Identification of disubstituted sulfonamide compounds as specific inhibitors of hepatitis B virus covalently closed circular DNA formation.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a central role in viral infection and persistence and is the basis for viral rebound after the cessation of therapy, as well as the elusiveness of a cure even after extended treatment. Therefore, there is an urgent need for the development of novel therapeutic agents that directly target cccDNA formation and maintenance. By employing an innovative cell-based cccDNA assay in which secreted HBV e antigen is a cccDNA-dependent surrogate, we screened an in-house small-molecule library consisting of 85,000 drug-like compounds. Two structurally related disubstituted sulfonamides (DSS), termed CCC-0975 and CCC-0346, emerged and were confirmed as inhibitors of cccDNA production, with low micromolar 50% effective concentrations (EC(50)s) in cell culture. Further mechanistic studies demonstrated that DSS compound treatment neither directly inhibited HBV DNA replication in cell culture nor reduced viral polymerase activity in the in vitro endogenous polymerase assay but synchronously reduced the levels of HBV cccDNA and its putative precursor, deproteinized relaxed circular DNA (DP-rcDNA). However, DSS compounds did not promote the intracellular decay of HBV DP-rcDNA and cccDNA, suggesting that the compounds interfere primarily with rcDNA conversion into cccDNA. In addition, we demonstrated that CCC-0975 was able to reduce cccDNA biosynthesis in duck HBV-infected primary duck hepatocytes. This is the first attempt, to our knowledge, to identify small molecules that target cccDNA formation, and DSS compounds thus potentially serve as proof-of-concept drug candidates for development into therapeutics to eliminate cccDNA from chronic HBV infection.

Clonal expansion of normal-appearing human hepatocytes during chronic hepatitis B virus infection.

Chronic hepatitis B virus (HBV) infections are associated with persistent immune killing of infected hepatocytes. Hepatocytes constitute a largely self-renewing population. Thus, immune killing may exert selective pressure on the population, leading it to evolve in order to survive. A gradual course of hepatocyte evolution toward an HBV-resistant state is suggested by the substantial decline in the fraction of infected hepatocytes that occurs during the course of chronic infections. Consistent with hepatocyte evolution, clones of >1,000 hepatocytes develop postinfection in the noncirrhotic livers of chimpanzees chronically infected with HBV and of woodchucks infected with woodchuck hepatitis virus (W. S. Mason, A. R. Jilbert, and J. Summers, Proc. Natl. Acad. Sci. U. S. A. 102:1139-1144, 2005; W. S. Mason et al., J. Virol. 83:8396-8408, 2009). The present study was carried out to determine (i) if extensive clonal expansion of hepatocytes also occurred in human HBV carriers, particularly in the noncirrhotic liver, and (ii) if clonal expansion included normal-appearing hepatocytes, not just hepatocytes that appear premalignant. Host DNA extracted from fragments of noncancerous liver, collected during surgical resection of hepatocellular carcinoma (HCC), was analyzed by inverse PCR for randomly integrated HBV DNA as a marker of expanding hepatocyte lineages. This analysis detected extensive clonal expansion of hepatocytes, as previously found in chronically infected chimpanzees and woodchucks. Tissue sections were stained with hematoxylin and eosin (H&E), and DNA was extracted from the adjacent section for inverse PCR to detect integrated HBV DNA. This analysis revealed that clonal expansion can occur among normal-appearing human hepatocytes.

Detection of clonally expanded hepatocytes in chimpanzees with chronic hepatitis B virus infection.

During a hepadnavirus infection, viral DNA integrates at a low rate into random sites in the host DNA, producing unique virus-cell junctions detectable by inverse nested PCR (invPCR). These junctions serve as genetic markers of individual hepatocytes, providing a means to detect their subsequent proliferation into clones of two or more hepatocytes. A previous study suggested that the livers of 2.4-year-old woodchucks (Marmota monax) chronically infected with woodchuck hepatitis virus contained at least 100,000 clones of >1,000 hepatocytes (W. S. Mason, A. R. Jilbert, and J. Summers, Proc. Natl. Acad. Sci. USA 102:1139-1144, 2005). However, possible correlations between sites of viral-DNA integration and clonal expansion could not be explored because the woodchuck genome has not yet been sequenced. In order to further investigate this issue, we looked for similar clonal expansion of hepatocytes in the livers of chimpanzees chronically infected with hepatitis B virus (HBV). Liver samples for invPCR were collected from eight chimpanzees chronically infected with HBV for at least 20 years. Fifty clones ranging in size from approximately 35 to 10,000 hepatocytes were detected using invPCR in 32 liver biopsy fragments (approximately 1 mg) containing, in total, approximately 3 x 10(7) liver cells. Based on searching the analogous human genome, integration sites were found on all chromosomes except Y, approximately 30% in known or predicted genes. However, no obvious association between the extent of clonal expansion and the integration site was apparent. This suggests that the integration site per se is not responsible for the outgrowth of large clones of hepatocytes.

The amount of hepatocyte turnover that occurred during resolution of transient hepadnavirus infections was lower when virus replication was inhibited with entecavir.

Transient hepadnavirus infections can involve spread of virus to the entire hepatocyte population. In this situation hepatocytes present following recovery are derived from infected hepatocytes. During virus clearance antiviral cytokines are thought to block virus replication and formation of new covalently closed circular DNA (cccDNA), the viral transcriptional template. It remains unclear if existing cccDNA is eliminated noncytolytically or if hepatocyte death and proliferation, to compensate for killing of some of the infected hepatocytes, are needed to remove cccDNA from surviving infected hepatocytes. Interpreting the relationship between hepatocyte death and cccDNA elimination requires knowing both the amount of hepatocyte turnover and whether cccDNA synthesis is effectively blocked during the period of immune destruction of infected hepatocytes. We have addressed these questions by asking if treatment of woodchucks with the nucleoside analog inhibitor of viral DNA synthesis entecavir (ETV) reduced hepatocyte turnover during clearance of transient woodchuck hepatitis virus (WHV) infections. To estimate hepatocyte turnover, complexity analysis was carried out on virus-cell DNA junctions created by integration of WHV and present following recovery in the livers of WHV-infected control or ETV-treated woodchucks. We estimated that, on average, 2.2 to 4.8 times less hepatocyte turnover occurred during immune clearance in the ETV-treated woodchucks. Computer modeling of the complexity data suggests that mechanisms in addition to hepatocyte death were responsible for elimination of cccDNA during recovery from transient infections.

The liver of woodchucks chronically infected with the woodchuck hepatitis virus contains foci of virus core antigen-negative hepatocytes with both altered and normal morphology.

The livers of woodchucks chronically infected with woodchuck hepatitis virus (WHV) contain foci of morphologically altered hepatocytes (FAH) with "basophilic", "amphophilic" and "clear cell" phenotypes, which are possibly pre-neoplastic in nature. Interestingly, most fail to express detectable levels of WHV proteins and nucleic acids. We studied sections of WHV-infected liver tissue to determine if all foci of hepatocytes that failed to express detectable levels of WHV, as assessed by immunoperoxidase staining for WHV core antigen, could be classified morphologically as FAH. We found that at least half of the foci of WHV core antigen-negative hepatocytes did not show clear morphological differences in either H&E or PAS (periodic acid Schiff) stained sections from surrounding hepatocytes, and were therefore not designated as FAH. In the second approach, we assayed core antigen-negative foci for the presence of fetuin B, a serum protein produced by normal hepatocytes, but not by neoplastic hepatocytes in hepatocellular carcinomas. Basophilic and amphophilic FAH had reduced levels of fetuin B compared to hepatocytes present in the surrounding liver; fetuin B staining was detected in clear cell FAH but the level could not be accurately assessed because of the displacement of fetuin B to the cell periphery by accumulated glycogen. The foci of morphologically normal WHV core antigen-negative hepatocytes had similar levels of fetuin B to that of the surrounding hepatocytes. The co-existence of at least four types of WHV core antigen-negative foci, including those with no obvious morphologic changes, raises the possibility that the different foci arise from distinct primary events. We hypothesize that a common event is loss of the ability to express WHV, allowing these hepatocytes to escape immune mediated cell death and to undergo clonal expansion to form distinct foci.

The insertion domain of the duck hepatitis B virus core protein plays a role in nucleocapsid assembly.

Synthesis of hepadnaviral DNA is dependent upon both the viral DNA polymerase and the viral core protein, the subunit of the nucleocapsids in which viral DNA synthesis takes place. In a study of natural isolates of duck hepatitis B virus (DHBV), we cloned full-length viral genomes from a puna teal. One of the clones failed to direct viral DNA replication in transfected cells, apparently as a result of a 3 nt inframe deletion of histidine 107 in the core protein. Histidine 107 is located in the center of a predicted helical region of the "insertion domain", a stretch of 45 amino acids which appears to be at the tip of a spike on the surface of the nucleocapsid. The mutation was introduced into a well-characterized strain of DHBV for further analysis. Core protein accumulated in cells transfected with the mutant DHBV but was partially degraded, suggesting that it was unstable. Assembled nucleocapsids were not detected by capsid gel electrophoresis. Interestingly, the mutant protein appeared to form chimeric nucleocapsids with wild-type core protein. The chimeric nucleocapsids supported viral DNA replication. These results suggest that the insertion domain of the spike may play a role either in assembly of stable nucleocapsids, possibly in formation of the dimer subunits, or in triggering nucleocapsid disintegration, required during initiation of new rounds of infection.

Identification and characterization of avihepadnaviruses isolated from exotic anseriformes maintained in captivity.

Five new hepadnaviruses were cloned from exotic ducks and geese, including the Chiloe wigeon, mandarin duck, puna teal, Orinoco sheldgoose, and ashy-headed sheldgoose. Sequence comparisons revealed that all but the mandarin duck viruses were closely related to existing isolates of duck hepatitis B virus (DHBV), while mandarin duck virus clones were closely related to Ross goose hepatitis B virus. Nonetheless, the S protein, core protein, and functional domains of the Pol protein were highly conserved in all of the new isolates. The Chiloe wigeon and puna teal hepatitis B viruses, the two new isolates most closely related to DHBV, also lacked an AUG start codon at the beginning of their X open reading frame (ORF). But as previously reported for the heron, Ross goose, and stork hepatitis B viruses, an AUG codon was found near the beginning of the X ORF of the mandarin duck, Orinoco, and ashy-headed sheldgoose viruses. In all of the new isolates, the X ORF ended with a stop codon at the same position. All of the cloned viruses replicated when transfected into the LMH line of chicken hepatoma cells. Significant differences between the new isolates and between these and previously reported isolates were detected in the pre-S domain of the viral envelope protein, which is believed to determine viral host range. Despite this, all of the new isolates were infectious for primary cultures of Pekin duck hepatocytes, and infectivity in young Pekin ducks was demonstrated for all but the ashy-headed sheldgoose isolate.

Adenovirus-based gene therapy during clevudine treatment of woodchucks chronically infected with woodchuck hepatitis virus.

Interferon-alpha (IFN-alpha) is a potent suppressor of hepatitis B virus (HBV) replication in the HBV-transgenic mouse, depleting virus replication intermediates from infected hepatocytes via pathways mediated by interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). It has also been hypothesized that cytokines induce curing of infected hepatocytes via non-cytolytic pathways during resolution of transient hepadnavirus infections. We have therefore evaluated therapy of chronic woodchuck hepatitis virus (WHV) infections using treatment with the nucleoside analog clevudine [L-FMAU; 1-(2-fluoro-5-methyl-b-L-arabinofuranosyl) uracil] and therapy with adenovirus vectors expressing INF-gamma, TNF-alpha, and beta-galactosidase. Before their use in vivo, expression of IFN-gamma and TNF-alpha from the adenovirus vectors was evaluated in vitro. Conditioned media from adenovirus-infected WC-3 cells was shown to inhibit WHV replication in baculovirus-transduced cells. Adenovirus super-infection of the liver in woodchucks led to declines in the percentage of hepatocytes with detectable core antigen and nucleic acids, and in levels of covalently closed circular DNA (cccDNA) and total WHV DNA, but a major long-term benefit of adenovirus super-infection during clevudine treatment was not demonstrated. Moreover, the effect took at least 2 weeks to develop suggesting that the declines in the percentage of WHV-infected cells, ccc, and total WHV DNA resulted from induction of the adaptive immune response by the adenovirus super-infection, and only indirectly from the expression of cytokines by the vectors.

Hepatocyte turnover during resolution of a transient hepadnaviral infection.

We estimated the amount of hepatocyte turnover in the livers of three woodchucks undergoing clearance of a transient woodchuck hepatitis infection by determining the fate of integrated viral DNA as a genetic marker of the infected cell population. Integrated viral DNA was found to persist in liver tissue from recovered animals at essentially undiminished levels of 1 viral genome per 1,000-3,000 liver cells, suggesting that the hepatocytes in the recovered liver were derived primarily from the infected cell population. We determined the single and multicopy distribution of distinct viral cell junctions isolated from small pieces of liver after clearance of the infection to determine the cumulative amount of hepatocyte proliferation that had occurred during recovery. We estimated that proliferation was equivalent to a minimum of 0.7-1 complete random turnovers of the hepatocyte population of the liver. Our results indicated that during resolution of the transient infections a large fraction of the infected hepatocyte population was killed and replaced by hepatocyte cell division.