Michael Nassal

Hepatitis B and C virus coinfection: A novel model system reveals the absence of direct viral interference

Coinfection with hepatitis B virus (HBV) and hepatitis C virus (HCV) has been associated with severe liver disease and frequent progression to cirrhosis and hepatocellular carcinoma. Clinical evidence suggests reciprocal replicative suppression of the two viruses, or viral interference. However, interactions between HBV and HCV have been difficult to study due to the lack of appropriate model systems. We have established a novel model system to investigate interactions between HBV and HCV. Stable Huh‐7 cell lines inducibly replicating HBV were transfected with selectable HCV replicons or infected with cell culture–derived HCV. In this system, both viruses were found to replicate in the same cell without overt interference. Specific inhibition of one virus did not affect the replication and gene expression of the other. Furthermore, cells harboring replicating HBV could be infected with cell culture–derived HCV, arguing against superinfection exclusion. Finally, cells harboring replicating HBV supported efficient production of infectious HCV. Conclusion: HBV and HCV can replicate in the same cell without evidence for direct interference in vitro. Therefore, the viral interference observed in coinfected patients is probably due to indirect mechanisms mediated by innate and/or adaptive host immune responses. These findings provide new insights into the pathogenesis of HBV–HCV coinfection and may contribute to its clinical management in the future. (HEPATOLOGY 2009.)

Hepatitis B virus mutations associated with fulminant hepatitis induce apoptosis in primary Tupaia hepatocytes.

Hepatitis B virus (HBV) core promoter mutations have been implicated in the pathogenesis of fulminant hepatitis B. Due to the limited availability of primary human hepatocytes, the functional characterization of HBV mutants has been performed predominantly in transformed cells, which may not represent ideal model systems for studying virus–cell interactions. We and others have shown that primary hepatocytes of the tree shrew Tupaia belangeri support HBV infection and replication. In this study, we used primary Tupaia hepatocytes to analyze the phenotype of two HBV core promoter mutations that have been associated with a clinical outbreak of fatal fulminant hepatitis. Similar to previous findings in human hepatoma cells, the HBV core promoter mutations resulted in enhanced viral replication and core expression. Surprisingly, however, the presence of the mutations had a marked effect on hepatocyte viability not previously observed in hepatoma cells. Reduced cell viability was found to be due to the induction of apoptosis, as evidenced by caspase‐3 activation and nuclear fragmentation. In conclusion, HBV mutants exhibit a novel phenotype in primary hepatocytes distinctly different from previous findings in hepatoma cell lines. This phenotype may have important implications for the understanding of the fulminant clinical course associated with HBV mutations. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005.)41:247–256

A targeted functional RNA interference screen uncovers glypican 5 as an entry factor for hepatitis B and D viruses

Chronic hepatitis B and D infections are major causes of liver disease and hepatocellular carcinoma worldwide. Efficient therapeutic approaches for cure are absent. Sharing the same envelope proteins, hepatitis B virus and hepatitis delta virus use the sodium/taurocholate cotransporting polypeptide (a bile acid transporter) as a receptor to enter hepatocytes. However, the detailed mechanisms of the viral entry process are still poorly understood. Here, we established a high‐throughput infectious cell culture model enabling functional genomics of hepatitis delta virus entry and infection. Using a targeted RNA interference entry screen, we identified glypican 5 as a common host cell entry factor for hepatitis B and delta viruses. Conclusion: These findings advance our understanding of virus cell entry and open new avenues for curative therapies. As glypicans have been shown to play a role in the control of cell division and growth regulation, virus–glypican 5 interactions may also play a role in the pathogenesis of virus‐induced liver disease and cancer. (Hepatology 2016;63:35–48)

New insights into HBV replication: new opportunities for improved therapies

HBV is one of the smallest animal viruses yet one of the most successful human pathogens, inflicting potentially life-threatening liver disease on more than 5% of the global population. While infection is preventable by prophylactic vaccination, few options for the treatment of established chronic hepatitis B are available and none are effective in more than a fraction of patients. Moreover, interferons may cause severe side effects and the, as of August 2008, five approved nucleos(t)ide analogs suffer from rapid emergence of viral resistance. Hence, improved and sustained therapeutic success will require new treatments based on alternative modes of action. The minimalistic genetic outfit of HBV offers few obvious targets, and virus-specific restrictions have hampered understanding, in particular, of the early infection steps. However, recently established in vitro reconstitution systems for initation of reverse transcription and capsid assembly are ready to be exploited for mechanism–based screens for ne...

Host-targeting agents for treatment of hepatitis B virus infection

Hepatitis B virus (HBV) infection is a major cause of chronic liver disease, including liver cirrhosis, liver failure and hepatocellular carcinoma (HCC)-the second leading and fastest rising cause of cancer death world-wide. While de novo infection can be efficiently prevented by vaccination and chronic infection can be controlled using antivirals targeting the viral polymerase, the development of efficient antiviral strategies to eliminate the virus and thus to cure infection remains a key unmet medical need. The recent progress in the development of robust infectious HBV cell culture models now enables the investigation of the full viral life cycle, including a more detailed study of the molecular mechanisms of virus-host interactions responsible for viral persistence. The understanding of these virus-host interactions will be instrumental for the development of curative treatments. Host-dependency factors have recently emerged as promising candidates to treat and prevent infection by various pathogens. This review focuses on the potential of host-targeting agents (HTAs) as novel antivirals to treat and cure HBV infection. These include HTAs that inhibit de novo and re-infection, synthesis and spread of cccDNA as well as development of immune-based approaches eliminating or curing infected hepatocytes, including the eradication of viral cccDNA.

Structural Studies of Self-Assembled Subviral Particles: Combining Cell-Free Expression with 110 kHz MAS NMR Spectroscopy

Viral membrane proteins are prime targets in combatting infection. Still, the determination of their structure remains a challenge, both with respect to sample preparation and the need for structural methods allowing for analysis in a native-like lipid environment. Cell-free protein synthesis and solid-state NMR spectroscopy are promising approaches in this context, the former with respect to its great potential in the native expression of complex proteins, and the latter for the analysis of membrane proteins in lipids. Herein, we show that milligram amounts of the small envelope protein of the duck hepatitisu2005B virus (DHBV) can be produced by cell-free expression, and that the protein self-assembles into subviral particles. Proton-detected 2D NMR spectra recorded at a magic-angle-spinning frequency of 110u2005kHz on <500u2005μg protein show a number of isolated peaks with line widths comparable to those of model membrane proteins, paving the way for structural studies of this protein that is homologous to a potential drug target in HBV infection.

[Hepatitis B and C: From molecular virology to new antiviral therapies (part 1)].

Institut 1 Klinik für Innere Medizin II, Gastroenterologie, Hepatologie, Endokrinologie und Infektiologie, Universitätsklinikum Freiburg 2 Abteilung Gastroenterologie und Hepatologie, Universitätsspital Basel 3 Inserm U1110, Forschungsinstitut für Virusund Leberkrankheiten, Universität Strasbourg 4 Zentrum für Hepatologie und Gastroenterologie, Universitätsklinik Strasbourg 5 Abteilung Gastroenterologie und Hepatologie, Universitätsklinik Lausanne (CHUV)

Localizing Conformational Hinges by NMR: Where Do Hepatitis B Virus Core Proteins Adapt for Capsid Assembly?

The hepatitis B virus (HBV) icosahedral nucleocapsid is assembled from 240 chemically identical core protein molecules and, structurally, comprises four groups of symmetrically nonequivalent subunits. We show here that this asymmetry is reflected in solid-state NMR spectra of the capsids, in which peak splitting is observed for a subset of residues. We compare this information to dihedral angle variations from available 3D structures and also to computational predictions of dynamic domains and molecular hinges. We find that although, at the given resolution, dihedral angles variations directly obtained from the X-ray structures are not precise enough to be interpreted, the chemical-shift information from NMR correlates, and interestingly goes beyond, information from bioinformatics approaches. Our study reveals the high sensitivity with which NMR can detect the residues allowing the subtle conformational adaptations needed in lattice formation. Our findings are important for understanding the formation and modulation of protein assemblies in general.

Solid-state [13C–15N] NMR resonance assignment of hepatitis B virus core protein

Each year, nearly 900,000 deaths are due to serious liver diseases caused by chronic hepatitis B virus infection. The viral particle is composed of an outer envelope and an inner icosahedral nucleocapsid formed by multiple dimers of au2009~u200920xa0kDa self-assembling core protein (Cp). Here we report the solid-state 13C and 15N resonance assignments of the assembly domain, Cp149, of the core protein in its capsid form. A secondary chemical shift analysis of the 140 visible residues suggests an overall alpha-helical three-dimensional fold matching that derived for Cp149 from the X-ray crystallography of the capsid, and from solution-state NMR of the Cp149 dimer. Interestingly, however, at three distinct regions the chemical shifts in solution differ significantly between core proteins in the capsid state versus in the dimer state, strongly suggesting the respective residues to be involved in capsid assembly.