The hepatitis delta virus (HDV), composed of ~1,700nt, is the smallest circular RNA pathogen known to infect humans. Understanding the mode of replication of HDV implies on investigating the host proteins that bind to its genome. The polypyrimidine tract-binding protein-associated splicing factor (PSF), an HDV interacting protein, was found to interact with the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), and to facilitate the interaction of RNA transcripts with the CTD of RNAPII. Both PSF and RNAPII were found to interact with both polarities of the terminal stem loop domains of HDV RNA, which possess RNA promoter activity in vitro. Furthermore, PSF and RNAPII were found to simultaneously interact with HDV RNA in vitro. Together, the above experiments suggest that PSF acts as a transcription factor during HDV RNA replication by interacting with both the CTD of RNAPII and HDV RNA simultaneously. PSF knockdown experiments were performed to indicate that PSF is required for HDV RNA accumulation. Mutagenesis experiments of PSF revealed that HDV RNA accumulation might require the N terminal domain, and the RNA recognition motifs RRM1 and RRM2. I propose that the RRM1 and RRM2 domains might interact with HDV RNA, while the N-terminal domain might interact with the CTD of RNAPII for HDV RNA accumulation. Together, the above experiments provide a better understanding of how an RNA promoter might be recognized by RNAPII.

Hepatitis C Virus (HCV) is one of the most important causes of chronic liver disease, affecting more than 170 million people worldwide. The mechanisms of hepatitis C pathogenesis are unknown. Viral cytotoxicity and immune mediated mechanisms might play an important role in its pathogenesis. HCV infection and alcohol abuse frequently coexist and together lead to more rapid progression of liver disease, increasing the incidence and prevalence of cirrhosis and hepatocellular carcinoma. The cytopathic effect of HCV proteins, especially the core, E1 and E2 structural proteins, which induce liver steatosis, oxidative stress and cell transformation may be amplified by alcohol abuse. The purpose of this study was to characterize the liver damage mechanisms induced by HCV structural proteins and alcohol and to determine the potential molecular mechanism(s) that may promote chronic, progressive liver damage. A transgenic mouse model expressing HCV core, E1 and E2 was used to investigate whether alcohol increased HCV RNA expression. Real-time RT-PCR analysis of genes involved in lipid metabolism and transport confirmed their abnormal expression in the alcohol-fed transgenic mice. In addition, light and electron microscopy analysis were performed on liver tissues of transgenic mice on an alcoholic diet versus those on a normal diet, in order to identify histological changes. The severe hepatopathy in HCV transgenic mice was exacerbated by alcohol. Mitochondria and endoplasmic reticulum had severe abnormalities in the electron microscopy analysis. The second part of this study focused on adaptive immune responses, which may also play an important role in HCV pathogenesis. I focused my analysis on dendritic cells (DC), which have been the main suspects to explain immune impairment in HCV infection. Their powerful antigen-presenting function allows them to stimulate the antiviral response of CD4+ and CD8+ T cells, the effector cells of the immune system. This unique function of the DC makes them possible targets for immune evasion by the Hepatitis C virus. In this study, DCs were generated from mouse bone marrow cells. I investigated their maturation capacity in the presence of structural proteins of HCV. The impact of HCV core/E1/E2 polyprotein on DCs cytokine expression and ability to activate T-cell lymphocytes was also analyzed. A dysfunctional CD4 T cell response was observed after exposure of DCs to core/E1/E2 polyprotein, indicating inefficient CD4 priming, which might lead to chronic HCV infection in humans. The presence of the core/E1/E2 polyprotein reduced the DC maturation capacity and the expression of certain cytokines (IL-12, IFNg, IL-6, MCP-1) important for stimulation and chemotaxis of T cells and other immune cells. My studies contribute to the understanding of HCV pathogenesis and may have implications to the development of better therapies for HCV infection.

The hepatitis delta virus (HDV), composed of ~1,700nt, is the smallest circular RNA pathogen known to infect humans. Understanding the mode of replication of HDV implies on investigating the host proteins that bind to its genome. The polypyrimidine tract-binding protein-associated splicing factor (PSF), an HDV interacting protein, was found to interact with the carboxy terminal domain (CTD) of RNA polymerase II (RNAPII), and to facilitate the interaction of RNA transcripts with the CTD of RNAPII. Both PSF and RNAPII were found to interact with both polarities of the terminal stem loop domains of HDV RNA, which possess RNA promoter activity in vitro. Furthermore, PSF and RNAPII were found to simultaneously interact with HDV RNA in vitro. Together, the above experiments suggest that PSF acts as a transcription factor during HDV RNA replication by interacting with both the CTD of RNAPII and HDV RNA simultaneously. PSF knockdown experiments were performed to indicate that PSF is required for HDV RNA accumulation. Mutagenesis experiments of PSF revealed that HDV RNA accumulation might require the N terminal domain, and the RNA recognition motifs RRM1 and RRM2. I propose that the RRM1 and RRM2 domains might interact with HDV RNA, while the N-terminal domain might interact with the CTD of RNAPII for HDV RNA accumulation. Together, the above experiments provide a better understanding of how an RNA promoter might be recognized by RNAPII.

Hepatitis C Virus (HCV) is one of the most important causes of chronic liver disease, affecting more than 170 million people worldwide. The mechanisms of hepatitis C pathogenesis are unknown. Viral cytotoxicity and immune mediated mechanisms might play an important role in its pathogenesis. HCV infection and alcohol abuse frequently coexist and together lead to more rapid progression of liver disease, increasing the incidence and prevalence of cirrhosis and hepatocellular carcinoma. The cytopathic effect of HCV proteins, especially the core, E1 and E2 structural proteins, which induce liver steatosis, oxidative stress and cell transformation may be amplified by alcohol abuse. The purpose of this study was to characterize the liver damage mechanisms induced by HCV structural proteins and alcohol and to determine the potential molecular mechanism(s) that may promote chronic, progressive liver damage. A transgenic mouse model expressing HCV core, E1 and E2 was used to investigate whether alcohol increased HCV RNA expression. Real-time RT-PCR analysis of genes involved in lipid metabolism and transport confirmed their abnormal expression in the alcohol-fed transgenic mice. In addition, light and electron microscopy analysis were performed on liver tissues of transgenic mice on an alcoholic diet versus those on a normal diet, in order to identify histological changes. The severe hepatopathy in HCV transgenic mice was exacerbated by alcohol. Mitochondria and endoplasmic reticulum had severe abnormalities in the electron microscopy analysis. The second part of this study focused on adaptive immune responses, which may also play an important role in HCV pathogenesis. I focused my analysis on dendritic cells (DC), which have been the main suspects to explain immune impairment in HCV infection. Their powerful antigen-presenting function allows them to stimulate the antiviral response of CD4+ and CD8+ T cells, the effector cells of the immune system. This unique function of the DC makes them possible targets for immune evasion by the Hepatitis C virus. In this study, DCs were generated from mouse bone marrow cells. I investigated their maturation capacity in the presence of structural proteins of HCV. The impact of HCV core/E1/E2 polyprotein on DCs cytokine expression and ability to activate T-cell lymphocytes was also analyzed. A dysfunctional CD4 T cell response was observed after exposure of DCs to core/E1/E2 polyprotein, indicating inefficient CD4 priming, which might lead to chronic HCV infection in humans. The presence of the core/E1/E2 polyprotein reduced the DC maturation capacity and the expression of certain cytokines (IL-12, IFNg, IL-6, MCP-1) important for stimulation and chemotaxis of T cells and other immune cells. My studies contribute to the understanding of HCV pathogenesis and may have implications to the development of better therapies for HCV infection.

Tzukuan is an HBV-, HCV-, HDV- endemic township in southern Taiwan. Based on a mass screening on 2909 residents age of 45 years or more in 1997, the prevalence rates of HBsAg and anti-HCV were 12.8% and 41.6% respectively. Of HBsAg carriers, 15.3% were positive for anti-HDV. Tzukaun was divided into coastal area and inland area. The prevalence of anti-HCV of coastal area was two times higher than that of inland area (61.4% v.s. 29.1%) and genotype 1b and 2a are the main two subtypes. We wish to find the causes of discrepancy in these nearby areas by phylogenetic analysis. Stratified by the living areas, coastal or inland, 27 samples were picked up (ingroup). HCV sequence of NS5B region could be detected by RT-PCR then a nested PCR in eight males and ninteen females with mean age of 54.8 years old (range: 45-70). None of these 27 residents came from the same family. Another 10 HCV infected persons whose living townships also in southern Taiwan but other than Tzukuan were enrolled as local controls. From GenBank, 30 different HCV isolates were included. Phylogenic analysis unequivocally confirmed the simultaneous spread of two different HCV strains in this township clusters according to their subtypes were noted. A trend of the spreading from coastal to land area or an ultra-aggregation phynomenon which according to their living area, as we suspected, were not noted between Tzukuan¡¦s residents. In ingroup, the short genetic distance between the isolates of C hepatitis virus which came from different villages might be caused from the wide-spreading of HCV in this endemic area (the maximal and minimal genetic distance in 1b or 2a isolates are 0.0869 vs. 0.0098 and 0.0996 vs. 0.0334). Besides, according to the contacting history to foreigner by our aborigine tribes, from genebank, all isolates from different countries were included and three possible origins of HCV genotype 1b were noted in Tzukuan. All these findings might be caused from frequently HCV inflow in this endemic area and wide-spreading of HCV between different countries.

Düsseldorf, University, Thesis (doctoral), 2000.

Thesis (doctoral)--Universität, Münster (Westfalen).

published_or_final_version / Medicine / Master / Doctor of Medicine

<p>Dengue viruses (DENV) are mosquito&#8208;borne flaviviruses that pose a continued and growing threat to global health. There are estimated to be 390 million DENV infections each year, and because there is no vaccine or approved therapeutic treatment, developing a small&#8208;molecule treatment is imperative. Possible small&#8208;molecule drug therapies for DENV could be immune system modulators, inhibitors of DENV&#8208;required host factor, or inhibitors of a viral gene product. In this study, we chose to take the latter approach and focused our drug discovery efforts on the most highly conserved flaviviral protein, non&#8208;structural protein 5 (NS5). NS5 contains two major domains, each with different enzymatic activities. The N&#8208;terminus has methyltransferase activity, and the C terminus, an RNA&#8208;dependent RNA polymerase (RdRp). The activities of both domains are purine&#8208;dependent, and therefore both domains contribute to the purine&#8208;binding properties of NS5. Inhibition of either of these domains in NS5 results in inadequate propagation of DENV, and the purine&#8208;binding domains present ideal drug targets for disrupting these activities. These factors make NS5 protein an ideal candidate target for our small&#8208;molecule library screen.</p><p>A high&#8208;throughput fluorescence&#8208;based screen was employed to identify anti&#8208;DENV compounds based on their ability to competitively bind NS5. The screen was performed by binding green fluorescent protein NS5 fusion protein (GFP&#8208;NS5) to immobilized ATP resin, and then performing parallel elutions using over 3,000 distinct compounds. One compound in particular, HS&#8208;205020, was able to competitively elute GFP&#8208;NS5 from the ATP resin and also exhibited antiviral activity in both the U937+DCSIGN human monocyte cell line and BHK&#8208;21 cells. Additionally, HS&#8208;205020 was able to inhibit DENV NS5 RNA polymerase activity in vitro. HS&#8208;205020 is chemically distinct from the majority of previously reported NS5 inhibitors, which are nucleoside analogs that can cause severe toxicity in animal studies. In contrast, over the concentration range that produced anti&#8208;DENV effects, HS&#8208;205020 showed comparable viabilities to ribavirin, an FDA approved hepatitis C virus (HCV) therapeutic. These findings support HS&#8208;205020 as a potential dengue antiviral candidate, and its chemical scaffold represents as an ideal starting compound for future structure&#8208;activity relationship studies.</p> / Dissertation

Hepatitis B virus (HBV) causes an estimated 600,000 deaths annually, largely due to hepatocellular carcinoma (HCC). HBx, a promiscuous transactivator, is a viral oncoprotein, but its exact functions are poorly understood. Many studies have suggested that NF-κB signaling mediates HBx functions, but the underlying molecular mechanisms remain yet to be elucidated. Here, we provide evidence that HBx-mediated NF-κB activation depends on the physical interaction between HBx and a transcription factor, p65. In the cytoplasm, HBx-p65 interaction may promote IκBα phosphorylation and subsequent p65 nuclear localization. A cytokine assay using qPCR and RT-PCR indicates that HBx is associated with a unique profile of cytokine mRNA expression. As shown by chromatin immunoprecipitation (ChIP), HBx in the nuclues can be recruited to the gene promoter by p65. These findings support the importance of HBx-p65 interaction and suggest that it is potentially a promising target of novel therapeutics for HBV-associated liver diseases, including HCC.