The middle hepatitis B virus envelope protein is not necessary for infectivity of hepatitis delta virus.

The hepatitis delta virus (HDV) envelope contains the large (L), middle (M), and small (S) surface proteins encoded by coinfecting hepatitis B virus. Although HDV-like particles can be assembled with only the S protein in the envelope, the L protein is essential for infectivity in vitro (C. Sureau, B. Guerra, and R. Lanford, J. Virol. 67:366-372, 1993). Here, we demonstrate that the M protein, previously described as carrying a site for binding to polymerized human albumin, is not necessary for infectivity. HDV-like particles coated with the S plus L or the S plus M plus L proteins are infectious in primary cultures of chimpanzee hepatocytes. We conclude that the S and L proteins serve two essential functions in the HDV replication cycle; the S protein ensures the export of the HDV genome from an infected cell by forming a particle, and the L protein ensures its import into a human hepatocyte.     

Primary human hepatocytes are susceptible to infection by hepatitis delta virus assembled with envelope proteins of woodchuck hepatitis virus

Hepatitis B virus (HBV) and hepatitis delta virus (HDV) share the HBV envelope proteins. When woodchucks chronically infected with woodchuck hepatitis virus (WHV) are superinfected with HDV, they produce HDV with a WHV envelope, wHDV. Several lines of evidence are provided that wHDV infects not only cultured primary woodchuck hepatocytes (PWH) but also primary human hepatocytes (PHH). Surprisingly, HBV-enveloped HDV (hHDV) and wHDV infected PHH with comparable efficiencies; however, hHDV did not infect PWH. The basis for these host range specificities was investigated using as inhibitors peptides bearing species-specific pre-S (where S is the small envelope protein) sequences. It was found that pre-S1 contributed to the ability of wHDV to infect both PHH and PWH. In addition, the inability of hHDV to infect PWH was not overcome using a chimeric form of hHDV containing WHV S protein, again supporting the essential role of pre-S1 in infection of target cells. One interpretation of these data is that host range specificity of HDV is determined entirely by pre-S1 and that the WHV and HBV pre-S1 proteins recognize different receptors on PHH.     

Cellular receptor traffic is essential for productive duck hepatitis B virus infection

We have investigated the mechanism of duck hepatitis B virus (DHBV) entry into susceptible primary duck hepatocytes (PDHs), using mutants of carboxypeptidase D (gp180), a transmembrane protein shown to act as the primary cellular receptor for avian hepatitis B virus uptake. The variant proteins were abundantly produced from recombinant adenoviruses and tested for the potential to functionally outcompete the endogenous wild-type receptor. Overexpression of wild-type gp180 significantly enhanced the efficiency of DHBV infection in PDHs but did not affect ongoing DHBV replication, an observation further supporting gp180 receptor function. A gp180 mutant deficient for endocytosis abolished DHBV infection, indicating endocytosis to be the route of hepadnaviral entry. With further gp180 variants, carrying mutations in the cytoplasmic domain and characterized by an accelerated turnover, the ability of gp180 to function as a DHBV receptor was found to depend on a wild-type-like sorting phenotype which largely avoids transport toward the endolysosomal compartment. Based on these data, we propose a model in which a distinct intracellular DHBV traffic to the endosome, but not beyond, is a prerequisite for completion of viral entry, i.e., for fusion and capsid release. Furthermore, the deletion of the two enzymatically active carboxypeptidase domains of gp180 did not lead to a loss of receptor function.     

Scavenger receptor class B type I and hepatitis C virus infection of primary tupaia hepatocytes

Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. The study of early steps during HCV infection has been hampered by the lack of suitable in vitro or in vivo models. Primary Tupaia hepatocytes (PTH) have been shown to be susceptible to HCV infection in vitro and in vivo. Human scavenger receptor class B type I (SR-BI) represents an HCV receptor candidate mediating the cellular binding of E2 glycoprotein to HepG2 hepatoma cells. However, the function of SR-BI for viral infection of hepatocytes is unknown. In this study, we used PTH to assess the functional role of SR-BI as a putative HCV receptor. Sequence analysis of cloned tupaia SR-BI revealed a high homology between tupaia and human SR-BI. Transfection of CHO cells with human or tupaia SR-BI but not mouse SR-BI cDNA resulted in cellular E2 binding, suggesting that E2-binding domains between human and tupaia SR-BI are highly conserved. Preincubation of PTH with anti-SR-BI antibodies resulted in marked inhibition of E2 or HCV-like particle binding. However, anti-SR-BI antibodies were not able to block HCV infection of PTH. In conclusion, our results demonstrate that SR-BI represents an important cell surface molecule for the binding of the HCV envelope to hepatocytes and suggest that other or additional cell surface molecules are required for the initiation of HCV infection. Furthermore, the structural and functional similarities between human and tupaia SR-BI indicate that PTH represent a useful model system to characterize the molecular interaction of the HCV envelope and SR-BI on primary hepatocytes.     

Duck hepatitis B virus infection of hepatocytes is not dependent on low pH.

The pH dependency for initiation of infection by the hepadnavirus duck hepatitis B virus (DHBV) was investigated in primary duck hepatocytes. First, an infection assay was developed using a radioimmunoblot to measure DHBV e antigen secreted into tissue culture fluid from infected hepatocytes. The quantity of this viral marker was proportional to the duration of inoculation and the amount of DHBV used as inoculum. The role of pH in initiation of DHBV infection was investigated by using this assay, but no dependence on low pH was found. DHBV was able to infect hepatocytes in the presence of NH4Cl and monensin, agents that raise the pH in intracellular vesicles and prevent penetration of viruses dependent on low pH in endosomes. In control experiments, infection by Semliki Forest virus, which is low pH dependent, was inhibited, whereas herpes simplex virus type 1 infection, which is pH independent, occurred. Attempts to trigger DHBV-cell fusion by exposure of DHBV prebound to hepatocytes to mildly acidic pH were unsuccessful. In these experiments, it was also observed that internalization of DHBV occurred only between pH 6.8 and 8.0. Additionally, in the absence of cells, infectivity of DHBV was stable at pH 4.6 to 4.8, which is lower than the pH encountered in endosomes (pH 5 to 6.6). Thus, no evidence for a role for mildly acidic pH in the initiation of DHBV infection was found. Therefore, we propose that the infection route followed by DHBV resembles that of the group of enveloped viruses, including herpesviruses, that fuse with their host cells at neutral pH.     

Efficient infection of primary tupaia hepatocytes with purified human and woolly monkey hepatitis B virus

The Asian tree shrew, Tupaia belangeri, has been proposed as a novel animal model for studying hepatitis B virus (HBV) infection. Here, we describe a protocol for efficient and reproducible infection of primary tupaia hepatocytes with HBV. We report that human serum interferes with HBV binding to the hepatocytes, thus limiting the maximum multiplicity of infection. Purification of HBV virions by gradient sedimentation greatly enhances virus binding and infectivity. Covalently closed circular DNA was clearly detectable by Southern blot analysis and newly synthesized single-stranded HBV DNA was visible 2 weeks postinoculation. Primary tupaia hepatocytes are also susceptible to infection with the recently discovered woolly monkey hepatitis B virus (WMHBV) but not to woodchuck hepatitis virus infection. Compared to HBV, WMHBV replicated at a higher rate with single-stranded DNA detectable within the first week postinoculation. Primary tupaia hepatocytes should represent a useful system for studying early steps of HBV and WMHBV infection.     

Tissue culture system for infection with human hepatitis delta virus.

An in vitro culture system was developed for assaying the infectivity of the human hepatitis delta virus (HDV). Hepatocytes were isolated from chimpanzee liver and grown in a serum-free medium. Cells were shown to be infectible by HDV and to remain susceptible to infection for at least 3 weeks in culture, as evidenced by the appearance of RNA species characteristic of HDV replication as early as 6 days postinfection. When repeated experiments were carried out on cells derived from an animal free of hepatitis B virus (HBV), HDV infection occurred in a consistent fashion but there was no indication of infection with the HBV that was present in the inoculum. Despite numerous attempts with different sources of HBV inocula free of HDV, there was no evidence that indicated susceptibility of these cells to HBV infection. This observation may indicate that HBV and HDV use different modes of entry into hepatocytes. When cells derived from an HBV-infected animal were exposed to HDV, synthesis and release of progeny HDV particles were obtained in addition to HBV replication and production of Dane particles. Although not infectible with HBV, primary cultures of chimpanzee hepatocytes are capable of supporting part of the life cycle of HBV and the entire life cycle of HDV.     

Serum-derived hepatitis C virus infection of primary human hepatocytes is tetraspanin CD81 dependent

Hepatitis C virus-positive serum (HCVser, genotypes 1a to 3a) or HCV cell culture (JFH1/HCVcc) infection of primary normal human hepatocytes was assessed by measuring intracellular HCV RNA strands. Anti-CD81 antibodies and siRNA-CD81 silencing markedly inhibited (>90%) HCVser infection irrespective of HCV genotype, viral load, or liver donor, while hCD81-large intracellular loop (LEL) had no effect. However, JFH1/HCVcc infection of hepatocytes was modestly inhibited (40 to 60%) by both hCD81-LEL and anti-CD81 antibodies. In conclusion, CD81 is involved in HCVser infection of human hepatocytes, and comparative studies of HCVser versus JFH1/HCVcc infection of human hepatocytes and Huh-7.5 cells revealed that the cell-virion combination is determinant of the entry process.     

Replication of human hepatitis delta virus in primary cultures of woodchuck hepatocytes.

We obtained two lines of evidence that monolayer cultures of primary woodchuck hepatocytes support replication of the genome of human hepatitis delta virus (HDV). (i) From a Northern (RNA blot) analysis of the HDV-related RNA in infected cultures, both genomic and antigenomic 1.7-kilobase RNA species were detected at 11 days after infection. The ratio of genomic RNA to antigenomic RNA was 2:1 to 10:1, comparable to that previously reported in studies of experimentally infected chimpanzees and woodchucks. (ii) Replication in culture was also demonstrated by in situ hybridization with a strand-specific probe. Such studies showed that only a small fraction of the cultured cells supported replication and that in such cells the relative and absolute levels of the HDV RNAs were comparable to those in liver cells infected in vivo. Furthermore, as with the in vivo studies, the HDV RNAs were predominantly localized to the nucleus. In summary, we demonstrated that cultured cells supported both the early events of HDV adsorption and penetration and the intermediate events of genome replication.     

Scavenger receptor class B is required for hepatitis C virus uptake and cross-presentation by human dendritic cells

Class B scavenger receptors (SR-Bs) bind lipoproteins and play an important role in lipid metabolism. Most recently, SR-B type I (SR-BI) and its splicing variant SR-BII have been found to mediate bacterial adhesion and cytosolic bacterial invasion in mammalian cells. In this study, we demonstrate that SR-BI is a key host factor required for hepatitis C virus (HCV) uptake and cross-presentation by human dendritic cells (DCs). Whereas monocytes and T and B cells were characterized by very low or undetectable SR-BI expression levels, human DCs demonstrated a high level of cell surface expression of SR-BI similar to that of primary human hepatocytes. Antibodies targeting the extracellular loop of SR-BI efficiently inhibited HCV-like particle binding, uptake, and cross-presentation by human DCs. Moreover, human high-density lipoprotein specifically modulated HCV-like particle binding to DCs, indicating an interplay of HCV with the lipid transfer function of SR-BI in DCs. Finally, we demonstrate that anti-SR-BI antibodies inhibit the uptake of cell culture-derived HCV (HCVcc) in DCs. In conclusion, these findings identify a novel function of SR-BI for viral antigen uptake and recognition and may have an important impact on the design of HCV vaccines and immunotherapeutic approaches aiming at the induction of efficient antiviral immune responses.     

Envelope protein-mediated down-regulation of hepatitis B virus receptor in infected hepatocytes

Entry of duck hepatitis B virus (DHBV) is initiated by specific interaction of its large envelope protein (L) with a cellular entry receptor, recently identified as carboxypeptidase D (CPD; historically gp180). In this report, we present evidence demonstrating that this receptor is down-regulated as a result of DHBV infection: (i) receptor levels determined by Western blot were much reduced in DHBV-infected duck livers and undetectable by immunostaining in infected cultured hepatocytes; (ii) results from metabolic labeling experiments indicate enhanced receptor protein turnover; (iii) the kinetics of receptor loss from newly infected cells correlated with the accumulation of newly synthesized viral protein; (iv) expression of DHBV L protein, transduced from a recombinant adenovirus, was sufficient to eliminate gp180/CPD from the Golgi compartment, its normal predominant location; (v) gp180/CPD remained absent from the Golgi compartment in infected hepatocytes, even after overexpression from a recombinant adenovirus, while residual amounts subsequently became detectable in a perinuclear compartment, containing DHBV L protein; (vi) expression of DHBV L protein in a HepG2 cell line, stably expressing gp180/CPD, leads to incomplete receptor maturation and induces its degradation. Taken together, these data are consistent with a model in which the virus receptor interacts early in the biosynthetic pathway with the viral L protein, leading to its retention in a pre-Golgi compartment and to subsequent degradation, thus preventing receptor interference with the export of DHBV via the secretory pathway which it shares with its receptor. Accordingly, and analogously with receptor down-regulation in retroviral systems, DHBV receptor down-modulation may account for the much-reduced efficiency of DHBV superinfection of preinfected hepatocytes.     

Entry of hepatitis B virus into immortalized human primary hepatocytes by clathrin-dependent endocytosis

The lack of a suitable in vitro hepatitis B virus (HBV) infectivity model has limited examination of the early stages of the virus-cell interaction. In this study, we used an immortalized cell line derived from human primary hepatocytes, HuS-E/2, to study the mechanism of HBV infection. HBV infection efficiency was markedly increased after dimethyl sulfoxide (DMSO)-induced differentiation of the cells. Transmission electron microscopy demonstrated the presence of intact HBV particles in DMSO-treated HBV-infected HuS-E/2 cells, which could be infected with HBV for up to at least 50 passages. The pre-S1 domain of the large HBsAg (LHBsAg) protein specifically interacted with clathrin heavy chain (CHC) and clathrin adaptor protein AP-2. Short hairpin RNA knockdown of CHC or AP-2 in HuS-E/2 cells significantly reduced their susceptibility to HBV, indicating that both are necessary for HBV infection. Furthermore, HBV entry was inhibited by chlorpromazine, an inhibitor of clathrin-mediated endocytosis. LHBsAg also interfered with the clathrin-mediated endocytosis of transferrin by human hepatocytes. This infection system using an immortalized human primary hepatocyte cell line will facilitate investigations into HBV entry and in devising therapeutic strategies for manipulating HBV-associated liver disorders.     

CD81 is a central regulator of cellular events required for hepatitis C virus infection of human hepatocytes

Infection with hepatitis C virus (HCV) is still a major public health problem, and the events leading to hepatocyte infection are not yet fully understood. Combining confocal microscopy with biochemical analysis and studies of infection requirements using pharmacological inhibitors and small interfering RNAs, we show here that engagement of CD81 activates the Rho GTPase family members Rac, Rho, and Cdc42 and that the block of these signaling pathways drastically reduces HCV infectivity. Activation of Rho GTPases mediates actin-dependent relocalization of the HCV E2/CD81 complex to cell-cell contact areas where CD81 comes into contact with the tight-junction proteins occludin, ZO-1, and claudin-1, which was recently described as an HCV coreceptor. Finally, we show that CD81 engagement activates the Raf/MEK/ERK signaling cascade and that this pathway affects postentry events of the virus life cycle. In conclusion, we describe a range of cellular events that are manipulated by HCV to coordinate interactions with its multiple coreceptors and to establish productive infections and find that CD81 is a central regulator of these events.     

The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus

We discovered that the hepatitis C virus (HCV) envelope glycoprotein E2 binds to human hepatoma cell lines independently of the previously proposed HCV receptor CD81. Comparative binding studies using recombinant E2 from the most prevalent 1a and 1b genotypes revealed that E2 recognition by hepatoma cells is independent from the viral isolate, while E2-CD81 interaction is isolate specific. Binding of soluble E2 to human hepatoma cells was impaired by deletion of the hypervariable region 1 (HVR1), but the wild-type phenotype was recovered by introducing a compensatory mutation reported previously to rescue infectivity of an HVR1-deleted HCV infectious clone. We have identified the receptor responsible for E2 binding to human hepatic cells as the human scavenger receptor class B type I (SR-BI). E2-SR-BI interaction is very selective since neither mouse SR-BI nor the closely related human scavenger receptor CD36, were able to bind E2. Finally, E2 recognition by SR-BI was competed out in an isolate-specific manner both on the hepatoma cell line and on the human SR-BI-transfected cell line by an anti-HVR1 monoclonal antibody.     

Apparent helper-independent infection of woodchucks by hepatitis delta virus and subsequent rescue with woodchuck hepatitis virus.

Hepatitis delta virus (HDV) is a subviral agent of humans which is dependent upon hepatitis B virus as a helper for transmission. HDV can be experimentally transmitted to woodchucks by using woodchuck hepatitis virus (WHV) as the helper. We used this model system to study two types of HDV infections: those of animals already chronically infected with WHV and those of animals without any evidence of prior exposure to WHV. At 5 to 10 days after infection with HDV, liver biopsies of these two groups of animals indicated that around 1% of the hepatocytes were infected (HDV antigen positive). Moreover, similar amounts of replicative forms of HDV RNA were detected. In contrast, by 20 days postinfection, the two groups of animals were quite different in the extent of the HDV infection. The animals chronically infected with WHV showed spread of the infection within the liver and the release of high titers of HDV into the serum. In contrast, the animals not previously exposed to WHV showed a progressive reduction in liver involvement, and at no time up to 165 days postinfection could we detect HDV particles in the serum. However, if these animals were inoculated with a relatively high titer of WHV at either 7 or even 33 days after the HDV infection, HDV viremia was observed. Our data support the interpretation that in these animals, hepatocytes were initially infected in the absence of helper virus, HDV genome replication took place, and ultimately these replicating genomes were rescued by the secondary WHV infection. The observation that HDV can survive in the liver for at least 33 days in the absence of coinfecting helper virus may be relevant to the reemergence of HDV infection following liver transplantation.     

Immunology of hepatitis B virus and hepatitis C virus infection

More than 500 million people worldwide are persistently infected with the hepatitis B virus (HBV) and/or hepatitis C virus (HCV) and are at risk of developing chronic liver disease, cirrhosis and hepatocellular carcinoma. Despite many common features in the pathogenesis of HBV- and HCV-related liver disease, these viruses markedly differ in their virological properties and in their immune escape and survival strategies. This review assesses recent advances in our understanding of viral hepatitis, contrasts mechanisms of virus-host interaction in acute hepatitis B and hepatitis C, and outlines areas for future studies.     

Innate host response in primary human hepatocytes with hepatitis C virus infection

Background and Aim

The interaction between hepatitis C virus (HCV) and innate antiviral defense systems in primary human hepatocytes is not well understood. The objective of this study is to examine how primary human hepatocytes response to HCV infection.

Methods

An infectious HCV isolate JFH1 was used to infect isolated primary human hepatocytes. HCV RNA or NS5A protein in the cells was detected by real-time PCR or immunofluorescence staining respectively. Apoptosis was examined with flow cytometry. Mechanisms of HCV-induced IFN-β expression and apoptosis were determined.

Results

Primary human hepatocytes were susceptible to JFH1 virus and released infectious virus. IFN-α inhibited viral RNA replication in the cells. IFN-β and interferon-stimulated genes were induced in the cells during acute infection. HCV infection induced apoptosis of primary human hepatocytes through the TRAIL-mediated pathway. Silencing RIG-I expression in primary human hepatocytes inhibited IFN-β and TRAIL expression and blocked apoptosis of the cells, which facilitated viral RNA replication in the cells. Moreover, HCV NS34A protein inhibited viral induced IFN-β expression in primary human hepatocytes.

Conclusion

Innate host response is intact in HCV-infected primary human hepatocytes. RIG-I plays a key role in the induction of IFN and TRAIL by viruses and apoptosis of primary human hepatocytes via activation of the TRAIL-mediated pathway. HCV NS34A protein appears to be capable of disrupting the innate antiviral host responses in primary human hepatocytes. Our study provides a novel mechanism by which primary human hepatocytes respond to natural HCV infection.     

Duck hepatitis B virus infection of Muscovy duck hepatocytes and nature of virus resistance in vivo.

To test the hypothesis that in vivo resistance to hepadnavirus infection was due to resistance of host hepatocytes, we isolated hepatocytes from Muscovy ducklings and chickens, birds that have been shown to be resistant to duck hepatitis B virus (DHBV) infection, and attempted to infect them in vitro with virus from congenitally infected Pekin ducks. Chicken hepatocytes were resistant to infection, but we were able to infect approximately 1% of Muscovy duck hepatocytes in culture. Infection requires prolonged incubation with virus at 37 degrees C. Virus spread occurs in the Muscovy cultures, resulting in 5 to 10% DHBV-infected hepatocytes by 3 weeks after infection. The relatively low rate of accumulation of DHBV DNA in infected Muscovy hepatocyte cultures is most likely due to inefficient spread of virus infection; in the absence of virus spread, the rates of DHBV replication in Pekin and Muscovy hepatocyte cultures are similar. 5-Azacytidine treatment can induce susceptibility to DHBV infection in resistant primary Pekin hepatocytes but appears to have no similar effect in Muscovy cultures. The relatively inefficient infection of Muscovy duck hepatocytes that we have described may account for the absence of a detectable viremia in Muscovy ducklings experimentally infected with DHBV.     

Production of hepatitis delta virus and suppression of helper hepatitis B virus in a human hepatoma cell line.

The hepatitis delta virus (HDV) is a defective virus with a coat composing of the surface antigen of its helper virus, hepatitis B virus (HBV). Replication of HDV in the absence of HBV has been shown in cell cultures by transient transfection of the HDV plasmid. However, the formation and release of HDV virions have not been observed. In this report, a human hepatoma cell line HuH-7 was transiently cotransfected with HDV and HBV plasmids. The production of monomeric and multimeric antigenomic RNAs of HDV in the transfected cells indicated replication of the HDV genome. The major 3.5- and 2.1-kb RNAs of HBV were also expressed. Virions of both HDV and HBV were released from the cotransfected cells, as shown by the detection of monomeric genomic HDV RNA and partially double-stranded HBV DNA in the culture medium. Thus, this is the first report that describes the assembly and the release of HDV viral particles in an in vitro cell culture. The HDV virions released possessed physicochemical properties identical to those of the HDV virions found in infected human serum. Furthermore, expression of both the 3.5- and 2.1-kb RNAs of HBV was shown to be dramatically decreased by the presence of HDV, indicating suppression of the expression of HBV genes by HDV. The amount of HBV virions released was similarly suppressed by HDV. Cotransfection of HBV with an expression plasmid of the HDV delta antigen remarkably reduced the levels of the 3.5- and 2.1-kb HBV RNAs, indicating that suppression of the expression of HBV RNAs by HDV occurs via the action of the delta antigen. This HBV- and HDV-cotransfected human hepatoma cell line should provide an excellent system for the study of the function of the delta antigen and the interaction between HDV and its helper, HBV.