Introducing Prof Guo - presenting cccDNA inhibitor at ISVHLD

StephenW

       

Haitao Guo                        Assistant Professor, Microbiology and Immunology                 Drexel Institute for Biotechnology and Virology Research                                Department: Microbiology and Immunology                         Research interests: Hepatitis B; Innate Immunity; Antiviral                         Education: Ph.D., 2001, Wuhan University, China                         Research Staff: Dawei Cai, Ph.D, Postdoctoral fellow of Hepatitis B Foundation, Hui (Daisy) Nie, Ph.D, Postdoctoral researcher and Ran Yan, Ph.D, Postdoctoral researcher

       

        Research
                                                                                The research in my laboratory focuses on hepatitis B virus (HBV), the pathogen causing a major public health burden affecting approximately 350 million individuals worldwide. Chronic hepatitis B patients have an elevated risk of liver cirrhosis, hepatocellular carcinoma (HCC), and other severe clinical sequelae. Therefore it is of importance to study HBV virology and develop efficient antiviral strategies to cure chronic HBV infection and prevent its dire consequences. Our research centers on the molecular biology of HBV replication and morphogenesis, innate immunity control of HBV replication, as well as antiviral drug screening and development. Three major ongoing projects are briefly described as below.
        1. Molecular pathway of HBV covalently closed circular (ccc) DNA formation.  
        HBV is a noncytopathic, liver tropic DNA virus belonging to the Hepadnaviridae family. Upon infection, the virus genome is transported into the cell nucleus and converted into episomal covalently closed circular (ccc) DNA, which is the transcription template for all the viral mRNAs. One species, pregenomic (pg) RNA, is exported into the cytoplasm and packaged into the nucleocapsid, then undergoes reverse transcription to viral minus strand DNA and then plus strand DNA to yield the rcDNA genome. The mature capsids will either be packaged with envelope proteins and egress as virion particles, or shuttle to the nucleus to amplify the cccDNA reservoir (see the illustration):
       
       
       
        HBV cccDNA is an essential component of the HBV life cycle, and is responsible for the establishment of infection and viral persistence.  Since cccDNA is converted from virus genomic relaxed circular (RC) DNA, removal of the viral polymerase covalently attached to the RCDNA is thought to be an essential step during HBV cccDNA formation. Recently, we identified a polymerase-free HBV DNA species from HBV-infected hepatocytes, which is termed deproteinized DNA (dpDNA) and confirmed it as the functional precursor of cccDNA. The cellular localization of dpDNA, dpDNA nuclear transportation, dpDNA-core protein association, the 5’ end sequence of minus strand of dpDNA, and in vitro deproteinization reaction have been systematically studied. Our studies suggest a model for the molecular pathway of HBV cccDNA formation: The completion of HBV RCDNA plus strand DNA triggers the deproteinization of RCDNA, followed by the structure shift of nucleocapsid, which exposes the nuclear localization signal (NLS) of capsid protein; the dpDNA containing capsid is subsequently transported into the nucleus mediated by the cellular karyopherins, and finally the dpDNA is converted into cccDNA by cellular DNA repair machinery. We are currently studying the molecular details in this work model.
       
       
        2. Screening and development of small molecule compounds that inhibit HBV cccDNA formation
        Currently there is no definitive cure for chronic hepatitis B.  The main limitation of current treatment (interferon or nucleoside analogues) is the failure to eliminate the preexisting cccDNA pool and/or prevent cccDNA formation from trace-level wild-type or drug-resistant virus. Thus, there is an urgent need for the development of novel therapeutic agents that directly target cccDNA formation and maintenance. However, cccDNA has not previously been targeted in high throughput screens of small molecule libraries, which is mainly due to the lack of sensitive cccDNA detection methods and/or practical culture systems that produce cccDNA derived surrogate markers. By employing a HepDE19 cell-based cccDNA high throughput assay, in which the secretory HBeAg is cccDNA dependent and thus serves as a surrogate maker for cccDNA, we have screened an in-house 80,300 small molecule library, and primary hits were counterscreened in HepG2.2.15 cells, in which HBeAg is predominantly produced in a cccDNA-independent manner. The confirmed hits were subjected to secondary assay in HepDES19 cells.  Two structurally related disubstituted-sulfonamides (DSS) emerged as inhibitors of cccDNA production with EC50 at the micromolar level. These compounds were resynthesized and were confirmed to inhibit cccDNA formation in HepDES19 cells in a dose dependent manner. Further mechanism studies revealed that DSS compounds inhibit HBV cccDNA formation by reducing the level of deproteinized relaxed circular DNA (DP-rcDNA), which serves as the intermediate precursor for cccDNA. In addition, DSS compound did not inhibit HBV polymerase activity in the endogenous polymerase assay, suggesting DSS is not a polymerase inhibitor. Therefore, the DSS is considered as novel drug candidate for development into HBV therapeutics, and the structure-activity relationship (SAR) study for compound optimization is currently under investigation in collaborations with other scientists (Dr. Andrea Cuconati, Institute for Hepatitis and Virus Research; Dr. Michael Xu, Enatagen Therapeutics, Inc.).   
       
       
        3.  Innate control of HBV infection
        HBV is considered a stealth virus that replicates in the liver and escapes the recognition of the innate immune system, which is commonly utilized by the host cell to limit infection by many other viruses. In order to investigate whether the liver cell innate immune system could inhibit the HBV when the virus becomes visible to the host cells, we attempted to manually turn on the cell's innate defense signaling pathway by overexpression of the cellular pattern recognition receptor associated adaptors (IPS-1, TRIF, and MyD88) in HBV replicating cells, and surprisingly found that HBV replication was dramatically inhibited.  Mechanistic studies revealed that the primary anti-HBV effect is to noncytopathically reduce the HBV RNA stability and promote its degradation, and that the activation of NFkB pathway plays an important role in such observed inhibition. Further understanding the nature of these antiviral mechanisms would lead to the development of novel therapeutics that evoke the innate antiviral response to eliminate HBV infection.
       
        Currently, alpha interferon (IFN-α) is widely used in the clinical treatment of chronic hepatitis B. It is known that interferon has an antiviral effect via binding to cellular receptors and turning on the production of Interferon-Stimulated Genes (ISGs) following the activation of the Jak-Stat pathway. Therefore, our interest is to identify the ISGs with direct anti-HBV effect and understand their antiviral mechanisms. Among the hundreds of ISGs, 46 candidates were selected for our study according to their high expression in hepatocyte cells following interferon stimulation. By co-transfecting the 1.3mer HBV replication-competent construct and plasmid expressing individual ISG into hepatocyte-derived cell cultures (HepG2 and Huh7), 8 ISGs significantly inhibited viral DNA replication in both cell lines.  Further studies categorized the “hits” into two subsets depending on whether the primary viral target was RNA or DNA. The detailed antiviral mechanism of individual indentified ISG is currently under investigation in the laboratory.
       
        Selected Research Publications:
        [url=]"Indoleamine 2, 3-dioxygenase mediates the antiviral effect of gamma interferon against hepatitis B virus in human hepatocyte-derived cells"[/url]
        Mao R, Zhang J, Jiang D, Cai D, Levy J, Cuconati A, Block TM, Guo JT, and Guo H
        The Journal of Virology, 85: 1048-57, 2011.
        [url=]"Alkylated Porphyrins Have Broad Antiviral Activity against Hepadnaviruses, Flaviviruses, Filoviruses, and Arenaviruses"[/url]
        Guo H, Pan XB, Mao R, Zhang X, Wang L, Lu X, Chang J, Guo J, Passic S, Krebs FC, Wigdahl B, Warren TB,. Retterer CJ, Bavari S, Xu X, Cuconati A, and Block MT
        Antimicrobial Agents and Chemotherapy, 55: 478-86, 2011.
        [url=]"Production and Function of the Cytoplasmic Deproteinized Relaxed Circular DNA of Hepadnaviruses"[/url]
        Guo H, Mao R, Block TM, and Guo JT
        The Journal of Virology, 84: 387-396, 2010.
        "West Nile Virus Differentially Inhibits the Signal Transduction Pathways of Type I and III Interferons"
        Ma D, Jiang D, Qu X, Kennedy J, Guo H, Chang J, Gu B, Shi PY, Block TM, and Guo JT
        Antiviral Research, 83: 53-60, 2009.
        [url=]"Activation of Pattern Recognition Receptor-Mediated Innate Immunity Inhibits the Replication of Hepatitis B Virus in Human Hepatocyte-Derived Cells"[/url]
        Guo H, Jiang D, Ma D, Chang J, Dougherty AM, Cuconati A, Block TM, and Guo JT
        The Journal of Virology, 83: 847-858, 2009.
        [url=]"Liver Specific microRNA, miR-122, Enhances the Replication of Hepatitis C Virus in Non-hepatic Cells"[/url]
        Chang J, Guo JT, Jiang D, Guo H, Taylor JM, and Block TM
        The Journal of Virology, 82: 8215-8223, 2008.
        [url=]"Identification of Three Interferon-Inducible Cellular Enzymes That Inhibit the Replication of Hepatitis C Virus"[/url]
        Jiang D, Guo H, Xu C, Chang J, Gu B, Wang L, Block TM, and Guo JT
        The Journal of Virology, 82: 1665-1678, 2008.
        [url=]"Characterization of the Intracellular Deproteinized Relaxed Circular DNA of Hepatitis B Virus: An Intermediate of Covalently Closed Circular DNA Formation"[/url]
        Guo H, Jiang D, Zhou T, Cuconati A, Block TM, Guo JT
        The Journal of Virology, 81: 12472-12484, 2007.
        [url=]"Regulation of Hepatitis B Virus Replication by Phosphoatidylinositol 3-kinase-Akt Signal Transduction Pathway"[/url]
        Guo H, Zhou T, Jiang D, Cuconati A, Xia GH, Block TM, and Guo JT
        The Journal of Virology, 81: 10072-10080, 2007.
        [url=]"Molecular Virology of Hepatitis B Virus for Clinicians (Review)"[/url]
        Block TM, Guo H, and Guo JT
        Clinics in Liver Disease, 11: 685-706, 2007.
        [url=]"A Substituted Tetrahydro-Tetrazolo-Pyrimidine is a Specific and Novel Inhibitor of Hepatitis B Virus Surface Antigen Secretion"[/url]
        Dougherty AM*, Guo H*, Westby G, Liu Y, Simsek E, Guo JT, Mehta A, Norton P, Gu B, Block TM, and Cuconati A (*equal contribution)
        Antimicrobial Agents and Chemotherapy, 51: 4427-4437, 2007.
        [url=]"Alpha interferon-induced antiviral response non-cytolytically reduces replication defective adenovirus DNA in MDBK cells"[/url]
        Guo JT, Zhou T, Guo H, and Block TM
        Antiviral Research, 76: 232-240, 2007.
        [url=]"Hepatitis B Virus e Antigen Production is Dependent upon Covalently Closed Circular (ccc) DNA in HepAD38 Cell Cultures and May Serve as a cccDNA Surrogate in Antiviral Screening Assays"[/url]
        Zhou T*, Guo H*, Guo JT, and Block TM (*equal contribution)
        Antiviral Research, 72: 116-124, 2006.
        [url=]"The Insertion Domain of the Duck Hepatitis B Virus Core Protein Plays a Role in Nucleocapsid Assembly"[/url]
        Guo H, Aldrich CE, Saputelli JR, Xu C, and Mason WS
        Virology, 353: 443-450, 2006.
        [url=]"Identification and characterization of avihepadnaviruses isolated from exotic anseriformes maintained in captivity"[/url]
        Guo H, Mason WS, Aldrich CE, Saputelli JR, Miller D, Jilbert AR. and Newbold JE
        The Journal of Virology, 79: 2729-2742, 2005

hoimes

郭海涛，美国Drexel病毒学与生物技术研究学院助理教授
2 筛选和开发阻止cccdna生成的小颗粒混合物
近期没有治愈乙肝的有效方法。目前的治疗手段（干扰素或核苷类药物）不能清除已存在的cccdna池以及阻止野生株或耐药病毒生成新的CCCDNA，因而急需新的治疗药物靶向cccdna的生成及其存活。由于缺乏检测手段及实验系统，在筛选小颗粒混合物中没有把cccdna作为目标，借助于HepDE19-基于细胞的cccdna大量生产实验,两种二基替代磺酰胺类混合物DSS在半数有效浓度微分子水平上表现为cccdna生成的抑制剂,人工合成的这两种混合物被证实确能在HepDES19细胞中阻止CCCDNA的形成并成剂量依赖性.进一步的原理研究证实,DSS降低了脱蛋白自由环状DNA即DP-rcDNA的水平从而阻止了CCCDNA的生成,脱蛋白自由环状DNA是CCCDNA的先导介质,DSS不是聚合酶抑制剂(核苷).作为侯选开发治疗乙肝的药物,正在进行优化等研究工作.
第2段大致翻译稿.