Hepatitis B virus (HBV) morphogenesis is characterized by a large over-production of subviral particles and recently described new forms in parallel of complete viral particles (VP). This study was designed to depict circulating viral forms in HBV infected patient plasmas, using velocity gradients and most sensitive viral markers. Plasmas from chronic hepatitis B (CHB) patients, HBeAg positive or negative, genotype D or E, were fractionated on velocity and equilibrium gradients with or without detergent treatment. Antigenic and molecular markers were measured in plasma and in each collected fraction. Fast Nycodenz velocity gradients revealed good reproducibility and provided additional information to standard equilibrium sucrose gradients. HBV-RNAs circulated as enveloped particles in all plasmas, except one, and at lesser concentrations than VP. Calculations based on standardized measurements and relative virion and subviral particle molecular stoichiometry allowed to refine the experimental approach. For the HBeAg-positive plasma, VP were accompanied by an overproduction of enveloped capsids, either containing HBs, likely corresponding to empty virions, or for the main part, devoid of this viral envelope protein. Similarly, in the HBeAg-negative sample, HBs enveloped capsids, likely corresponding to empty virions, were detected and the presence of enveloped capsids devoid of HBs protein was suspected but not clearly evidenced due to the presence of contaminating high-density subviral particles. While HBeAg largely influences HBcrAg measurement and accounts for two-thirds of HBcrAg reactivity in HBeAg-positive patients, it remains a 10 times more sensitive marker than HBsAg to characterize VP containing fractions. Using Nycodenz velocity gradients and standardized biomarkers, our study proposes a detailed characterization of circulating viral forms in chronically HBV infected patients. We provide evidence for an excess of capsids in fractions enriched in Dane particles, likely due to the presence of empty virions but also by capsids enveloped by an HBs free lipid layer. Identification of this new circulating viral particle sets the basis for studies around the potential role of these entities in hepatitis B pathogeny and their physiological regulation.

Hepatitis B virus (HBV) infection is a major public health problem. Recently, the human liver bile acid transporter Na+/taurocholate cotransporting polypeptide (NTCP) has been identified as an HBV specific receptor. NTCP expression is known to be strongly regulated by IL-6. This study was aimed at characterizing the effect of IL-6 on HBV entry. HBV entry was inhibited by up to 90% when cells were pretreated with IL-6 as shown by a strong inhibition of long term HBsAg secretion. This effect was confirmed by showing a severe reduction of intracellular HBV cccDNA. In parallel, we observed a 98% decrease in NTCP mRNA steady state level and an 80% reduction in NTCP-mediated taurocholate uptake. IL-6-mediated inhibition of NTCP-mediated taurocholate uptake and viral entry exhibited similar dose-dependence and kinetics while restoration of NTCP expression suppressed the inhibitory effect of IL-6. NTCP-mediated HBV entry is therefore markedly inhibited by IL-6.

To improve a previously constructed broadly neutralizing hepatitis B virus (HBV)‐specific preS1 humanized antibody (HzKR127), we further humanized it through specificity‐determining residue (SDR) grafting. Moreover, we improved affinity by mutating two residues in heavy‐chain complementarity‐determining regions (CDR), on the basis of the crystal structure of the antigen–antibody complex. HzKR127‐3.2 exhibited 2.5‐fold higher affinity and enhanced virus‐neutralizing activity compared to the original KR127 antibody and showed less immunogenic potential than HzKR127. Enhanced virus‐neutralizing activity was achieved by the increased association rate, providing insights into engineering potent antibody therapeutics for HBV immunoprophylaxis. HzKR127‐3.2 may be a good candidate for HBV immunoprophylaxis.

Le virus de l'hepatite B (HBV) est un agent pathogene humain, tres contagieux, responsable de pathologies hepatiques telles que la cirrhose ou le carcinome hepatocellulaire. Il appartient a la famille des Hepadnaviridae dont les membres infectent differentes especes animales et provoquent des hepatites aigues ou chroniques. Bien que les connaissances concernant la biologie moleculaire de ces virus aient beaucoup progresse durant les vingt dernieres annees, le processus d'entree du HBV dans les hepatocytes est toujours inconnu. En effet, les mecanismes permettant l'adsorption des virus sur la membrane plasmique, leur penetration dans la cellule, ainsi que la liberation de leur nucleocapside n'ont pas encore ete elucides. Dans ce contexte, l'objectif de cette revue est de faire un point sur les connaissances acquises sur l'etape d'entree virale du HBV en presentant les donnees recueillies sur l'attachement des virus, leur internalisation, la liberation des nucleocapsides et leur adressage jusqu'au noyau des cellules infectees.

The entry pathway of the hepatitis C virus (HCV), a major human pathogen, into the cell is incompletely defined. To better characterize this viral life cycle stage, we screened a small interfering RNA library dedicated to the membrane trafficking and remodeling with the infection model of Huh-7.5.1 cells by HCV pseudoparticles (HCVpp). Results showed that the down-regulation of different factors implied in clathrin-mediated endocytosis (CME) inhibits HCVpp cell infection. In addition, knockdown of the phosphatidylinositol 4-kinase type III-alpha (PI4KIII alpha) prevented infection by HCVpp or by cell-culture grown JFH-1-based HCV. Moreover, the replication activity of an HCV replicon was also affected by the PI4KIII alpha knockdown. Additional investigations on the different members of the PI4K family revealed that the presence of PI4KIII beta in the host cells influenced their susceptibility to HCVpp infection and their capacity to sustain the HCV replication. The PI4KIII involvement during the HCV life cycle seemed to occur by other ways than the control of the CME or of the membranous expression of HCV receptors. Finally, our library screening completed data on the CME-dependant entry route of HCV and identified 2 kinases, PI4KIII alpha and beta, as relevant potential therapeutic targets.-Trotard, M., Lepere-Douard, C., Regeard, M., Piquet-Pellorce, C., Lavillette, D., Cosset, F.-L., Gripon, P., Le Seyec, J. Kinases required in hepatitis C virus entry and replication highlighted by small interference RNA screening. FASEB J. 23, 3780-3789 (2009). www.fasebj.org

ABSTRACT The early steps of the hepatitis B virus (HBV) life cycle are still poorly understood. Indeed, neither the virus receptor at the cell surface nor the mechanism by which nucleocapsids are delivered to the cytosol of infected cells has been identified. Extensive mutagenesis studies in pre-S1, pre-S2, and most of the S domain of envelope proteins revealed the presence of two regions essential for HBV infectivity: the 77 first residues of the pre-S1 domain and a conformational motif in the antigenic loop of the S domain. In addition, at the N-terminal extremity of the S domain, a putative fusion peptide, partially overlapping the first transmembrane (TM1) domain and preceded by a PEST sequence likely containing several proteolytic cleavage sites, was identified. Since no mutational analysis of these two motifs potentially implicated in the fusion process was performed, we decided to investigate the ability of viruses bearing contiguous deletions or substitutions in the putative fusion peptide and PEST sequence to infect HepaRG cells. By introducing the mutations either in the L and M proteins or in the S protein, we demonstrated the following: (i) that in the TM1 domain of the L protein, three hydrophobic clusters of four residues were necessary for infectivity; (ii) that the same clusters were critical for S protein expression; and, finally, (iii) that the PEST sequence was dispensable for both assembly and infection processes.

Interleukin (IL) 1beta is a proinflammatory cytokine known to markedly alter expression of major organic anion transporters in rodent hepatocytes. However, its effects toward human hepatic transporters remain poorly characterized. Therefore, the present study was aimed at determining IL-1beta effects on expression of organic anion transporters in primary human hepatocytes and highly differentiated human hepatoma HepaRG cells. Exposure to 1 ng/ml IL-1beta was first shown to markedly repress mRNA expression of sodium-taurocholate cotransporting polypeptide (NTCP), a major sinusoidal transporter handling bile acids, in both human hepatocytes and HepaRG cells. It concomitantly reduced NTCP protein levels and NTCP-mediated cellular uptake of taurocholate in HepaRG cells. Other transporters such as the influx transporters organic anion transporting polypeptide (OATP)-B, OATP-C, and OATP8 and the efflux pumps multidrug resistance-associated protein (MRP) 2, MRP3, MRP4, and breast cancer resistance protein were also down-regulated at mRNA levels in human hepatocytes treated by IL-1beta for 24 h, and most of these transporters were similarly repressed in IL-1beta-exposed HepaRG cells; the cytokine also reduced bile salt export pump (BSEP) and OATP-C protein expression in human hepatocytes. IL-1beta was further shown to activate the extracellular signal-regulated protein kinase (ERK) in human hepatocytes and HepaRG cells; however, chemical inhibition of this kinase failed to counteract repressing effects of IL-1beta toward NTCP, BSEP, OATP-B, and OATP-C. Taken together, these data indicate that IL-1beta treatment reduced expression of major organic anion transporters in human hepatic cells in an ERK-independent manner. Such IL-1beta effects may likely participate in both cholestasis and alterations of hepatic detoxification pathways caused by inflammation in humans.

Hepatitis B virus (HBV) is an important human pathogen, which targets the liver extremely efficient, gaining access to hepatocytes by a so far unknown receptor and replicating in a hepatocyte-specific fashion. Cell differentiation seems to determine HBV replication. We here show that the level of hepatocyte differentiation, as indicated by hepatocyte polarization and metabolic activity, is closely correlated to the transcription of the HBV RNA pregenome. Pregenome transcription determined the level of HBV replication in various cell lines of hepatocellular origin and in primary human hepatocytes. A variety of hepatocyte-enriched nuclear factors have been described to regulate transcription of the pregenome, but it remained unknown which factors link HBV replication to hepatocyte differentiation. We determined that high expression levels of HNF4alpha but not its potential cofactors or other hepatocyte-enriched transcription factors were essential for efficient HBV replication, and link it to hepatocyte differentiation. HNF1alpha contributed to the control of HBV replication because it regulated the expression of HNF4alpha. Thus, a concerted action of HNF4alpha and HNF1alpha, which also determines morphological and functional differentiation of hepatocytes, links HBV replication to hepatocyte differentiation.

The recently described hepatic cell line HepaRG is the sole hepatoma cell line susceptible to hepatitis B virus (HBV) infection. It provides a unique tool for investigating some unresolved issues of the virus' biology, particularly the formation of the viral mini-chromosome believed to be responsible for the persistence of infection. In this study, we characterized the main features of HBV infection: it is restricted to a subpopulation of differentiated hepatocyte-like cells that express albumin as a functional marker and represents around 10 % of all differentiated HepaRG cells. Infection may persist for more than 100 days in cells maintained at the differentiated state. Even though infected cells continued to produce infectious viral particles, very limited or no spreading of infection was observed. Low genetic variation was also observed in the viral DNA from viruses found in the supernatant of infected cells, although this cannot explain the lack of reinfection. HBV infection of HepaRG cells appears to be a very slow process: viral replication starts at around day 8 post-infection and reaches a maximum at day 13. Analysis of viral DNA showed slow and inefficient conversion of the input relaxed circular DNA into covalently closed circular (CCC) DNA, but no further amplification. Continuous lamivudine treatment inhibited viral replication, but neither prevented viral infection nor initial formation of CCC DNA. In conclusion, HBV infection in differentiated HepaRG cells is characterized by long-term persistence without a key feature of hepadnaviruses, the so-called 'CCC DNA amplification' described in the duck hepatitis B model.

Entry of the hepatitis C virus (HCV) into the cell seems to be a complex multi-step process involving several cellular factors such as the scavenger class B type I receptor (SRBI). Until now, all investigations conducted to assess the involvement of SRBI have been based on in vitro infection models which use human hepatoma-derived cell lines. However, the HCV entry pathway may be altered in these types of cells because of the impairment of some hepatic characteristics. In this study, we showed that SRBI also plays an essential role in HCV entry into primary human hepatocytes with two distinct approaches: gene extinction and antibodies neutralization assays.

Hepatitis C virus is a human pathogen responsible for liver diseases including acute and chronic hepatitis, cirrhosis and hepatocellular carcinoma. Its high prevalence, the absence of a prophylactic vaccine and the poor efficiency of current therapies are huge medical problems. Since the discovery of the hepatitis C virus, our knowledge of its biology has been largely punctuated by the development of original models of research. At the end of the 1980s, the chimpanzee model led to cloning of the viral genome and the definition of infectious molecular clones. In 1999, a breakthrough was achieved with the development of a robust in vitro replication model named ‘replicon’. This system allowed intensive research into replication mechanisms and drug discovery. Later, in 2003, pseudotyped retroviruses harbouring surface proteins of hepatitis C virus were produced to specifically investigate the viral entry process. It was only in 2005 that infectious viruses were produced in vitro , enabling intensive investigations into the entire life cycle of the hepatitis C virus. This review describes the different in vitro models developed to study hepatitis C virus, their contribution to current knowledge of the virus biology and their future research applications.

ABSTRACT The early events of hepatitis B virus (HBV) infection remain unclear. In 2006, Stoeckl et al. proposed a new entry mechanism involving a translocation motif (TLM) present in the pre-S2 domain of envelope proteins (L. Stoeckl, A. Funk, A. Kopitzki, B. Brandenburg, S. Oess, H. Will, H. Sirma, and E. Hildt, Proc. Natl. Acad. Sci. USA 103:6730-6734, 2006). After receptor binding and internalization into the endosomal compartment, this motif would allow the translocation of HBV particles through the endosomal membrane into the cytosol. In this study we have used two different mutated viruses containing a truncated TLM and showed their ability to infect human hepatocytes in primary culture, thus demonstrating the dispensability of the TLM for HBV infectivity.