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.     

Effects of Entecavir on Hepatitis B Virus Covalently Closed Circular DNA in Hepatitis B e Antigen-Positive Patients with Hepatitis B

The aim of this study was to assess the effect of 48-week entecavir therapy on serum and intrahepatic hepatitis B virus, covalently closed circular DNA (HBV cccDNA) levels in hepatitis B e antigen (HBeAg)-positive patients. A total of 120 patients with HBeAg-positive chronic hepatitis were treated with entecavir for 48 weeks. Serum HBV markers, total HBV DNA, and HBV cccDNA levels were measured at baseline and week 48. Biopsies from 20 patients were available for both intrahepatic total HBV DNA and cccDNA testing at these timepoints. HBV cccDNA levels were decreased from a median level of 5.1×10⁶ copies/mL at baseline to a median level of 2.4×10³ copies/mL at week 48. Reduction magnitudes of HBV cccDNA in patients with normalized alanine aminotransferase levels and those undergoing HBeAg seroconversion were significantly greater than those in alanine aminotransferase-abnormal and HBeAg positive patients. Intrahepatic HBV cccDNA was decreased significantly after 48 weeks of treatment, but could not be eradicated. In conclusion, treatment of HBeAg-positive hepatitis B patients with entecavir for 48 weeks decreased serum and intrahepatic HBV cccDNA significantly, and the magnitude of HBV cccDNA reduction was related to total HBV DNA decrease, alanine aminotransferase normalization, and HBeAg seroconversion.     

Duck Hepatitis B Virus cccDNA Amplification Efficiency in Natural Infection Is Regulated by Virus Secretion Efficiency

Previous mutation based studies showed that ablating synthesis of viral envelope proteins led to elevated hepadnaviral covalently closed circular DNA (cccDNA) amplification, but it remains unknown how cccDNA amplification is regulated in natural hepadnaviral infection because of a lack of research system. In this study we report a simple procedure to prepare two identical duck hepatitis B virus inocula, but they possess 10-100-fold difference in cccDNA amplification in infected cell culture. We demonstrate that the infected cells with higher cccDNA amplification significantly reduce the virus secretion efficiency that results in higher accumulation of relaxed circular DNA (rcDNA) and DHBsAg in the cells. The infected cells with lower cccDNA amplification significantly increase the virus secretion efficiency that leads to lower intracellular rcDNA and DHBsAg accumulation. In contrast with the findings generated in the mutation based experimental system, the regulation of cccDNA amplification in natural hepadnaviral infection bypasses direct regulation of the cellular envelope proteins concentration, instead it modulates virus secretion efficiency that ultimately impacts the intracellular rcDNA concentration, an important factor determining the destination of the synthesized rcDNA in infected cells.     

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.Transient hepatitis B virus (HBV) infections, which generally last <6 months, do not cause cirrhosis and cause only minor increases in the risk of hepatocellular carcinoma (HCC) (3, 46). Chronic infections, typically lifelong, can cause cirrhosis and HCC (3). Of the ∼350 million HBV carriers now alive, ca. 60 million will die prematurely of cirrhosis and/or HCC. Cirrhosis, which usually develops late in infection, is a significant risk factor for HCC. Early reports stated that most HCCs occur on a background of cirrhosis. However, later studies suggested that as many as 50% of HCCs may occur in noncirrhotic liver (4), that is, in patients in whom the progression of liver disease still appears rather mild. Thus, liver damage that appears severe by histologic examination is not a prerequisite for HCC.Interestingly, during chronic HBV infections there is, in the face of persistent viremia, a decline over time in the fraction of infected hepatocytes, from 100% to as little as a few percent (5, 12-14, 16, 17, 22, 23, 27, 34, 37, 38). Along with HCC, this is perhaps the most surprising and unexplained outcome of chronic infection. The timing of this decline has not been systematically studied, but it is presumably gradual, occurring over years or decades, and dependent on persistent, albeit low-level, killing of infected hepatocytes by antiviral cytotoxic T lymphocytes (CTLs) (20). It is believed that the liver is largely a closed, self-renewing population. Such a population might be expected to evolve under any strong or persistent selective pressure. In HBV-infected patients, the earliest and most persistent selective pressure is immune killing of infected hepatocytes, which should initially constitute the entire hepatocyte population. Persistent killing of HBV-infected hepatocytes could lead to clonal expansion of mutant or epigenetically altered hepatocytes that had lost the ability to support infection and that were not, therefore, targeted by antiviral CTLs.Such a selective pressure may explain why foci of altered hepatocytes (FAH) and HCC are typically virus negative (1, 6, 11, 26, 29, 31, 35, 40, 41, 44). Normal or preneoplastic hepatocytes (e.g., in FAH) that have evaded the host immune response should undergo clonal expansion, because their death rate is lower than that of surrounding hepatocytes, even if they do not have a higher growth rate. Indeed, clonal expansion of hepatocytes has been detected, in the absence of cirrhosis, in woodchucks chronically infected with woodchuck hepatitis virus (WHV) (19) and in chimpanzees chronically infected with HBV (21). The presence of discrete foci of normal-appearing but virus-negative hepatocytes in chronically infected woodchuck livers (39) suggested, but did not prove, that normal-appearing hepatocytes that had lost the ability to support virus replication might clonally expand.The purpose of the present study was, therefore, to determine if normal-appearing hepatocytes undergo clonal expansion. To address this issue, we focused on noncirrhotic livers, because hepatocyte appearance and organization in many cirrhotic nodules are often considered to indicate premalignancy (7, 24, 25, 44), and this, together with the cellular environment in the cirrhotic liver, may explain why as many as 50% of cirrhotic nodules have been found to be made up of clonally expanded hepatocytes (2, 18, 24, 25, 28, 44). In older HBV patients, cirrhosis, the result of cumulative scarring due to ongoing tissue injury, presumably produces an evolutionary pressure on the hepatocyte population due to restricted blood flow and altered hepatic architecture.Clonal expansion was detected by assaying for integrated HBV DNA by inverse PCR (19, 21). Because integration occurs at random sites in host DNA, each integration event provides a unique genetic marker for the cell in which it occurred, and for any daughter cells. Thus, the clonal expansion of these tagged hepatocytes can be measured by determining how many times a given virus-cell DNA junction is repeated in a liver fragment. Analysis of fragments of nontumorous liver from noncirrhotic HCC patients revealed that at least 1% of hepatocytes are present as clones of >1,000 cells. Examination of 5-μm-thick sections of paraffin-embedded livers from the same patients revealed that clonally expanded hepatocytes were present in liver sections lacking preneoplastic lesions or changes. Therefore, normal-appearing hepatocytes must have undergone clonal expansion. Although clonal expansion was detected by analysis of integrated HBV DNA, the expansion did not appear to be due to the site of integration of the viral DNA into host DNA.These results are consistent with the hypothesis that immune selection and the later emergence of liver cirrhosis, with altered lobular organization and restricted blood flow, may constitute the two major selective pressures on the hepatocyte population that culminate in hepatocellular carcinoma. More-direct proof of the role, if any, of immune selection in hepatocyte evolution and HCC will require, first of all, an assay with a greater ability to detect clonally expanded hepatocytes. The present approach is limited by a number of factors, including a need for integration near a particular restriction endonuclease cleavage site in host DNA and for conservation of particular viral sequences so that the integrated DNA can be amplified using the PCR primers chosen. These issues may explain why the fraction of clonally expanded hepatocytes reported here is much less than that suggested by histologic data showing that more than 50% of hepatocytes appear negative for virus replication in long-term carriers. Further dissection of this issue will also require localization and determination of the virologic status of hepatocyte clones present in tissue sections.     

A Serum MicroRNA Signature Is Associated with the Immune Control of Chronic Hepatitis B Virus Infection

Background and Aims

The virus/host interplay mediates liver pathology in chronic HBV infection. MiRNAs play a pivotal role in virus/host interactions and are detected in both serum and HBsAg-particles, but studies of their dynamics during chronic infection and antiviral therapy are missing. We studied serum miRNAs during different phases of chronic HBV infection and antiviral treatment.

Methods

MiRNAs were profiled by miRCURY-LNA-Universal-RT-miRNA-PCR (Exiqon-A/S) and qPCR-panels-I/II-739-miRNA-assays and single-RT-q-PCRs. Two cohorts of well-characterized HBsAg-carriers were studied (median follow-up 34–52 months): a) training-panel (141 sera) and HBsAg-particles (32 samples) from 61 HBsAg-carriers and b) validation-panel (136 sera) from 84 carriers.

Results

Thirty-one miRNAs were differentially expressed in inactive-carriers (IC) and chronic-hepatitis-B (CHB) with the largest difference for miR-122-5p, miR-99a-5p and miR-192-5p (liver-specific-miRNAs), over-expressed in both sera and HBsAg-particles of CHB (ANOVA/U-test p-values: <0.000001/0.000001; <0.000001/0.000003; <0.000001/0.000005, respectively) and significantly down-regulated during- and after-treatment in sustained-virological-responders (SVR). MiRNA-profiles of IC and SVR clustered in the heatmap. Liver-miRNAs were combined with miR-335, miR-126 and miR-320a (internal controls) to build a MiR-B-Index with 100% sensitivity, 83.3% and 92.5% specificity (−1.7 cut-off) in both training and validation cohorts to identify IC. MiR-B-Index (−5.72, −20.43/14.38) correlated with ALT (49, 10/2056 U/l, ρ = −0.497, p<0.001), HBV-DNA (4.58, undetectable/>8.3 Log₁₀ IU/mL, ρ = −0.732, p<0.001) and HBsAg (3.40, 0.11/5.49 Log₁₀ IU/mL, ρ = −0.883, p<0.001). At multivariate analysis HBV-DNA (p = 0.002), HBsAg (p<0.001) and infection-phase (p<0.001), but not ALT (p = 0.360) correlated with MiR-B-Index. In SVR to Peg-IFN/NUCs MiR-B-Index improved during-therapy and post-treatment reaching IC-like values (5.32, −1.65/10.91 vs 6.68, 0.54/9.53, p = 0.324) beckoning sustained HBV-immune-control earlier than HBsAg-decline.

Conclusions

Serum miRNA profile change dynamically during the different phases of chronic HBV infection. We identified a miRNA signature associated with both natural-occurring and therapy-induced immune control of HBV infection. The MiR-B-Index might be a useful biomarker for the early identification of the sustained switch from CHB to inactive HBV-infection in patients treated with antivirals.     

利用乙型肝炎病毒共价闭合环状DNA构建慢性乙型肝炎病毒感染的小鼠模型

目的通过水动力法注射乙型肝炎病毒(HBV)共价闭合环状DNA(cccDNA)构建C57BL/6小鼠慢性乙型肝炎病毒感染的模型。方法取29只C57BL/6小鼠,分为实验组、对照组和空白组,应用水动力法分别注射HBV cccDNA、pAAV-HBV1.2及等渗盐水,于注射后收集不同时间点的血清和肝组织。利用放射免疫法检测血清样本中乙型肝炎病毒表面抗原(HBsAg)和乙型肝炎病毒e抗原(HBeAg);荧光定量PCR检测血清和肝组织中HBV DNA拷贝数;免疫组织化学法检测肝组织中HBsAg和乙型肝炎病毒核心抗原(HBc Ag)的表达;苏木精-伊红(HE)染色观察肝组织病理变化;使用SPSS 17.0对数据进行统计学分析。结果实验组HBsAg和HBeAg表达均呈现4个上升-下降曲线:HBsAg峰值分别出现在第3天、第3周、第7周和第9周;HBeAg峰值分别出现在第1天、第1周、第4周和第10周。对照组HBsAg和HBeAg表达分别呈现2个或3个明显的峰:HBsAg峰值分别出现在第3天和第8周;HBeAg峰值分别出现在第1天、第3周和第10周。空白组未检测出HBsAg和HBeAg。实验组HBV DNA拷贝数高于对照组的拷贝数(P<0.01);肝组织中HBV DNA拷贝数高于同期血清中的拷贝数(P<0.01);实验组和对照组的肝组织中均有HBsAg和HBc Ag的表达;实验组与对照组出现肝脏细胞炎症、肝细胞纤维化、肝细胞坏死等病理变化,而空白组正常。结论利用水动力法向C57BL/6小鼠体内转入HBV cccDNA,成功建立了慢性乙型肝炎病毒感染的小鼠模型,与对照组比较,新建立的小鼠乙肝模型具有更高的HBV表达,动物模型为研究乙型肝炎病毒HBV cccDNA的感染及其引起肝损伤的机制奠定了基础。     

A Mechanism To Explain the Selection of the Hepatitis e Antigen-Negative Mutant during Chronic Hepatitis B Virus Infection

Hepatitis B virus (HBV) expresses two structural forms of the nucleoprotein, the intracellular nucleocapsid (hepatitis core antigen [HBcAg]) and the secreted nonparticulate form (hepatitis e antigen [HBeAg]). The aim of this study was to evaluate the ability of HBcAg- and HBeAg-specific genetic immunogens to induce HBc/HBeAg-specific CD4⁺/CD8⁺ T-cell immune responses and the potential to induce liver injury in HBV-transgenic (Tg) mice. Both the HBcAg- and HBeAg-specific plasmids primed comparable immune responses. Both CD4⁺ and CD8⁺ T cells were important for priming/effector functions of HBc/HBeAg-specific cytotoxic T-lymphocyte (CTL) responses. However, a unique two-step immunization protocol was necessary to elicit maximal CTL priming. Genetic vaccination did not prime CTLs in HBe- or HBc/HBeAg-dbl-Tg mice but elicited a weak CTL response in HBcAg-Tg mice. When HBc/HBeAg-specific CTLs were adoptively transferred into HBc-, HBe-, and HBc/HBeAg-dbl-Tg mice, the durations of the liver injury and inflammation were significantly greater in HBeAg-Tg recipient mice than in HBcAg-Tg mice. Importantly, liver injury in HBc/HBeAg-dbl-Tg mice was similar to the injury observed in HBeAg-Tg mice. Loss of HBeAg synthesis commonly occurs during chronic HBV infection; however, the mechanism of selection of HBeAg-negative variants is unknown. The finding that hepatocytes expressing wild-type HBV (containing both HBcAg and HBeAg) are more susceptible to CTL-mediated clearance than hepatocytes expressing only HBcAg suggest that the HBeAg-negative variant may have a selective advantage over wild-type HBV within the livers of patients with chronic infection during an immune response and may represent a CTL escape mutant.     

Isolation of in Vitro Synthesized Covalently Closed Circular Double-Stranded DNA by Selective Denaturation and Filtration

Based upon their resistance to irreversible denaturation, covalently closed circular (CCC) DNA (non-nicked, double-stranded circular molecules) can be purified by alkaline denaturation, neutralization, and filtration through a nitrocellulose membrane. This procedure offers a simple means of isolating in vitro synthesized CCC DNA molecules. The preparations of molecules obtained by this method consisted of 91-97% CCC DNA and contained no detectable inhibitors of biological activity or enzymatic digestion.     

Protective Efficacy of DNA Vaccines against Duck Hepatitis B Virus Infection

The efficacy of DNA vaccines encoding the duck hepatitis B virus (DHBV) pre-S/S and S proteins were tested in Pekin ducks. Plasmid pcDNA I/Amp DNA containing the DHBV pre-S/S or S genes was injected intramuscularly three times, at 3-week intervals. All pre-S/S and S-vaccinated ducks developed total anti-DHBs and specific anti-S antibodies with similar titers reaching 1/10,000 to 1/50,000 and 1/2,500 to 1/4,000, respectively, after the third vaccination. However, following virus challenge, significant differences in the rate of virus removal from the bloodstream and the presence of virus replication in the liver were found between the groups. In three of four S-vaccinated ducks, 90% of the inoculum was removed between <5 and 15 min postchallenge (p.c.) and no virus replication was detected in the liver at 4 days p.c. In contrast, in all four pre-S/S-vaccinated ducks, 90% of the inoculum was removed between 60 and 90 min p.c. and DHBsAg was detected in 10 to 40% of hepatocytes. Anti-S serum abolished virus infectivity when preincubated with DHBV before inoculation into 1-day-old ducklings and primary duck hepatocyte cultures, while anti-pre-S/S serum showed very limited capacity to neutralize virus infectivity in these two systems. Thus, although both DNA vaccines induced high titers of anti-DHBs antibodies, anti-S antibodies induced by the S-DNA construct were highly effective in neutralizing virus infectivity while similar levels of anti-S induced by the pre-S/S-DNA construct conferred only very limited protection. This phenomenon requires further clarification, particularly in light of the development of newer HBV vaccines containing pre-S proteins and a possible discrepancy between anti-HBs titers and protective efficacy.