Inhibition of the HCV Core Protein on the Immune Response to HBV Surface Antigen and on HBV Gene Expression and Replication In Vivo

The hepatitis C virus (HCV) core protein is a multifunctional protein that can interfere with the induction of an immune response. It has been reported that the HCV core protein inhibits HBV replication in vitro. In this study, we test the effect of the HCV core gene on the priming of the immune response to hepatitis B surface antigen (HBsAg) and on the replication of HBV in vivo. Our results showed that the full-length HCV core gene inhibits the induction of an immune response to the heterogeneous antigen, HBsAg, at the site of inoculation when HCV core (pC191) and HBsAg (pHBsAg) expression plasmids are co-administered as DNA vaccines into BALB/c mice. The observed interference effect of the HCV core occurs in the priming stage and is limited to the DNA form of the HBsAg antigen, but not to the protein form. The HCV core reduces the protective effect of the HBsAg when the HBsAg and the HCV core are co-administered as vaccines in an HBV hydrodynamic mouse model because the HCV core induces immune tolerance to the heterogeneous HBsAg DNA antigen. These results suggest that HCV core may play an important role in viral persistence by the attenuation of host immune responses to different antigens. We further tested whether the HCV core interfered with the priming of the immune response in hepatocytes via the hydrodynamic co-injection of an HBV replication-competent plasmid and an HCV core plasmid. The HCV core inhibited HBV replication and antigen expression in both BALB/c (H-2d) and C57BL/6 (H-2b) mice, the mouse models of acute and chronic hepatitis B virus infections. Thus, the HCV core inhibits the induction of a specific immune response to an HBsAg DNA vaccine. However, HCV C also interferes with HBV gene expression and replication in vivo, as observed in patients with coinfection.     

Phosphonylmethoxyalkyl derivatives of purine as inhibitors of human hepatitis B virus DNA synthesis

A selected number of antiviral compounds which have been previously shown to inhibit the replication of DNA viruses or retroviruses were examined for their inhibitory effects on human hepatitis B virus (HBV) DNA synthesis. The assay system was based on the use of a human hepatoblastoma cell line (HB611) that continuously synthesizes HBV DNA. The following phosphonylmethoxyalkyl-purine derivatives were found to inhibit HBV DNA synthesis: 9-(2-phosphonyl-methoxyethyl)-2',6'-diaminopurine (PMEDAP), (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA) and 9-(2-phosphonylmethoxyethyl)adenine (PMEA). PMEDAP, HPMPA and PMEA not only inhibit HBV DNA synthesis in HB611 cells but also duck hepatitis B virus (DHBV) DNA and core antigen synthesis in primary duck hepatocytes.     

HBc and HBe antigenicity and DNA-binding activity of major core protein P22 in hepatitis B virus core particles isolated from the cytoplasm of human liver cells.

Highly purified hepatitis B virus core particles were obtained in large amounts from the cytoplasm of infected human liver cells. This DNA polymerase-negative core preparation had only hepatitis B core antigen-specific antigenicity and showed a surprising stability. Two forms of a single protein of 22,000 molecular weight, P22, were resolved electrophoretically; the slower moving species, P22a, appeared to be a reduced form of the protein, and the faster moving species, P22b, could have represented a conformational isomer containing an intramolecular disulfide bond(s). The immunological properties and DNA-binding activity of the reduced form, P22a, were examined following separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by transfer onto nitrocellulose membranes (Western blotting). We found that the hepatitis B virus C gene protein shared the antigenic site responsible for both hepatitis B core and e antigen reactivity. We also demonstrated that the core protein(s) bound specifically the genomic hepatitis B virus DNA in comparison with a plasmid DNA (pBR322). This last observation was further substantiated by a radioimmunological method. P22a was also found to be phosphorylated in vitro by the endogenous protein kinase activity, copurified with the hepatitis B core antigen particles. These findings suggest that P22 is a multifunctional protein which is incorporated into core particles within the cytoplasm of the host cell before DNA encapsidation. A critical role of this protein in hepatitis B virus assembly is suggested.     

Hepatitis B Virus-Specific miRNAs and Argonaute2 Play a Role in the Viral Life Cycle

Disease-specific serum miRNA profiles may serve as biomarkers and might reveal potential new avenues for therapy. An HBV-specific serum miRNA profile associated with HBV surface antigen (HBsAg) particles has recently been reported, and AGO2 and miRNAs have been shown to be stably associated with HBsAg in serum. We identified HBV-associated serum miRNAs using the Toray 3D array system in 10 healthy controls and 10 patients with chronic hepatitis B virus (HBV) infection. 19 selected miRNAs were then measured by quantitative RT-PCR in 248 chronic HBV patients and 22 healthy controls. MiRNA expression in serum versus liver tissue was also compared using biopsy samples. To examine the role of AGO2 during the HBV life cycle, we analyzed intracellular co-localization of AGO2 and HBV core (HBcAg) and surface (HBsAg) antigens using immunocytochemistry and proximity ligation assays in stably transfected HepG2 cells. The effect of AGO2 ablation on viral replication was assessed using siRNA. Several miRNAs, including miR-122, miR-22, and miR-99a, were up-regulated at least 1.5 fold (P<2E-08) in serum of HBV-infected patients. AGO2 and HBcAg were found to physically interact and co-localize in the ER and other subcellular compartments. HBs was also found to co-localize with AGO2 and was detected in multiple subcellular compartments. Conversely, HBx localized non-specifically in the nucleus and cytoplasm, and no interaction between AGO2 and HBx was detected. SiRNA ablation of AGO2 suppressed production of HBV DNA and HBs antigen in the supernatant.

Conclusion

These results suggest that AGO2 and HBV-specific miRNAs might play a role in the HBV life cycle.     

C-Terminal Substitution of HBV Core Proteins with Those from DHBV Reveals That Arginine-Rich (167)RRRSQSPRR(175) Domain Is Critical for HBV Replication

To investigate the contributions of carboxyl-terminal nucleic acid binding domain of HBV core (C) protein for hepatitis B virus (HBV) replication, chimeric HBV C proteins were generated by substituting varying lengths of the carboxyl-terminus of duck hepatitis B virus (DHBV) C protein for the corresponding regions of HBV C protein. All chimeric C proteins formed core particles. A chimeric C protein with 221-262 amino acids of DHBV C protein, in place of 146-185 amino acids of the HBV C protein, supported HBV pregenomic RNA (pgRNA) encapsidation and DNA synthesis: 40% amino acid sequence identity or 45% homology in the nucleic-acid binding domain of HBV C protein was sufficient for pgRNA encapsidation and DNA synthesis, although we predominantly detected spliced DNA. A chimeric C protein with 221-241 and 251-262 amino acids of DHBV C, in place of HBV C 146-166 and 176-185 amino acids, respectively, could rescue full-length DNA synthesis. However, a reciprocal C chimera with 242-250 of DHBV C ((242)RAGSPLPRS(250)) introduced in place of 167-175 of HBV C ((167)RRRSQSPRR(175)) significantly decreased pgRNA encapsidation and DNA synthesis, and full-length DNA was not detected, demonstrating that the arginine-rich (167)RRRSQSPRR(175) domain may be critical for efficient viral replication. Five amino acids differing between viral species (underlined above) were tested for replication rescue; R169 and R175 were found to be important.     

High-level hepatitis B virus replication in transgenic mice.

Hepatitis B virus (HBV) transgenic mice whose hepatocytes replicate the virus at levels comparable to that in the infected livers of patients with chronic hepatitis have been produced, without any evidence of cytopathology. High-level viral gene expression was obtained in the liver and kidney tissues in three independent lineages. These animals were produced with a terminally redundant viral DNA construct (HBV 1.3) that starts just upstream of HBV enhancer I, extends completely around the circular viral genome, and ends just downstream of the unique polyadenylation site in HBV. In these animals, the viral mRNA is more abundant in centrilobular hepatocytes than elsewhere in the hepatic lobule. High-level viral DNA replication occurs inside viral nucleocapsid particles that preferentially form in the cytoplasm of these centrilobular hepatocytes, suggesting that an expression threshold must be reached for nucleocapsid assembly and viral replication to occur. Despite the restricted distribution of the viral replication machinery in centrilobular cytoplasmic nucleocapsids, nucleocapsid particles are detectable in the vast majority of hepatocyte nuclei throughout the hepatic lobule. The intranuclear nucleocapsid particles are empty, however, suggesting that viral nucleocapsid particle assembly occurs independently in the nucleus and the cytoplasm of the hepatocyte and implying that cytoplasmic nucleocapsid particles do not transport the viral genome across the nuclear membrane into the nucleus during the viral life cycle. This model creates the opportunity to examine the influence of viral and host factors on HBV pathogenesis and replication and to assess the antiviral potential of pharmacological agents and physiological processes, including the immune response.     

DNA-binding activity of hepatitis B e antigen polypeptide lacking the protaminelike sequence of nucleocapsid protein of human hepatitis B virus.

The characteristics of binding of hepatitis B core antigen (HBcAg) and hepatitis B e antigen (HBeAg) polypeptides to hepatitis B virus (HBV) DNA were analyzed. HBcAg polypeptide from recombinant HBV core particles and HBeAg polypeptide from partially purified serum HBeAg were prepared and verified to have molecular weights of 21,500 (P21.5) and of 17,000 (P17) and 18,000 (P18), respectively, by immunoblot analysis. By reaction of these proteins on a nitrocellulose membrane with cloned 32P-HBV DNA, it was revealed that the HBeAg polypeptide, which lacks the C-terminal 34 amino acids of P21.5, as well as the HBcAg polypeptide, bound to the DNA. The secondary structures of nucleocapsid proteins of HBV, woodchuck hepatitis virus, and ground squirrel hepatitis virus were predicted by the Garnier algorithm. Amino acid sequences which, in addition to those of the C-terminal regions, may contribute to binding were proposed to be the 21-amino-acid residues located at amino acids 100 to 120 of the nucleocapsid proteins of these hepadnaviruses.     

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.     

Replication and gene expression of hepatitis B virus in a transgenic mouse that contains the complete viral genome.

We have sought to address the problem of the host and tissue specificity of the hepatitis B virus (HBV) by using transgenic mice obtained after injection of head-to-tail dimers of the HBV genome. Viral DNA replication and protein synthesis were obtained in one of nine transgenic mice containing integrated HBV DNA. The RNAs encoding the HBV surface antigen and the core antigen were synthesized in the liver, the kidney, and the heart. In these organs, DNA replicative intermediates similar to those found during normal infection were associated with corelike structures. Large amounts of core polypeptides and capsids were detected in the nuclei in the absence of any pathological effect. These results show that the different steps of HBV multiplication can take place in nonliver nonhuman cells once the problem of entry into the host cell is overcome. In the absence of a small laboratory animal infectable by HBV, such transgenic mice should be helpful for the study of many aspects of viral multiplication.     

Production of hepatitis B virus in vitro by transient expression of cloned HBV DNA in a hepatoma cell line.

Transfection of human hepatoma cell lines with cloned HBV DNA resulted in the secretion of large amounts of hepatitis B surface antigen (HBsAg) and core-related antigens (HBc/HBeAg) if well-differentiated cell lines were employed. Synthesis of both viral antigens was the highest in cell line HuH-7 and continued for approximately 25 days. Particles resembling hepatitis B virions (Dane particles) by morphology, density and by the presence of the preS1 surface antigen were released from the transfected HuH-7 cells into the culture medium. These particles produced in vitro were also indistinguishable from the naturally occurring hepatitis B virions in containing the virus-associated DNA polymerase and mature HBV genomes. Restriction analysis of these DNA molecules was compatible with the nucleotide sequence of the transfecting HBV DNA sequence. Viral surface antigens and core proteins present in the culture medium were fractionated and characterized by immunoprecipitation and SDS--PAGE after labeling with [35S]methionine. Antisera specific for X-gene products identified in cell extracts two hitherto unknown HBV gene products. This system thus provides a new approach to open questions regarding HBV-related gene function and HBV replication.     

Role of immunoproteasome catalytic subunits in the immune response to hepatitis B virus

Inhibition of hepatitis B virus (HBV) replication and viral clearance from an infected host requires both the innate and adaptive immune responses. Expression of interferon (IFN)-inducible proteasome catalytic and regulatory subunits correlates with the IFN-alpha/beta- and IFN-gamma-mediated noncytopathic inhibition of HBV in transgenic mice and hepatocytes, as well as with clearance of the virus in acutely infected chimpanzees. The immunoproteasome catalytic subunits LMP2 and LMP7 alter proteasome specificity and influence the pool of peptides available for presentation by major histocompatibility complex class I molecules. We found that these subunits influenced both the magnitude and specificity of the CD8 T-cell response to the HBV polymerase and envelope proteins in immunized HLA-A2-transgenic mice. We also examined the role of LMP2 and LMP7 in the IFN-alpha/beta- and IFN-gamma-mediated inhibition of virus replication using HBV transgenic mice and found that they do not play a direct role in this process. These results demonstrate the ability of the IFN-induced proteasome catalytic subunits to shape the HBV-specific CD8 T-cell response and thus potentially influence the progression of infection to acute or chronic disease. In addition, these studies identify a potential key role for IFN in regulating the adaptive immune response to HBV through alterations in viral antigen processing.     

Naturally occurring missense mutation in the polymerase gene terminating hepatitis B virus replication.

A hepatitis B virus (HBV) genome was cloned from human liver. Numerous mutations in all viral genes define this HBV DNA as a mutant, divergent from all known HBV DNA sequences. Functional analyses of this mutant demonstrated a defect blocking viral DNA synthesis. The genetic basis of this defect was identified as a single missense mutation in the 5' region of the viral polymerase gene, resulting in the inability to package pregenomic RNA into core particles. The replication defect could be trans-complemented by a full-length wild-type, but not by a full-length mutant or 3'-truncated wild-type, polymerase gene construct. Our findings indicate a critical role of the 5' polymerase gene region in the life cycle of the virus and suggest that introducing missense mutations in this region can be a strategy to terminate viral replication in vivo.     

The hepatitis B virus (HBV) precore protein inhibits HBV replication in transgenic mice.

In this study, we examined the ability of the hepatitis B virus (HBV) precore, envelope, and X gene products to modulate HBV replication in the livers of transgenic mice that replicate the virus. Hepatic HBV replication was not affected by overexpression of the envelope or X gene products when these animals were crossed with transgenic mice that express the corresponding viral genes in the hepatocyte. Overexpression of the precore protein, however, eliminated nucleocapsid particles from the cytoplasm of the hepatocytes and abolished HBV replication without affecting the hepatic steady-state content of pregenomic HBV RNA. These observations suggest that the precore protein can exert a dominant negative effect on HBV replication, presumably at the level of nucleocapsid particle maturation or stability, suggesting an important role for this enigmatic viral protein in the HBV life cycle.