Browsing by Author "Mao, Richeng"

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    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.
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    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.
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    Hepatitis B e antigen induces the expansion of monocytic myeloid-derived suppressor cells to dampen T-cell function in chronic hepatitis B virus infection
    (PLOS, 2019-04-18) Yang, Feifei; Yu, Xueping; Zhou, Chenliang; Mao, Richeng; Zhu, Mengqi; Zhu, Haoxiang; Ma, Zhenxuan; Mitra, Bidisha; Zhao, Gan; Huang, Yuxian; Guo, Haitao; Wang, Bin; Zhang, Jiming; Microbiology and Immunology, School of Medicine
    Chronic hepatitis B virus (HBV) infection is associated with functionally impaired virus-specific T cell responses. Although the myeloid-derived suppressor cells (MDSCs) are known to play a critical role in impairing antiviral T cell responses, viral factors responsible for the expansion of MDSCs in chronic hepatitis B (CHB) remain obscure. In order to elucidate the mechanism of monocytic MDSCs (mMDSCs) expansion and T cell function suppression during persistent HBV infection, we analyzed the circulation frequency of mMDSCs in 164 CHB patients and 70 healthy donors, and found that the proportion of mMDSCs in HBeAg (+) CHB patients was significantly increased compared to that in HBeAg (-) patients, which positively correlated with the level of HBeAg. Furthermore, exposure of peripheral blood mononuclear cells (PBMCs) isolated from healthy donors to HBeAg led to mMDSCs expansion and significant upregulation of IL-1β, IL-6 and indoleamine-2, 3-dioxygenase (IDO), and depletion of the cytokines abrogated HBeAg-induced mMDSCs expansion. Moreover, HBeAg-induced mMDSCs suppressed the autologous T-cell proliferation in vitro, and the purified mMDSCs from HBeAg (+) subjects markedly reduced the proliferation of CD4+ and CD8+ T cells and IFN-γ production, which could be efficiently restored by inhibiting IDO. In summary, HBeAg-induced mMDSCs expansion impairs T cell function through IDO pathway and favors the establishment of a persistent HBV infection, suggesting a mechanism behind the development of HBeAg-induced immune tolerance.
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    Hepatitis B Virus Precore Protein p22 Inhibits Alpha Interferon Signaling by Blocking STAT Nuclear Translocation
    (American Society for Microbiology, 2019-07-01) Mitra, Bidisha; Wang, Jinyu; Kim, Elena S.; Mao, Richeng; Dong, Dong; Liu, Yuanjie; Zhang, Jiming; Guo, Haitao; Microbiology and Immunology, School of Medicine
    Antagonism of host immune defenses against hepatitis B virus (HBV) infection by the viral proteins is speculated to cause HBV persistence and the development of chronic hepatitis. The circulating hepatitis B e antigen (HBeAg, p17) is known to manipulate host immune responses to assist in the establishment of persistent viral infection, and HBeAg-positive (HBeAg+) patients respond less effectively to IFN-α therapy than do HBeAg-negative (HBeAg−) patients in clinical practice. However, 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 report that the cytosolic p22 protein, but not the secreted HBeAg, significantly reduces interferon-stimulated response element (ISRE) activity and the expression of interferon-stimulated genes (ISGs) upon alpha interferon (IFN-α) stimulation in cell cultures. In line with this, HBeAg+ patients exhibit weaker induction of ISGs in their livers than do HBeAg− patients upon IFN-α therapy. Mechanistically, while p22 does not alter the total STAT1 or pSTAT1 levels in cells treated with IFN-α, it blocks the nuclear translocation of pSTAT1 by interacting with the nuclear transport factor karyopherin α1 through its C-terminal arginine-rich domain. In summary, our study suggests that HBV precore protein, specifically the p22 form, impedes JAK-STAT signaling to help the virus evade the host innate immune response and, thus, causes resistance to IFN therapy. IMPORTANCE Chronic hepatitis B virus (HBV) infection continues to be a major global health concern, and patients who fail to mount an efficient immune response to clear the virus will develop a life-long chronic infection that can progress to chronic active hepatitis, cirrhosis, and primary hepatocellular carcinoma. There is no definite cure for chronic hepatitis B, and alpha interferon (IFN-α) is the only available immunomodulatory drug, to which only a minority of chronic patients are responsive, with hepatitis B e antigen (HBeAg)-negative patients responding better than HBeAg-positive patients. We herein report that the intracellular HBeAg, also known as precore or p22, inhibits the antiviral signaling of IFN-α, which sheds light on the enigmatic function of precore protein in shaping HBV chronicity and provides a perspective toward areas that need to be further studied to make the current therapy better until a cure is achieved.
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    Interferon-inducible ribonuclease ISG20 inhibits hepatitis B virus replication through directly binding to the epsilon stem-loop structure of viral RNA
    (PLOS, 2017-04-11) Liu, Yuanjie; Nie, Hui; Mao, Richeng; Mitra, Bidisha; Cai, Dawei; Yan, Ran; Guo, Ju-Tao; Block, Timothy M.; Mechti, Nadir; Guo, Haitao; Microbiology and Immunology, School of Medicine
    Hepatitis B virus (HBV) replicates its DNA genome through reverse transcription of a viral RNA pregenome. We report herein that the interferon (IFN) stimulated exoribonuclease gene of 20 KD (ISG20) inhibits HBV replication through degradation of HBV RNA. ISG20 expression was observed at basal level and was highly upregulated upon IFN treatment in hepatocytes, and knock down of ISG20 resulted in elevation of HBV replication and attenuation of IFN-mediated antiviral effect. The sequence element conferring the susceptibility of HBV RNA to ISG20-mediated RNA degradation was mapped at the HBV RNA terminal redundant region containing epsilon (ε) stem-loop. Furthermore, ISG20-induced HBV RNA degradation relies on its ribonuclease activity, as the enzymatic inactive form ISG20D94G was unable to promote HBV RNA decay. Interestingly, ISG20D94G retained antiviral activity against HBV DNA replication by preventing pgRNA encapsidation, resulting from a consequence of ISG20-ε interaction. This interaction was further characterized by in vitro electrophoretic mobility shift assay (EMSA) and ISG20 was able to bind HBV ε directly in absence of any other cellular proteins, indicating a direct ε RNA binding capability of ISG20; however, cofactor(s) may be required for ISG20 to efficiently degrade ε. In addition, the lower stem portion of ε is the major ISG20 binding site, and the removal of 4 base pairs from the bottom portion of ε abrogated the sensitivity of HBV RNA to ISG20, suggesting that the specificity of ISG20-ε interaction relies on both RNA structure and sequence. Furthermore, the C-terminal Exonuclease III (ExoIII) domain of ISG20 was determined to be responsible for interacting with ε, as the deletion of ExoIII abolished in vitro ISG20-ε binding and intracellular HBV RNA degradation. Taken together, our study sheds light on the underlying mechanisms of IFN-mediated HBV inhibition and the antiviral mechanism of ISG20 in general.
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    RNA Helicase DDX17 Inhibits Hepatitis B Virus Replication by Blocking Viral Pregenomic RNA Encapsidation
    (American Society for Microbiology, 2021) Mao, Richeng; Dong, Minhui; Shen, Zhongliang; Zhang, Hu; Liu, Yuanjie; Cai, Dawei; Mitra, Bidisha; Zhang, Jiming; Guo, Haitao; Microbiology and Immunology, School of Medicine
    DDX17 is a member of the DEAD-box helicase family proteins involved in cellular RNA folding, splicing, and translation. It has been reported that DDX17 serves as a cofactor of host zinc finger antiviral protein (ZAP)-mediated retroviral RNA degradation and exerts direct antiviral function against Raft Valley fever virus through binding to specific stem-loop structures of viral RNA. Intriguingly, we have previously shown that ZAP inhibits hepatitis B virus (HBV) replication through promoting viral RNA decay, and the ZAP-responsive element (ZRE) of HBV pregenomic RNA (pgRNA) contains a stem-loop structure, specifically epsilon, which serves as the packaging signal for pgRNA encapsidation. In this study, we demonstrated that the endogenous DDX17 is constitutively expressed in human hepatocyte-derived cells but dispensable for ZAP-mediated HBV RNA degradation. However, DDX17 was found to inhibit HBV replication primarily by reducing the level of cytoplasmic encapsidated pgRNA in a helicase-dependent manner. Immunofluorescence assay revealed that DDX17 could gain access to cytoplasm from nucleus in the presence of HBV RNA. In addition, RNA immunoprecipitation and electrophoretic mobility shift assays demonstrated that the enzymatically active DDX17 competes with HBV polymerase to bind to pgRNA at the 5' epsilon motif. In summary, our study suggests that DDX17 serves as an intrinsic host restriction factor against HBV through interfering with pgRNA encapsidation. IMPORTANCE Hepatitis B virus (HBV) chronic infection, a long-studied but yet incurable disease, remains a major public health concern worldwide. Given that HBV replication cycle highly depends on host factors, deepening our understanding of the host-virus interaction is thus of great significance in the journey of finding a cure. In eukaryotic cells, RNA helicases of the DEAD box family are highly conserved enzymes involved in diverse processes of cellular RNA metabolism. Emerging data have shown that DDX17, a typical member of the DEAD box family, functions as an antiviral factor through interacting with viral RNA. In this study, we, for the first time, demonstrate that DDX17 inhibits HBV through blocking the formation of viral replication complex, which not only broadens the antiviral spectrum of DDX17 but also provides new insight into the molecular mechanism of DDX17-mediated virus-host interaction.