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, the undisputed “holy grail” of chronic hepatitis B therapy is the elimination of viral cccDNA, which is not satisfactorily achieved by the available medications. 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 HBeAg 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 EC50 values at low micromolar levels 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 primarily interfere with rcDNA conversion into cccDNA. In addition, we demonstrated that CCC-0975 was able to reduce cccDNA biosynthesis in duck hepatitis B virus infected primary duck hepatocytes. To our knowledge, this effort is the first attempt at identifying 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. The structure-activity relationship (SAR) study for compound optimization is currently under investigation in collaborations with Drs. Andrea Cuconati (Institute for Hepatitis and Virus Research) and 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:
"Identification of the Disubstituted Sulfonamides as Specific Inhibitors of Hepatitis B Virus Covalently Closed Circular DNA Formation"
Cai D, Mills C, Yu W, Yan R, Aldrich C, Saputelli J, Mason WS, Xu X, Guo JT, Block TM, Cuconati A, and H Guo
Antimicrob. Agents Chemother., 56: 4277-4288, 2012.
"Characterization of the Host Factors Required for Hepadnavirus Covalently Closed Circular (ccc) DNA Formation"
Guo H, Xu C, Zhou T, Block TM, and JT Guo
PLoS One 7: e43270, 2012.
"Indoleamine 2, 3-dioxygenase mediates the antiviral effect of gamma interferon against hepatitis B virus in human hepatocyte-derived cells"
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.
"Alkylated Porphyrins Have Broad Antiviral Activity against Hepadnaviruses, Flaviviruses, Filoviruses, and Arenaviruses"
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.
"Production and Function of the Cytoplasmic Deproteinized Relaxed Circular DNA of Hepadnaviruses"
Guo H, Mao R, Block TM, and Guo JT
The Journal of Virology, 84: 387-396, 2010.
"Activation of Pattern Recognition Receptor-Mediated Innate Immunity Inhibits the Replication of Hepatitis B Virus in Human Hepatocyte-Derived Cells"
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.
"Liver Specific microRNA, miR-122, Enhances the Replication of Hepatitis C Virus in Non-hepatic Cells"
Chang J, Guo JT, Jiang D, Guo H, Taylor JM, and Block TM
The Journal of Virology, 82: 8215-8223, 2008.
"Characterization of the Intracellular Deproteinized Relaxed Circular DNA of Hepatitis B Virus: An Intermediate of Covalently Closed Circular DNA Formation"
Guo H, Jiang D, Zhou T, Cuconati A, Block TM, Guo JT
The Journal of Virology, 81: 12472-12484, 2007.
"Regulation of Hepatitis B Virus Replication by Phosphoatidylinositol 3-kinase-Akt Signal Transduction Pathway"
Guo H, Zhou T, Jiang D, Cuconati A, Xia GH, Block TM, and Guo JT
The Journal of Virology, 81: 10072-10080, 2007.
"Molecular Virology of Hepatitis B Virus for Clinicians (Review)"
Block TM, Guo H, and Guo JT
Clinics in Liver Disease, 11: 685-706, 2007.
"A Substituted Tetrahydro-Tetrazolo-Pyrimidine is a Specific and Novel Inhibitor of Hepatitis B Virus Surface Antigen Secretion"
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.
"Alpha interferon-induced antiviral response non-cytolytically reduces replication defective adenovirus DNA in MDBK cells"
Guo JT, Zhou T, Guo H, and Block TM
Antiviral Research, 76: 232-240, 2007.
"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"
Zhou T*, Guo H*, Guo JT, and Block TM (*equal contribution)
Antiviral Research, 72: 116-124, 2006.
"The Insertion Domain of the Duck Hepatitis B Virus Core Protein Plays a Role in Nucleocapsid Assembly"
Guo H, Aldrich CE, Saputelli JR, Xu C, and Mason WS
Virology, 353: 443-450, 2006.
"Identification and characterization of avihepadnaviruses isolated from exotic anseriformes maintained in captivity"
Guo H, Mason WS, Aldrich CE, Saputelli JR, Miller D, Jilbert AR. and Newbold JE
The Journal of Virology, 79: 2729-2742, 2005