Lipid-mediated introduction of hepatitis B virus capsids into nonsusceptible cells allows highly efficient replication and facilitates the study of early infection events

The hepatitis B virus (HBV) is an enveloped DNA virus which is highly infectious in vivo. In vitro, only primary hepatocytes of humans and Tupaia belangeri or the novel HepaRG cell line are susceptible to HBV, but infection is inefficient and study of early infection events in single cells is unsatisfactory. Since hepatoma cells replicate the virus efficiently after transfection, this limited infection efficiency must be related to the initial entry phase. Here, we describe the lipid-based delivery of HBV capsids into nonsusceptible cells, circumventing the natural entry pathway. Successful infection was monitored by observing the emergence of the nuclear viral covalently closed circular DNA and the production of progeny virus and subviral particles. Lipid-mediated transfer initiated productive infection that was at least 100-fold more effective than infection of permissive cell cultures. High-dose capsid transfer showed that the uptake was not receptor limited and allowed the intracellular transport of capsids and genomes to be examined microscopically. The addition of inhibitors confirmed an entry pathway by fusion of the lipid with the plasma membrane. By indirect immune fluorescence and native fluorescence in situ hybridization, we followed the pathway of capsids and viral genomes in individual cells. We observed an active microtubule-dependent capsid transfer to the nucleus and a subsequent release of the viral genomes exclusively into the karyoplasm. Lipid-mediated transfer of viral capsids thus appears to allow efficient introduction of genetic information into target cells, facilitating studies of early infection events which are otherwise impeded by the small number of viruses entering the cell.     

Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner

Persistence of hepatitis B virus (HBV) infection requires covalently closed circular (ccc)DNA formation and amplification, which can occur via intracellular recycling of the viral polymerase-linked relaxed circular (rc) DNA genomes present in virions. Here we reveal a fundamental difference between HBV and the related duck hepatitis B virus (DHBV) in the recycling mechanism. Direct comparison of HBV and DHBV cccDNA amplification in cross-species transfection experiments showed that, in the same human cell background, DHBV but not HBV rcDNA converts efficiently into cccDNA. By characterizing the distinct forms of HBV and DHBV rcDNA accumulating in the cells we find that nuclear import, complete versus partial release from the capsid and complete versus partial removal of the covalently bound polymerase contribute to limiting HBV cccDNA formation; particularly, we identify genome region-selectively opened nuclear capsids as a putative novel HBV uncoating intermediate. However, the presence in the nucleus of around 40% of completely uncoated rcDNA that lacks most if not all of the covalently bound protein strongly suggests a major block further downstream that operates in the HBV but not DHBV recycling pathway. In summary, our results uncover an unexpected contribution of the virus to cccDNA formation that might help to better understand the persistence of HBV infection. Moreover, efficient DHBV cccDNA formation in human hepatoma cells should greatly facilitate experimental identification, and possibly inhibition, of the human cell factors involved in the process.     

Production of infectious duck hepatitis B virus in a human hepatoma cell line.

The differentiated human hepatoma cell line Hep-G2 was transfected with cloned duck hepatitis B virus (DHBV) DNA. Introduction of closed circular DNA into the human liver cells resulted in the production of viral proteins: core antigen was detected in the cytoplasm, and e antigen, a related product, was secreted into the medium. Moreover, viral particles were released into the tissue culture medium which were indistinguishable from authentic DHBV by density, antigenicity, DNA polymerase activity, and morphology. Intravenous injection of tissue culture-derived DHBV particles into Pekin ducks established DHBV infection. In conclusion, transfection of human hepatoma cells with cloned DHBV DNA results in the production of infectious virus, as occurs with cloned human hepatitis B virus DNA. Human liver cells are therefore competent to support production of the avian and mammalian hepadnaviruses, indicating that liver-specific viral gene expression is controlled by evolutionarily conserved mechanisms. This new DHBV transfection system offers the opportunity to rapidly produce mutated DHBV which then can be further investigated in Pekin ducks.     

Protease-induced infectivity of hepatitis B virus for a human hepatoblastoma cell line.

The human hepatoblastoma cell line HepG2 produces and secretes hepatitis B virus (HBV) after transfection of cloned HBV DNA. Intact virions do not infect these cells, although they attach to the surface of the HepG2 cell through binding sites in the pre-S1 domain. Entry of enveloped virions into the cell often requires proteolytic cleavage of a viral surface protein that is involved in fusion between the cell membrane and the viral envelope. Recently, we observed pre-S-independent, nonspecific binding between hepatitis B surface (HBs) particles and HepG2 cells after treatment of HBs antigen particles with V8 protease, which cleaves next to a putative fusion sequence. Chymotrypsin removed this fusion sequence and did not induce binding. In this study, we postulate that lack of a suitable fusion-activating protease was the reason why the HepG2 cells were not susceptible to HBV. To test this hypothesis, virions were partially purified from the plasma of HBV carriers and treated with either staphylococcal V8 or porcine chymotrypsin protease. Protease-digested virus lost reactivity with pre-S2-specific antibody but remained morphologically intact as determined by electron microscopy. After separation from the proteases, virions were incubated with HepG2 cells at pH 5.5. Cultures inoculated with either intact or chymotrypsin-digested virus did not contain detectable levels of intracellular HBV DNA at any time following infection. However, in cultures inoculated with V8-digested virions, HBV-specific products, including covalently closed circular DNA, viral RNA, and viral pre-S2 antigen, could be detected in a time-dependent manner following infection. Immunofluorescence analysis revealed that 10 to 30% of the infected HepG2 cells produced HBV antigen. Persistent secretion of virus by the infected HepG2 cells lasted at least 14 days and was maintained during several reseeding steps. The results show that V8-digested HBV can productively infect tissue cultures of HepG2 cells. It is suggested that proteolysis-dependent exposure of a fusion domain within the envelope protein of HBV is necessary during natural infection.     

Production of hepatitis B virus by a differentiated human hepatoma cell line after transfection with cloned circular HBV DNA

Closed-circular HBV DNA was introduced into cells of the established human hepatoma culture HepG2. The culture medium of one of 40 single-cell clones contained HBV surface antigen (HBsAg), core-related antigens (HBc/eAg), and HBV DNA sequences. HBV DNA and DNA polymerase activity were detected in particles resembling both nucleocapsids and complete virions (Dane particles). Intracellular integrated and extrachromosomal HBV DNA sequences were detected. Relaxed-circular and single-stranded forms of viral DNA were identified as likely replicative intermediates of the HBV genome. In conclusion, in vitro production of Dane-like particles by transformed human hepatocytes has been achieved. This model should be valuable as a cell culture system for studying virus replication and virus-host cell interactions.     

Specific uptake of asialofetuin-tacked liposomes encapsulating interferon-gamma by human hepatoma cells and its inhibitory effect on hepatitis B virus replication.

Asialofetuin-tacked liposomes (AF-liposomes) encapsulating interferon (IFN)-gamma were bound and internalized into a human hepatoma cell line, HepG2 cells, selectively through asialoglycoprotein receptor, but not non-tacked liposomes (N-liposomes). AF-liposomal IFN-gamma was more effective for inhibition of viral DNA replication in hepatitis B virus (HBV)-producing clone from HepG2 cells transfected with HBV-DNA than N-liposomal IFN-gamma. AF-liposomes may increase the therapeutic potential of IFN-gamma through asialoglycoprotein receptor in treating HBV-infected hepatocytes.     

Replication advantage and host factor-independent phenotypes attributable to a common naturally occurring capsid mutation (I97L) in human hepatitis B virus

Mutations of human hepatitis B virus (HBV) occur frequently within the capsid (core) protein in natural infections. The most frequent mutation of the core protein in HBV from Southeast Asia occurs at amino acid 97, changing an isoleucine (I) to a leucine (L). In our systematic study of virus-host interactions, we have examined the replication efficiency of a site-directed mutant, I97L, and its parental wild-type HBV in several different hepatoma cell lines. Interestingly, we found that this capsid variant replicated in human Huh7 hepatoma cells approximately 4.8-fold better than its parental wild-type HBV. A similar phenomenon was observed in another hepatoma cell line, J3. In addition, the level of encapsidated RNA pregenome in mutant I97L was about 5.7-fold higher than that of the wild-type HBV in Huh7 cells. Unlike Huh7 cells, no significant difference in viral DNA replication between the same I97L mutant and its parental wild-type HBV was observed in HepG2, a human hepatoblastoma cell line. This finding of a profound replication advantage for mutant I97L in Huh7 and J3 cells but not in HepG2 cells may have important implications for the emergence of this mutant in chronic HBV carriers. We speculate here that the mutation confers a host factor-independent growth advantage for the survival of HBV variants in gradually dedifferentiating hepatocytes and thus helps prolong viral persistence.     

Evidence for a protein-trafficking gene that rescues the defective glucocorticoid-regulated transport and Golgi retention of mouse mammary tumor virus glycoproteins in a rat hepatoma cell-sorting variant.

Glucocorticoids regulate the trafficking of cell surface mouse mammary tumor virus (MMTV) glycoproteins in the virus-infected rat hepatoma cell line M1.54. The CR4 rat hepatoma sorting variant, which is derived from M1.54 cells by immunoselection, is uniquely defective in the glucocorticoid-regulated transport of MMTV glycoproteins. Indirect immunofluorescence of fixed permeabilized cells and subcellular fractionation of isolated microsomes revealed that variant CR4 cells retain the MMTV glycoproteins in Golgi-like membranes after glucocorticoid treatment. The variant CR4 phenotype can be complemented by interspecies cell fusions with human HepG2 hepatoma cells and by DNA rescue with genomic fragments isolated from either human or rat hepatoma cells. Transfected wild-type genomic fragments rescue the sorting defect in CR4 at a frequency consistant with a single genetic locus, whereas homologous transfection with CR4 genomic DNA has no effect. Thus, complementation of a rat hepatoma cell-sorting variant supports the existence of a novel protein-trafficking activity encoded by the human or rat genomes that acts in trans in the Golgi to selectively mediate the sorting of cell surface MMTV glycoproteins in glucocorticoid-treated cells.     

Hepatitis B virus Core protein nuclear interactome identifies SRSF10 as a host RNA-binding protein restricting HBV RNA production

Despite the existence of a preventive vaccine, chronic infection with Hepatitis B virus (HBV) affects more than 250 million people and represents a major global cause of hepatocellular carcinoma (HCC) worldwide. Current clinical treatments, in most of cases, do not eliminate viral genome that persists as a DNA episome in the nucleus of hepatocytes and constitutes a stable template for the continuous expression of viral genes. Several studies suggest that, among viral factors, the HBV core protein (HBc), well-known for its structural role in the cytoplasm, could have critical regulatory functions in the nucleus of infected hepatocytes. To elucidate these functions, we performed a proteomic analysis of HBc-interacting host-factors in the nucleus of differentiated HepaRG, a surrogate model of human hepatocytes. The HBc interactome was found to consist primarily of RNA-binding proteins (RBPs), which are involved in various aspects of mRNA metabolism. Among them, we focused our studies on SRSF10, a RBP that was previously shown to regulate alternative splicing (AS) in a phosphorylation-dependent manner and to control stress and DNA damage responses, as well as viral replication. Functional studies combining SRSF10 knockdown and a pharmacological inhibitor of SRSF10 phosphorylation (1C8) showed that SRSF10 behaves as a restriction factor that regulates HBV RNAs levels and that its dephosphorylated form is likely responsible for the anti-viral effect. Surprisingly, neither SRSF10 knock-down nor 1C8 treatment modified the splicing of HBV RNAs but rather modulated the level of nascent HBV RNA. Altogether, our work suggests that in the nucleus of infected cells HBc interacts with multiple RBPs that regulate viral RNA metabolism. Our identification of SRSF10 as a new anti-HBV restriction factor offers new perspectives for the development of new host-targeted antiviral strategies.     

A molecularly cloned hepatitis B virus produced in vitro is infectious in a chimpanzee.

The infectivity of hepatitis B virus (HBV) produced in vitro by HepG2 cells transfected with HBV DNA (HepG2T14) has been assayed in a chimpanzee. Following inoculation, the chimpanzee underwent a typical course of type B hepatitis infection, characterized by elevation of serum aminotransferases and by histological identification of hepatic damage. Hepatitis B surface antigen and core-related antigen appeared in the serum at weeks 5 and 7, respectively, after infection. HBV DNA was detected in serum samples, and replicative forms of the HBV genome were identified in liver biopsies. Subtype identification of hepatitis B surface antigen and restriction enzyme analysis of HBV DNA in both the inoculum and the serum of the infected chimpanzee confirmed that the hepatitis B infection observed in this animal was caused by viral particles produced by HepG2T14 cells. These findings indicate that, although HepG2 cells do not seem to be susceptible to infection by HBV in vitro, they can produce biologically active infectious virions after transfection with cloned HBV DNA.