IU Indianapolis ScholarWorks :: Browsing by Author "Guo, Haitao"

Browsing by Author "Guo, Haitao"

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Biogenesis and molecular characteristics of serum hepatitis B virus RNA
(Public Library of Science, 2020-10-20) Shen, Sheng; Xie, Zhanglian; Cai, Dawei; Yu, Xiaoyang; Zhang, Hu; Kim, Elena S.; Zhou, Bin; Hou, Jinlin; Zhang, Xiaoyong; Huang, Qi; Sun, Jian; Guo, Haitao; Medicine, School of Medicine
HBV is an enveloped DNA virus that replicates its DNA genome via reverse transcription of a pregenomic (pg) RNA intermediate in hepatocytes. Interestingly, HBV RNA can be detected in virus-like particles in chronic hepatitis B (CHB) patient serum and has been utilized as a biomarker for intrahepatic cccDNA activity in treated patients. However, the biogenesis and molecular characteristics of serum HBV RNA remain to be fully defined. In this study, we found that the encapsidated serum HBV RNA predominately consists of pgRNA, which are detergent- and ribonuclease-resistant. Through blocking HBV DNA replication without affecting pgRNA encapsidation by using the priming-defective HBV mutant Y63D or 3TC treatment, we demonstrated that the cell culture supernatant contains a large amount of pgRNA-containing nonenveloped capsids and a minor population of pgRNA-containing virions. The formation of pgRNA-virion requires both capsid assembly and viral envelope proteins, which can be inhibited by capsid assembly modulators and an envelope–knockout mutant, respectively. Furthermore, the pgRNA-virion utilizes the multivesicular body pathway for egress, in a similar way as DNA-virion morphogenesis. Northern blotting, RT-PCR, and 3’ RACE assays revealed that serum/supernatant HBV pgRNA are mainly spliced and devoid of the 3’-terminal sequences. Furthermore, pgRNA-virion collected from cells treated with a reversible HBV priming inhibitor L-FMAU was unable to establish infection in HepG2-NTCP cells. In summary, serum HBV RNA is secreted in noninfectious virion-like particle as spliced and poly(A)-free pgRNA. Our study will shed light on the molecular biology of serum HBV RNA in HBV life cycle, and aid the development of serum HBV RNA as a novel biomarker for CHB diagnosis and treatment prognosis.
Biological Functions of Intracellular Hepatitis B e Antigen
(2019-09) Mitra, Bidisha; Guo, Haitao; Androphy, Elliot J.; Kaplan, Mark; Yu, Andy; Lu, Tao
The function(s) of the intracellular form of HBeAg, previously reported as the preCore protein intermediate (p22) without the N-terminal signal peptide, remains elusive. Here, we propose to elucidate the translocation of p22 during its formation from endoplasmic reticulum (ER) to cytosol, how it differs from core in its inability to form a capsid and the biological functions of cytoplasmic and nuclear p22. Firstly, we have identified that a portion of p22, after the cleavage of its signal peptide in ER, is released back into the cytosol through an ERAD-independent mechanism, as neither wildtype nor dominant-negative p97 affected the ER-to-cytosol translocation of p22 or ER-Golgi secretion of HBeAg. Secondly, despite sharing the same sequence with core protein except for the extended 10 amino acid precore region at the N-terminus, we observed that p22 wildtype and C-7Q mutant are unable to form a capsid. Thirdly, we report that p22 but not the secreted HBeAg significantly reduced interferon stimulated response element (ISRE) activity and expression of interferon stimulated genes (ISGs) upon interferon-alpha (IFN- α) stimulation. Furthermore, in line with this, RNA-seq analysis of ISG induction profile from IFN-α treated patients showed that HBeAg(+) patients exhibited reduced and weak antiviral ISG upregulations compared to HBeAg(-) patients. Further, mechanistic study indicated that while p22 did not alter the total STAT1 or p-STAT1 levels in IFN-α treated cells, it blocked the nuclear translocation of p-STAT1 by interacting with karyopherin α1, indicating that the cytoplasmic p22 may impede JAK-STAT signaling to help the virus evade host innate immune response and cause resistance to IFN therapy in patients. Additionally, nuclear p22 and nuclear core were found to interact with the promoter regions (ISRE – containing) of ISGs, suggesting a new mechanism of inhibition of ISG expression upon stimulation. Finally, we found that the nuclear p22 can bind to cccDNA minichromosome and affects cccDNA maintenance and/or transcription. Thus, our results indicate that there is a novel ER sorting mechanism for the distribution of the intracellular and secretory HBeAg, and the intracellular HBeAg may contribute to HBV persistence by interfering with IFN-α elicited JAK-STAT signaling and regulating cccDNA metabolism.
The butterfly effect in viral infection: From a host DNA single nucleotide change to HBV episome steadiness
(Elsevier, 2019-02-10) Kim, Elena S.; Guo, Haitao; Microbiology and Immunology, School of Medicine
Cholesterol 25-hydroxylase suppresses SARS-CoV-2 replication by blocking membrane fusion
(NAS, 2020-12) Zang, Ruochen; Case, James Brett; Yutuc, Eylan; Ma, Xiucui; Shen, Sheng; Gomez Castro, Maria Florencia; Liu, Zhuoming; Zeng, Qiru; Zhao, Haiyan; Son, Juhee; Rothlauf, Paul W.; Kreutzberger, Alex J. B.; Hou, Gaopeng; Zhang, Hu; Bose, Sayantan; Wang, Xin; Vahey, Michael D.; Mani, Kartik; Griffiths, William J.; Kirchhausen, Tom; Fremont, Daved H.; Guo, Haitao; Diwan, Abhinav; Wang, Yuqin; Diamond, Michael S.; Whelan, Sean P. J.; Ding, Siyuan; Microbiology and Immunology, School of Medicine
Cholesterol 25-hydroxylase (CH25H) is an interferon (IFN)-stimulated gene that shows broad antiviral activities against a wide range of enveloped viruses. Here, using an IFN-stimulated gene screen against vesicular stomatitis virus (VSV)-SARS-CoV and VSV-SARS-CoV-2 chimeric viruses, we identified CH25H and its enzymatic product 25-hydroxycholesterol (25HC) as potent inhibitors of SARS-CoV-2 replication. Internalized 25HC accumulates in the late endosomes and potentially restricts SARS-CoV-2 spike protein catalyzed membrane fusion via blockade of cholesterol export. Our results highlight one of the possible antiviral mechanisms of 25HC and provide the molecular basis for its therapeutic development.
Cyclin-dependent Kinase Inhibitor 3 (CDKN3) Mediates the Antiviral Effect of Alpha Interferon against HBV Replication through Inhibition of Pregenomic RNA Encapsidation
(Office of the Vice Chancellor for Research, 2015-04-17) Cai, Dawei; Yan, Ran; Mao, Richeng; Block, Timothy; Cuconati, Andrea; Guo, Haitao
HBV capsid (core) protein is a phosphoprotein that contains three major serine phosphoacceptor sites in its C-terminal domain. In our effort to investigate the potential site-specific and combinational roles of serine phosphorylation in HBV DNA replication, we found that the primary effect of core phosphorylation on HBV replication was on the pregenomic (pg) RNA encapsidation step. Further mechanistic studies revealed that the core phosphorylation state-dependent interaction between viral core and polymerase (pol) plays a critical role in HBV pgRNA encapsidation. It has been well documented that IFN-α prevents HBV pgRNA encapsidation in cell cultures, however, the underlying molecular mechanisms remain unclear. We report herein that IFN-α-elicited inhibition of HBV pgRNA encapsidation is associated with a loss of core/pol interaction without affecting the steady state level of either protein, indicating that IFN-α inhibits HBV pgRNA encapsidation through blocking core phosphorylation-dependent interaction with pol. Since cyclin-dependent kinase 2 (CDK2) was identified as a kinase for HBV core, we next analyzed the inductivity of CDK2 and its associated regulatory factors in IFN-α-treated cells. We found that a cellular CDK2 inhibitor, cyclin-dependent Kinase Inhibitor 3 (CDKN3), was significantly upregulated by IFN-α. We further demonstrated that overexpression of CDKN3 inhibited core/pol interaction and subsequent pgRNA encapsidation and DNA replication, which is reminiscent of IFN-α’s anti-HBV activity. What’s more, knockdown of CDKN3 in HBV replicating cells completely attenuated IFN-α-mediated inhibition of HBV core/pol interaction and pgRNA encapsidation. Taken together, CDKN3 is a host restriction factor for HBV replication through inhibition of viral nucleocapsid formation, and it plays a dominant role in IFN-α-elicited antiviral activity against HBV in cell cultures. The detailed profile of CDKN3-mediated alteration of HBV core phosphorylation in the context of IFN-α treatment is currently under investigation.
Epigenetic Regulation of Hepatitis B Virus Covalently Closed Circular DNA: Implications for Epigenetic Therapy against Chronic Hepatitis B
(Wiley, 2017) Hong, Xupeng; Kim, Elena S.; Guo, Haitao; Department of Microbiology and Immunology, School of Medicine
Hepatitis B virus (HBV) infection represents a significant public health burden worldwide. Although current therapeutics manage to control the disease progression, lifelong treatment and surveillance are required because drug resistance develops during treatment and reactivations frequently occur following medication cessation. Thus, the occurrence of hepatocellular carcinoma (HCC) is decreased but not eliminated. One major reason for the treatment failure is the inability to eradicate or inactivate the viral covalently closed circular DNA (cccDNA) which is a stable episomal form of viral genome decorated with host histones and non-histone proteins. Accumulating evidence suggests that epigenetic modifications of cccDNA contribute to viral replication and the outcome of chronic HBV infection. Here, we summarize the progress on HBV epigenetics research and the therapeutic implications for chronic HBV infection by learning from the epigenetic therapies for cancer and other viral diseases, which may open a new venue to cure the chronic hepatitis B.
Establishment of an Inducible HBV Stable Cell Line that Expresses cccDNA-dependent Epitope-tagged HBeAg for Screening of cccDNA Modulators
(Elsevier, 2016-08) Cai, Dawei; Wang, Xiaohe; Yan, Ran; Mao, Richeng; Liu, Yuanjie; Ji, Changhua; Cuconati, Andrea; Guo, Haitao; Microbiology and Immunology, School of Medicine
Hepatitis B virus (HBV) covalently closed circular (ccc) DNA is essential to the virus life cycle, its elimination during chronic infection is considered critical to a durable therapy but has not been achieved by current antivirals. Despite being essential, cccDNA has not been the major target of high throughput screening (HTS), largely because of the limitations of current HBV tissue culture systems, including the impracticality of detecting cccDNA itself. In response to this need, we have previously developed a proof-of-concept HepDE19 cell line in which the production of wildtype e antigen (HBeAg) is dependent upon cccDNA. However, the existing assay system is not ideal for HTS because the HBeAg ELISA cross reacts with a viral HBeAg homologue, which is the core antigen (HBcAg) expressed largely in a cccDNA-independent fashion in HepDE19 cells. To further improve the assay specificity, we report herein a “second-generation” cccDNA reporter cell line, termed HepBHAe82. In the similar principle of HepDE19 line, an in-frame HA epitope tag was introduced into the precore domain of HBeAg open reading frame in the transgene of HepBHAe82 cells without disrupting any cis-element critical for HBV replication and HBeAg secretion. A chemiluminescence ELISA assay (CLIA) for the detection of HA-tagged HBeAg with HA antibody serving as capture antibody and HBeAb serving as detection antibody has been developed to eliminate the confounding signal from HBcAg. The miniaturized HepBHAe82 cell based assay system exhibits high level of cccDNA-dependent HA-HBeAg production and high specific readout signals with low background. We have also established a HepHA-HBe4 cell line expressing transgene-dependent HA-HBeAg as a counter screen to identify HBeAg inhibitors. The HepBHAe82 system is amenable to antiviral HTS development, and can be used to identify host factors that regulate cccDNA metabolism and transcription.
The Eukaryotic SMC5/6 Complex Represses the Replicative Program of High-Risk Human Papillomavirus
(2020-10) Gibson, Ryan Taylor; Androphy, Elliot; Guo, Haitao; Yu, Andy; Mayo, Lindsey
Human papillomaviruses (HPVs) are non-enveloped, circular double-stranded DNA viruses that infect basal keratinocytes of stratified squamous epithelia. High-risk HPV (HR-HPV) infection causes nearly all cervical cancers and an increasing number of head and neck cancers. While prophylactic vaccinations have reduced the incidence of HPV infection and attributable cancers, currently there is no cure for pre-existing HPV infection. As such, HPV remains a global health threat and a better understanding of HPV biology remains of significant medical importance for identification of novel therapeutic targets. The multi-subunit structural maintenance of chromosomes 5/6 complex (SMC5/6) is comprised of SMC5, SMC6 and NSE1-4. SMC5/6 is essential for homologous recombination DNA repair and reportedly functions as an antiviral factor during hepatitis B and herpes simplex-1 viral infections. Intriguingly, SMC5/6 has been found to associate with HR-HPV E2 proteins, which are multifunctional transcription factors essential to regulation of viral replication and transcription. The function of SMC5/6 associations with E2, as well as its role during HR-HPV infection remain unclear and we explored this question in the context of HR-HPV- 31. SMC6 interacted with HPV-31 E2 and co-immunoprecipitation of SMC6/E2 complexes required the E2 transactivation domain, inferring SMC6 association is limited to the full-length E2 isoform. Depletion of SMC6 and NSE3 increased HPV replication and transcription in keratinocytes stably maintaining episomal HPV-31, suggesting that the SMC5/6 complex represses these processes. Neither SMC6 nor NSE3 co-IP the viral E1 DNA helicase alone or E1/E2 complexes but the association of SMC6 with E2 was reduced in the presence of E1, indicating that SMC6 competes with E1 for E2 binding. This infers that SMC6 repression of the viral replicative program may involve inhibiting initiation of viral replication by disrupting E2 interactions with E1. Chromatin immunoprecipitation determined that SMC6 is present on episomal HPV-31 genomes, alluding to a possible role for SMC5/6 in modifying the chromatin state of viral DNA. Taken together, these findings describe a novel function for SMC5/6 as a repressor of the HPV-31 replicative program.
Functional association of cellular microtubules with viral capsid assembly supports efficient hepatitis B virus replication
(Nature Publishing group, 2017-09-06) Iwamoto, Masashi; Cai, Dawei; Sugiyama, Masaya; Suzuki, Ryosuke; Aizaki, Hideki; Ryo, Akihide; Ohtani, Naoko; Tanaka, Yasuhito; Mizokami, Masashi; Wakita, Takaji; Guo, Haitao; Watashi, Koichi; Microbiology and Immunology, School of Medicine
Viruses exploit host factors and environment for their efficient replication. The virus-host interaction mechanisms for achieving an optimal hepatitis B virus (HBV) replication have been largely unknown. Here, a single cell cloning revealed that HepAD38 cells, a widely-used HBV-inducible cell line, contain cell clones with diverse permissiveness to HBV replication. The HBV permissiveness was impaired upon treatment with microtubule inhibitor nocodazole, which was identified as an HBV replication inhibitor from a pharmacological screening. In the microtubule-disrupted cells, the efficiency of HBV capsid assembly was remarkably decreased without significant change in pre-assembly process. We further found that HBV core interacted with tubulin and co-localized with microtubule-like fibriforms, but this association was abrogated upon microtubule-disassembly agents, resulting in attenuation of capsid formation. Our data thus suggest a significant role of microtubules in the efficient capsid formation during HBV replication. In line with this, a highly HBV permissive cell clone of HepAD38 cells showed a prominent association of core-microtubule and thus a high capacity to support the capsid formation. These findings provide a new aspect of virus-cell interaction for rendering efficient HBV replication.
A global scientific strategy to cure hepatitis B
(Elsevier, 2019-07) Revill, Peter A.; Chisari, Francis V.; Block, Joan M.; Dandri, Maura; Gehring, Adam J.; Guo, Haitao; Hu, Jianming; Kramvis, Anna; Lampertico, Pietro; Janssen, Harry L. A.; Levrero, Massimo; Li, Wenhui; Liang, T. Jake; Lim, Seng-Gee; Lu, Fengmin; Penicaud, M. Capucine; Tavis, John E.; Thimme, Robert; Zoulim, Fabien; Microbiology and Immunology, School of Medicine
Chronic hepatitis B virus (HBV) infection is a global public health challenge on the same scale as tuberculosis, HIV, and malaria. The International Coalition to Eliminate HBV (ICE-HBV) is a coalition of experts dedicated to accelerating the discovery of a cure for chronic hepatitis B. Following extensive consultation with more than 50 scientists from across the globe, as well as key stakeholders including people affected by HBV, we have identified gaps in our current knowledge and new strategies and tools that are required to achieve HBV cure. We believe that research must focus on the discovery of interventional strategies that will permanently reduce the number of productively infected cells or permanently silence the covalently closed circular DNA in those cells, and that will stimulate HBV-specific host immune responses which mimic spontaneous resolution of HBV infection. There is also a pressing need for the establishment of repositories of standardised HBV reagents and protocols that can be accessed by all HBV researchers throughout the world. The HBV cure research agenda outlined in this position paper will contribute markedly to the goal of eliminating HBV infection worldwide.

Browsing by Author "Guo, Haitao"

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