNA polymerase (RdRp) encoded by positive-strand RNA viruses is critical to the replication of viral RNA genome. Like other positive-strand RNA viruses, replication of hepatitis C virus (HCV) RNA is mediated through a negative-strand intermediate, which is generated through copying the positive-strand genomic RNA. Although it has been demonstrated that HCV NS5B alone can direct RNA replication through a copy-back primer at the 3′ end, de novo initiation of RNA synthesis is likely to be the mode of RNA replication in infected cells. In this study, we demonstrate that a recombinant HCV NS5B protein has the ability to initiate de novo RNA synthesis in vitro. The NS5B used HCV 3′ X-tail RNA (98 nucleotides) as the template to synthesize an RNA product of monomer size, which can be labeled by [γ-32P]nucleoside triphosphate. The de novo initiation activity was further confirmed by using small synthetic RNAs ending with dideoxynucleotides at the 3′ termini. In addition, HCV NS5B preferred GTP as the initiation nucleotide. The optimal conditions for the de novo initiation activity have been determined. Identification and characterization of the de novo priming or initiation activity by HCV NS5B provides an opportunity to screen for inhibitors that specifically target the initiation step.

Ribavirin inhibits West Nile virus replication and cytopathic effect in neural cells

West Nile virus (WNV) is an emerging mosquito-borne pathogen that was reported for the first time in the Western hemisphere in August 1999, when an encephalitis outbreak in New York City resulted in 62 clinical cases and 7 deaths. WNV, for which no antiviral therapy has been described, was recently recovered from a pool of mosquitoes collected in New York City. In anticipation of the recurrence of WNV during the summer of 2000, an analysis was made of the efficacy of the nucleoside analogue ribavirin, a broad-spectrum antiviral compound with activity against several RNA viruses, for treatment of WNV infection. High doses of ribavirin were found to inhibit WNV replication and cytopathogenicity in human neural cells in vitro.

Hepatitis C Virus Internal Ribosome Entry Site (IRES) Stem Loop IIId Contains a Phylogenetically Conserved GGG Triplet Essential for Translation and IRES Folding

ABSTRACT The hepatitis C virus (HCV) internal ribosome entry site (IRES) is a highly structured RNA element that directs cap-independent translation of the viral polyprotein. Morpholino antisense oligonucleotides directed towards stem loop IIId drastically reduced HCV IRES activity. Mutagenesis studies of this region showed that the GGG triplet (nucleotides 266 through 268) of the hexanucleotide apical loop of stem loop IIId is essential for IRES activity both in vitro and in vivo. Sequence comparison showed that apical loop nucleotides (UUGGGU) were absolutely conserved across HCV genotypes and the GGG triplet was strongly conserved among related Flavivirus andPestivirus nontranslated regions. Chimeric IRES elements with IIId derived from GB virus B (GBV-B) in the context of the HCV IRES possess translational activity. Mutations within the IIId stem loop that abolish IRES activity also affect the RNA structure in RNase T1-probing studies, demonstrating the importance of correct RNA folding to IRES function.

Template/Primer Requirements and Single Nucleotide Incorporation by Hepatitis C Virus Nonstructural Protein 5B Polymerase

ABSTRACT Nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) possesses an RNA-dependent RNA polymerase activity responsible for viral genome RNA replication. Despite several reports on the characterization of this essential viral enzyme, little is known about the reaction pathway of NS5B-catalyzed nucleotide incorporation due to the lack of a kinetic system offering efficient assembly of a catalytically competent polymerase/template/primer/nucleotide quaternary complex. In this report, specific template/primer requirements for efficient RNA synthesis by HCV NS5B were investigated. For intramolecular copy-back RNA synthesis, NS5B utilizes templates with an unstable stem-loop at the 3′ terminus which exists as a single-stranded molecule in solution. A template with a stable tetraloop at the 3′ terminus failed to support RNA synthesis by HCV NS5B. Based on these observations, a number of single-stranded RNA templates were synthesized and tested along with short RNA primers ranging from two to five nucleotides. It was found that HCV NS5B utilized di- or trinucleotides efficiently to initiate RNA replication. Furthermore, the polymerase, template, and primer assembled initiation-competent complexes at the 3′ terminus of the template RNA where the template and primer base paired within the active site cavity of the polymerase. The minimum length of the template is five nucleotides, consistent with a structural model of the NS5B/RNA complex in which a pentanucleotide single-stranded RNA template occupies a groove located along the fingers subdomain of the polymerase. This observation suggests that the initial docking of RNA on NS5B polymerase requires a single-stranded RNA molecule. A unique β-hairpin loop in the thumb subdomain may play an important role in properly positioning the single-stranded template for initiation of RNA synthesis. Identification of the template/primer requirements will facilitate the mechanistic characterization of HCV NS5B and its inhibitors.

Hepatitis C virus therapies: current treatments, targets and future perspectives.

Chronic hepatitis C virus (HCV) infection is the cause of an emerging global epidemic of chronic liver disease. Current combination therapies are at best 80% efficacious and are often poorly tolerated. Strategies to improve the therapeutic response include the development of novel interferons, nucleoside analogues with reduced haemolysis compared with ribavirin and inosine 5′-monophosphate dehydrogenase inhibitors. Compounds in preclinical or early clinical trials include small molecules that inhibit virus-specific enzymes (such as the serine proteases, RNA polymerase and helicase) or interfere with translation (including anti-sense molecules, iRNA and ribozymes). Advances in understanding HCV replication, obtaining a sub-genomic replicon and contriving potential small animal models, in addition to solving crystallographic structures for the replication enzymes, have improved prospects for developing novel therapies. This review summarizes current and evolving treatments for chronic hepatitis C infection. In addition, progress in HCV targets and drug discovery tools valuable in the search for novel anti-HCV agents is detailed.

Generation and Characterization of a Hepatitis C Virus NS3 Protease-Dependent Bovine Viral Diarrhea Virus

ABSTRACT Unique to pestiviruses, the N-terminal protein encoded by the bovine viral diarrhea virus (BVDV) genome is a cysteine protease (Npro) responsible for a self-cleavage that releases the N terminus of the core protein (C). This unique protease is dispensable for viral replication, and its coding region can be replaced by a ubiquitin gene directly fused in frame to the core. To develop an antiviral assay that allows the assessment of anti-hepatitis C virus (HCV) NS3 protease inhibitors, a chimeric BVDV in which the coding region of Npro was replaced by that of an NS4A cofactor-tethered HCV NS3 protease domain was generated. This cofactor-tethered HCV protease domain was linked in frame to the core protein of BVDV through an HCV NS5A-NS5B junction site and mimicked the proteolytic function of Npro in the release of BVDV core for capsid assembly. A similar chimeric construct was built with an inactive HCV NS3 protease to serve as a control. Genomic RNA transcripts derived from both chimeric clones, PH/B(wild-type HCV NS3 protease) and PH/B(S139A) (mutant HCV NS3 protease) were then transfected into bovine cells (MDBK). Only the RNA transcripts from the PH/B clone yielded viable viruses, whereas the mutant clone, PH/B(S139A), failed to produce any signs of infection, suggesting that the unprocessed fusion protein rendered the BVDV core protein defective in capsid assembly. Like the wild-type BVDV (NADL), the chimeric virus was cytopathic and formed plaques on the cell monolayer. Sequence and biochemical analyses confirmed the identity of the chimeric virus and further revealed variant viruses due to growth adaptation. Growth analysis revealed comparable replication kinetics between the wild-type and the chimeric BVDVs. Finally, to assess the genetic stability of the chimeric virus, an Npro-null BVDV (BVDV−Npro in which the entire Npro coding region was deleted) was produced. Although cytopathic, BVDV−Npro was highly defective in viral replication and growth, a finding consistent with the observed stability of the chimeric virus after serial passages.

Amantadine and Rimantadine Have No Direct Inhibitory Effects against Hepatitis C Viral Protease, Helicase, ATPase, Polymerase, and Internal Ribosomal Entry Site-Mediated Translation

Amantadine, a drug known to inhibit influenza A viral matrix (M2) protein function, was reported to be an effective treatment in some patients with chronic hepatitis C virus (HCV) infection. Sequence comparison shows no homology between M2 and any of the HCV proteins. The effects of amantadine and a related analogue, rimantadine, on viral protease, helicase, ATPase, RNA-dependent RNA polymerase, and HCV internal ribosomal entry site (IRES) translation were tested by established in vitro biochemical assays. No inhibition (>15%) of HCV protease, helicase, ATPase, and polymerase was observed with concentrations up to 400 microgram/mL. IRES-specific inhibition was not observed at clinically relevant concentrations, but both cap and IRES reporter genes were suppressed at higher levels, suggesting nonspecific translation inhibition. In conclusion, amantadine and rimantadine have no direct and specific inhibitory effects against HCV protease, helicase, ATPase, polymerase, and IRES in vitro.

TT virus infection in patients with chronic hepatitis C virus infection - effect of primers, prevalence, and clinical significance

BACKGROUND/AIM A novel DNA virus, TT virus (TTV), was recently identified in patients with post-transfusion non-A-G hepatitis. The aim of this study was to determine the prevalence and clinical significance of TTV infection in patients with chronic hepatitis C virus (HCV) infection. METHODS We analyzed pretreatment serum samples from 171 United States and European patients who relapsed after interferon-alpha treatment and were recruited into an interferon-alpha-2b/ribavirin combination treatment trial. TTV DNA was detected by PCR using two different set of primers (TTV-A and TTV-B) derived from open reading frames 1 and 2, respectively. RESULTS TTV was detected in 29.2% of the patients with the TTV-A primer set, 70.8% with the TTV-B primer-set, and 72.5% if positive by either/both sets of the primers. The amplicons generated by primer set A were sequenced and a phylogenetic tree was constructed. The 50 isolates belonged to group la (n=8), 1b (n=17), 2a (n=21), 2b (n=3), and 4 (n=1). There was no difference in demographic (age, sex distribution, estimated duration of HCV infection), biochemical (serum ALT levels), virologic (serum HCV RNA levels, HCV genotype distribution), or histologic scores, and their subsequent response to either interferon-alpha-2b or interferon-alpha-2b/ribavirin combination treatment. CONCLUSIONS The prevalence of TTV infection reported previously may have been significantly underestimated, based on the primers originally described and used by most studies. Although TTV infection is very common in patients with chronic HCV infection, it has no identifiable clinical significance.

24 Development of novel therapies for hepatitis C

Publisher Summary This chapter discusses the development therapies for of novel hepatitis C. Hepatitis C virus (HCV) is an elusive pathogen. Given the flexibility of HCV to adapt through its genetic evolution, the development of resistance to therapies targeted at HCV enzymes is a given. Combination therapies attacking various steps of the viral life cycle and boostering the host immune response represent the most attractive approach at present. This combination therapy improves the complete and sustained virological response rate to 49% for patients who relapsed after a previous course of interferon-a therapy and 41% for treatment of naive patients. Despite the improved efficacy of the combination therapy, 50–60% of the patients failed to respond in a sustained fashion. The current combination therapy also requires a treatment period of 6–12 months. Hence, newer and better anti-HCV therapies are still needed. In chronic HCV infection, the current understanding is that the host immune response is ineffective in controlling and eliminating HCV infection. Hence, it is possible that augmentation of the HCV specific immune response may assist the host in clearing the viral infection.