Ultra-deep pyrosequencing analysis of the hepatitis B virus preCore region and main catalytic motif of the viral polymerase in the same viral genome

Hepatitis B virus (HBV) pregenomic RNA contains a hairpin structure (ϵ) located in the preCore region, essential for viral replication. ϵ stability is enhanced by the presence of preCore variants and ϵ is recognized by the HBV polymerase (Pol). Mutations in the retrotranscriptase domain (YMDD) of Pol are associated with treatment resistance. The aim of this study was to analyze the preCore region and YMDD motif by ultra-deep pyrosequencing (UDPS). To evaluate the UDPS error rate, an internal control sequence was inserted in the amplicon. A newly developed technique enabled simultaneous analysis of the preCore region and Pol in the same viral genome, as well as the conserved sequence of the internal control. Nucleotide errors in HindIII yielded a UDPS error rate <0.05%. UDPS study confirmed the possibility of simultaneous detection of preCore and YMDD mutations, and demonstrated the complexity of the HBV quasispecies and cooperation between viruses. Thermodynamic stability of the ϵ signal was found to be the main constraint for selecting main preCore mutations. Analysis of ϵ-signal variability suggested the essential nature of the ϵ structural motif and that certain nucleotides may be involved in ϵ signal functions

HBV quasispecies composition in Lamivudine-failed chronic hepatitis B patients and its influence on virological response to Tenofovir-based rescue therapy

The present study sought to evaluate the structure of HBV quasispecies in Lamivudine (LMV)-failed chronic hepatitis B (CHB) patients and its impact in defining the subsequent virological responses to Tenofovir (TDF)-based rescue-therapy. By analyzing HBV clones encompassing reverse transcriptase (RT) and surface (S) region from LMV-failed and treatment-naïve CHB patients, we identified 5 classical and 12 novel substitutions in HBV/RT and 9 substitutions in immune-epitopes of HBV/S that were significantly associated with LMV failure. In silico analysis showed spatial proximity of some of the newly-identified, mutated RT residues to the RT catalytic centre while most S-substitutions caused alteration in epitope hydrophobicity. TDF administration resulted in virological response in 60% of LMV-failed patients at 24-week but non-response in 40% of patients even after 48-weeks. Significantly high frequencies of 6 S-substitutions and one novel RT-substitution, rtH124N with 6.5-fold-reduced susceptibility to TDF in vitro, were noted at baseline in TDF non-responders than responders. Follow-up studies depicted greater evolutionary drift of HBV quasispecies and significant decline in frequencies of 3 RT and 6 S-substitutions in responder-subgroup after 24-week TDF-therapy while most variants persisted in non-responders. Thus, we identified the HBV-RT/S variants that could potentially predict unfavorable response to LMV/TDF-therapy and impede immune-mediated viral clearance

Receptor Complementation and Mutagenesis Reveal SR-BI as an Essential HCV Entry Factor and Functionally Imply Its Intra- and Extra-Cellular Domains

HCV entry into cells is a multi-step and slow process. It is believed that the initial capture of HCV particles by glycosaminoglycans and/or lipoprotein receptors is followed by coordinated interactions with the scavenger receptor class B type I (SR-BI), a major receptor of high-density lipoprotein (HDL), the CD81 tetraspanin, and the tight junction protein Claudin-1, ultimately leading to uptake and cellular penetration of HCV via low-pH endosomes. Several reports have indicated that HDL promotes HCV entry through interaction with SR-BI. This pathway remains largely elusive, although it was shown that HDL neither associates with HCV particles nor modulates HCV binding to SR-BI. In contrast to CD81 and Claudin-1, the importance of SR-BI has only been addressed indirectly because of lack of cells in which functional complementation assays with mutant receptors could be performed. Here we identified for the first time two cell types that supported HCVpp and HCVcc entry upon ectopic SR-BI expression. Remarkably, the undetectable expression of SR-BI in rat hepatoma cells allowed unambiguous investigation of human SR-BI functions during HCV entry. By expressing different SR-BI mutants in either cell line, our results revealed features of SR-BI intracellular domains that influence HCV infectivity without affecting receptor binding and stimulation of HCV entry induced by HDL/SR-BI interaction. Conversely, we identified positions of SR-BI ectodomain that, by altering HCV binding, inhibit entry. Finally, we characterized alternative ectodomain determinants that, by reducing SR-BI cholesterol uptake and efflux functions, abolish HDL-mediated infection-enhancement. Altogether, we demonstrate that SR-BI is an essential HCV entry factor. Moreover, our results highlight specific SR-BI determinants required during HCV entry and physiological lipid transfer functions hijacked by HCV to favor infection

Direct Infection and Replication of Naturally Occurring Hepatitis C Virus Genotypes 1, 2, 3 and 4 in Normal Human Hepatocyte Cultures

Hepatitis C virus (HCV) infection afflicts about 170 million individuals worldwide. However, the HCV life cycle is only partially understood because it has not been possible to infect normal human hepatocytes in culture. The current Huh-7 systems use cloned, synthetic HCV RNA expressed in hepatocellular carcinoma cells to produce virions, but these cells cannot be infected with naturally occurring HCV obtained from infected patients.Here, we describe a human hepatocyte culture permissible to the direct infection with naturally occurring HCV genotypes 1, 2, 3 and 4 in the blood of HCV-infected patients. The culture system mimics the biology and kinetics of HCV infection in humans, and produces infectious virions that can infect naïve human hepatocytes.This culture system should complement the existing systems, and may facilitate the understanding of the HCV life cycle, its effects in the natural host cell, the hepatocyte, as well as the development of novel therapeutics and vaccines

The impact of currently licensed therapies on viral and immune responses in Chronic Hepatitis B: considerations for future novel therapeutics.

Despite the availability of a preventative vaccine, chronic hepatitis B (CHB) remains a global healthcare challenge with the risk of disease progression due to cirrhosis and hepatocellular carcinoma. Although current treatment strategies, interferon and nucleos(t)ide analogues have contributed to reducing morbidity and mortality related to CHB, these therapies are limited in providing functional cure. The treatment paradigm in CHB is rapidly evolving with a number of new agents in the developmental pipeline. However, until novel agents with functional cure capability are available in the clinical setting, there is a pressing need to optimize currently licensed therapies. Here, we discuss current agents used alone and/or in combination strategies along with the impact of these therapies on viral and immune responses. Novel treatment strategies are outlined, and the potential role of current therapies in the employment of pipeline agents is discussedWellcome Trust Clinical Research Training Fellowship (107389/Z/15/Z)NIHR Academic Clinical LectureshipBarts Charity Project Grants (723/1795 and MGU/0406NIHR Research for patient benefit award (PB‐PG‐0614‐34087) to PTF

The bovine viral diarrhoea virus: a model for the study of antiviral molecules interfering with N-glycosylation and folding of envelope glycoprotein

The current treatment of chronic hepatitis C combines interferon alpha and ribavirin andis effective in only half of the patients treated. Considerable efforts are being made to developnovel anti-HVC molecules with a better efficacy particularly for refractory patients. Molecules targeting specifically viral activities are the most studied. However, an antiviral strategy based uniquely on the utilisation of this type of molecules is expected to encounter problems caused by the emergence of viral escape mutants, as already widely described for HIV and HBV. Alternative approaches and molecules are needed to complement antiviral strategies based on inhibitors of viral enzyme. Ideally, new molecules should target steps of the viral cycle that are potentially less likely to give rise to resistance. The assembly and morphogenesis of HCV belong to these yet untargeted steps of the life cycle

Antiviral effect of N-butyldeoxynojirimycin against bovine viral diarrhea virus correlates with misfolding of E2 envelope proteins and impairment of their association into E1-E2 heterodimers.

The iminosugar N-butyldeoxynojirimycin (NB-DNJ), an endoplasmic reticulum alpha-glucosidase inhibitor, has an antiviral effect against bovine viral diarrhea virus (BVDV). In this report, we investigate the molecular mechanism of this inhibition by studying the folding pathway of BVDV envelope glycoproteins in the presence and absence of NB-DNJ. Our results show that, while the disulfide-dependent folding of E2 glycoprotein occurs rapidly (2.5 min), the folding of E1 occurs slowly (30 min). Both BVDV envelope glycoproteins associate rapidly with calnexin and dissociate with different kinetics. The release of E1 from the interaction with calnexin coincides with the beginning of E1 and E2 association into disulfide-linked heterodimers. In the presence of NB-DNJ, the interaction of E1 and E2 with calnexin is prevented, leading to misfolding of the envelope glycoproteins and inefficient formation of E1-E2 heterodimers. The degree of misfolding and the lack of association of E1 and E2 into disulfide-linked complexes in the presence of NB-DNJ correlate with the dose-dependent antiviral effect observed for this iminosugar

Mechanism of action of ribavirin in anti-HCV regimens: New insights for an age-old question?

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HBV infection and HCC: the 'dangerous liaisons'.

Singapore Translational Research (STaR) Investigator Award (NMRC/STaR/013/2012)