Chungming Chang

Evaluation of Transcriptional Efficiency of Hepatitis B Virus Covalently Closed Circular DNA by Reverse Transcription-PCR Combined with the Restriction Enzyme Digestion Method

ABSTRACT Virus persistence in chronic hepatitis B patients is due to the sustaining level of covalently closed circular DNA (cccDNA) within the nuclei of infected hepatocytes. In this study, we used a modified 1.3-fold hepatitis B virus (HBV) genome, with a BclI genetic marker embedded in the redundancy region, to examine the transcriptional activity of cccDNA and the effect of the HBx protein on transcriptional regulation. After harvesting total RNA from transfected cells or stable lines, we specifically identified and monitored the transcripts from cccDNA by using reverse transcription-PCR (RT-PCR) combined with the restriction enzyme digestion method. In this approach, we have found that (i) RT-PCR combined with detection of the BclI marker is a highly specific method for distinguishing cccDNA-derived transcripts from the original integrated viral genome, (ii) the transcriptional ability of cccDNA was less efficient than that from the integrated viral genome, and (iii) the transcriptional activity of cccDNA was significantly regulated by the HBx protein, a potential transcription activator. In conclusion, we provided a tool with which to elucidate the transcriptional regulation of cccDNA and clarified the transcriptional regulation mechanism of HBx on cccDNA. The results obtained may be helpful in the development of a clinical intervention for patients with chronic HBV infections.

HBV replication is significantly reduced by IL-6

Interleukin-6 (IL-6) is a pleiotropic cytokine with pivotal functions in the regulation of the biological responses of several target cells including hepatocytes. The level of serum IL-6 has been reported to be elevated in patients with chronic hepatitis B, cirrhosis and hepatocellular carcinoma and represents the best marker of HBV-related clinical progression as compared with several other cytokines. In this study, we found that IL-6 was able to effectively suppress hepatitis B virus (HBV) replication and prevent the accumulation of HBV covalently closed circular DNA (cccDNA) in a human hepatoma cell line. We also demonstrated that the suppression of HBV replication by IL-6 requires concurrently a moderate reduction of viral transcripts/core proteins and a marked decrease in viral genome-containing nucleocapsids. Studies on the stability of existing viral capsids suggest that the IL-6 effect on the reduction of genome-containing nucleocapsids is mediated through the prevention of the formation of genome-containing nucleocapsids, which is similar to the effect of interferons. However, IFN-α/β and IFN-γ did not participate in the IL-6-induced suppression of HBV replication. Taken together, our results will provide important information to better understand the role of IL-6 in the course of HBV infection.

Transforming growth factor‐β1 suppresses hepatitis B virus replication primarily through transcriptional inhibition of pregenomic RNA

Transforming growth factor–beta1 (TGF‐β1) is a pleiotropic cytokine with pivotal roles in the regulation of cellular functions and immune responses. In this study, we found that TGF‐β1 was able to effectively suppress hepatitis B virus (HBV) replication. In the presence of TGF‐β1, the level of viral replicative intermediates was dramatically decreased, both in actively dividing cells and in confluent cells. At the same time, the levels of viral transcripts, core protein, and nucleocapsid were significantly diminished by TGF‐β1 treatment. Interestingly, the inhibitory activity of TGF‐β1 was associated with preferential reduction of the level of pregenomic RNA compared with pre‐C mRNA. Further analysis indicated that TGF‐β1 might exert its antiviral effect primarily through reducing expression of the HBV core protein by transcriptional regulation instead of posttranscriptional modification. Conclusion: TGF‐β1 may play a dual role in HBV infection, in the suppression of immune responses against viral infection and in the direct inhibition of viral replication, resulting in minimization of liver damage in patients with chronic hepatitis. (HEPATOLOGY 2007.)

Transforming Growth Factor-β1 Suppresses Hepatitis B Virus Replication by the Reduction of Hepatocyte Nuclear Factor-4α Expression

Several studies have demonstrated that cytokine-mediated noncytopathic suppression of hepatitis B virus (HBV) replication may provide an alternative therapeutic strategy for the treatment of chronic hepatitis B infection. In our previous study, we showed that transforming growth factor-beta1 (TGF-β1) could effectively suppress HBV replication at physiological concentrations. Here, we provide more evidence that TGF-β1 specifically diminishes HBV core promoter activity, which subsequently results in a reduction in the level of viral pregenomic RNA (pgRNA), core protein (HBc), nucleocapsid, and consequently suppresses HBV replication. The hepatocyte nuclear factor 4alpha (HNF-4α) binding element(s) within the HBV core promoter region was characterized to be responsive for the inhibitory effect of TGF-β1 on HBV regulation. Furthermore, we found that TGF-β1 treatment significantly repressed HNF-4α expression at both mRNA and protein levels. We demonstrated that RNAi-mediated depletion of HNF-4α was sufficient to reduce HBc synthesis as TGF-β1 did. Prevention of HNF-4α degradation by treating with proteasome inhibitor MG132 also prevented the inhibitory effect of TGF-β1. Finally, we confirmed that HBV replication could be rescued by ectopic expression of HNF-4α in TGF-β1-treated cells. Our data clarify the mechanism by which TGF-β1 suppresses HBV replication, primarily through modulating the expression of HNF-4α gene.

Encapsidation of truncated human hepatitis b virus genomes through trans-complementation of the core protein and polymerase

Mutational analyses and complementation tests were used to analyze the strategy of packaging and of replication of human hepatitis B virus (HBV). By creating new restriction enzyme sites and by varying the genome length of HBV mutants, we identified that the mutated genomes could be encapsidated through trans-complementation of the polymerase and/or core protein. This study demonstrates that the polymerase of HBV, similar to that of duck hepatitis B virus (DHBV), is synthesized de novo instead of through a core-polymerase fusion protein. The results also indicate that both the polymerase and the core protein can be supplied in trans during viral packaging, and that the complementation is not due to recombination between the cotransfected plasmids. Furthermore, HBV genome deleted down to 2.4 kb is still able to be encapsidated, as measured by the endogenous polymerase reaction. Taken together, these results provide a basis for using HBV as a vector to deliver foreign genes into hepatocytes and for defining the location of the packaging signal on the HBV genome.

Dynamics of HBV cccDNA expression and transcription in different cell growth phase

BackgroundThe covalently closed-circular DNA (cccDNA) of hepatitis B virus (HBV) is associated with viral persistence in HBV-infected hepatocytes. However, the regulation of cccDNA and its transcription in the host cells at different growth stages is not well understood.MethodsWe took advantages of a stably HBV-producing cell line, 1.3ES2, and examine the dynamic changes of HBV cccDNA, viral transcripts, and viral replication intermediates in different cellular growth stages.ResultsIn this study, we showed that cccDNA increased suddenly in the initial proliferation phase of cell growth, probably attributable to its nuclear replenishment by intracellular nucleocapsids. The amount of cccDNA then decreased dramatically in the cells during their exponential proliferation similar to the loss of extrachromosomal plasmid DNA during cell division, after which it accumulated gradually while the host cells grew to confluency. We found that cccDNA was reduced in dividing cells and could be removed when proliferating cells were subjected to long term of lamivudine (3TC) treatment. The amounts of viral replicative intermediates were rapidly reduced in these proliferating cells and were significantly increased after cells reaching confluency. The expression levels of viral transcripts were increased in parallel with the elevated expression of hepatic transcription factors (HNF4α, CEBPα, PPARα, etc.) during cell growth confluency. The HBV transcripts were transcribed from both integrated viral genome and cccDNA, however the transcriptional abilities of cccDNA was less efficient then that from integrated viral genome in all cell growth stages. We also noted increases in the accumulation of intracellular viral particles and the secretion of mature virions as the cells reached confluency and ceased to grow.ConclusionsBased on the dynamics of HBV replication, we propose that HBV replication is modulated differently in the different stages of cell growth, and can be divided into three phases (initial proliferation phase, exponential proliferation phase and growth confluency phase) according to the cell growth curve. The regulation of cccDNA in different cell growth phase and its importance regarding HBV replication are discussed.

Effects of Genomic Length on Translocation of Hepatitis B Virus Polymerase-Linked Oligomer

ABSTRACT Accurate translocation of the polymerase-linked oligomer to the acceptor site (DR1*) in reverse transcription is crucial for maintaining the correct size of the hepatitis B virus (HBV) genome. Various sizes of foreign sequences were inserted at different sites of the HBV genome, and their effects on accurate translocation of polymerase-linked oligomer to DR1* were tested. Three types of replicate DNA products were observed in these insertion mutants: RC (relaxed circle) and type I and type II DL (duplex linear) DNA. Our results indicated that the minus strand of RC and type I DL form was elongated from DR1*, while the minus strand of the type II DL form was elongated from multiple internal acceptor sites (IAS), such as IAS2. These IASs were also found to be used by wild-type HBV but with a very low frequency. Mutation of IAS2 by base substitution abrogated polymerase-linked oligomer transferring to IAS2, demonstrating that base pairing also plays an important role in the function of IAS2 as a polymerase-linked oligomer acceptor site. Data obtained from our insertion mutants also demonstrate that the distance between the polymerase-linked oligomer priming site and the acceptor is important. The polymerase-linked oligomer prefers to translocate to an acceptor, DR1* or IAS2, which are ca. 3.2 kb apart. However, it will translocate to both DR1* and IAS2 if they are not located 3.2 kb apart. These results suggest that the polymerase-linked oligomer may be able to scan bidirectionally for appropriate acceptor sites at a distance of 3.2 kb. A model is proposed to discuss the possible mechanism of polymerase-linked oligomer translocation.

One single nucleotide difference alters the differential expression of spliced RNAs between HBV genotypes A and D

Hepatitis B virus (HBV) is generally classified into eight genotypes (A to H) based on genomic sequence divergence. The sequence variation among the different HBV genotypes suggests that the spliced RNAs should be different from genotype to genotype. However, the cis-acting element involved in the modulation of the distinct expression profiles of spliced HBV RNAs remains unidentified. Moreover, the biological role of splicing in the life cycle of HBV is not yet understood. In this study, spliced RNAs generated from genotypes A and D were carefully characterized in transfected HepG2 cells. The species and frequency of the spliced RNAs were dramatically different in the two genotypes. Of note, a population of multiply spliced RNAs with intron 2067-2350 excision was identified in HBV genotype A-transfected HepG2 cells, but not in genotype D transfected HepG2 cells. Further, we found a single nucleotide difference (2335) located within the polypyrimidine tract of the splice acceptor site 2350 between the two genotypes, and a single base substitution at 2335 was able to convert the splicing pattern of genotype D (or genotype A) to that of genotype A (or genotype D). These findings suggest that different unique splice sites may be preferentially used in different HBV genotypes resulting in distinct populations of spliced RNAs. The possible significance of the distinct spliced RNAs generated from the different HBV genotypes in HBV infection is discussed.

Decreased Expression of UK114 Is Related to the Differentiation Status of Human Hepatocellular Carcinoma

Previous studies have identified that the expression of UK114 is tissue specific and the protein has been found to be most abundant in liver and kidney. However, the expression of UK114 in human hepatocellular carcinoma and its relationship to differentiation and transformation of hepatocellular carcinoma have not been studied. In this study, the expression of UK114 in human hepatocellular carcinoma was examined by Northern and Western blot analyses. We found that UK114 was significantly down-regulated in most of hepatocellular carcinoma tissues compared with adjacent nontumor tissues (72.7%) at both mRNA and protein levels. We looked into the possibility that this decreased expression of UK114 in the hepatocellular carcinoma tissues may play a role in the differentiation or tumorigenicity of hepatocellular carcinoma. Immunohistochemical staining showed that the reduced expression of UK114 in hepatocellular carcinoma tissues was correlated with the tumor differentiation status as graded by the Edmondson-Steiner classification. On the other hand, overexpression of UK114 was not able to suppress the proliferation of human hepatoma cells and tumorigenicity in nude mice. These results suggest that UK114 does not seem to act as a tumor suppressor gene; however, it may useful as a biomarker that will assist in the grading of the differentiation status of hepatocellular carcinoma samples. (Cancer Epidemiol Biomarkers Prev 2008;17(3):535–42)

Advances in Biomedical Research

The National Institute of Health (NItl)/US Food and Drug Administration (FDA) and Chinese American Association (CAA) and the Washington DC Chapter of Society of Chinese Bioscientists in America (SCBA) have succesfully sponsored five consecutive annual joint scientific symposia since 1994. Leading experts at the forefront of various biomedical fields have been invited to present their research findings. These joint symposia have sparkled spirited discussions and led to productive collaborations among Chinese bioscientists in the great Washington DC metropolitan area. The sixth symposium was held in the NIH Bethesda campus (Bldg. 10, Lippsett Amphitheater) on October 9, 1999. This years symposium highlighted recent advances in biomedical sciences, especially the HIV and tumor biology that has attracted attention from both the scientific community as well as the popular press. HIV infection has been a serious worldwide health concern. Prevention, intervention and treatment of this deadly diseae represent major challenges and opportunities to biomedical researchers. Three talks were devoted to this issue. The first two presentations by Drs. Sylvia Lee-Huang (NYU) and Hao Chia Chen (NIH) described the exciting discovery of new anti-HIV agents of promising clinical potentials. Several new anti-HIV agents including (1) MAP30 and GAP31 from medical plants and (2) AVL and AVR from urine of pregnant women and (3) RNase U and urinary lysozyme C from pregnant women were described. The third talk by Dr. Kuan-Teh Jeang (NIH) focused on the pivotal role of CXCR4 in the treatment of HIV infection. The EGF signaling pathway plays an important role in tumorigenesis and angiogenesis. A new regulatory protein CAIR-1 that can control the releasing PLC-g in response to growth tactor stimulation was presented by Dr. Howard Doong (NIH). Defects in the mismatch repair genes have been identified in various tumors. The human MYH, a homologue of Escherichia coli MutY, have been identified. Dr. A-lien Lu (University of Maryland) presented the linkage between inactivation of hMYH and tumor progression. Immunological regulation and response to diseases via an intricate network of pathways in the human body: three presentations addressed this topic; Dr. XiaoDong Li (Johns Hopkins University) presented the rapid identification of differentially expressed genes in human TH2 cells and their functional significance in allergic asthma. Dr. J. Qian (American Red Cross) focused on the prevention and treatment of heraophilic inhibitors by exploiting the CD40L/CD40 and B7/CD28 pathways to develop an anti-hemophilic inhibitor in the murine model. Dr. Tiang-Li Wang (Johns Hopkins University) described the use of nucleic acid vaccine to prevent human papiltomavirus-induced cervical cancer. Linkage E7 antigen with targeting signal of the MCH II pathway of LAMP-1 significantly enhances the potency of DNA vaccine. Dr. Yufan Shi, Dr. Andrew Chang, Dr. T.-C. Wu, and Dr. Yunbo Shi provided valuable advice in organizing this symposium. We would like to thank Dr. M.K. Jeang, Cardiovascular Center, University of Texas of Health Sciences Center at Houston, the Science Division of Taipei Economic and Cultural Representative Office in US, and generous support from a number of companies. We especially thank Dr. K.-T. Jeang, a former president ofNIH/FDA CAA, for his insights and enthusiastic and continuing support for this joint symposium.