Kinetic Analysis of Wild-Type and YMDD Mutant Hepatitis B Virus Polymerases and Effects of Deoxyribonucleotide Concentrations on Polymerase Activity

ABSTRACT Mutations in the YMDD motif of the hepatitis B virus (HBV) DNA polymerase result in reduced susceptibility of HBV to inhibition by lamivudine, at a cost in replication fitness. The mechanisms underlying the effects of YMDD mutations on replication fitness were investigated using both a cell-based viral replication system and an in vitro enzyme assay to examine wild-type (wt) and YMDD-mutant polymerases. We calculated the affinities of wt and YMDD-mutant polymerases for each natural deoxyribonucleoside triphosphate (dNTP) and determined the intracellular concentrations of each dNTP in HepG2 cells under conditions that support HBV replication. In addition, inhibition constants for lamivudine triphosphate were determined for wt and YMDD-mutant polymerases. Relative to wt HBV polymerase, each of the YMDD-mutant polymerases showed increased apparent Km values for the natural dNTP substrates, indicating decreased affinities for these substrates, as well as increased Ki values for lamivudine triphosphate, indicating decreased affinity for the drug. The effect of the differences in apparent Km values between YMDD-mutant polymerase and wt HBV polymerase could be masked by high levels of dNTP substrates (>20 μM). However, assays using dNTP concentrations equivalent to those measured in HepG2 cells under physiological conditions showed decreased enzymatic activity of YMDD-mutant polymerases relative to wt polymerase. Therefore, the decrease in replication fitness of YMDD-mutant HBV strains results from the lower affinities (increased Km values) of the YMDD-mutant polymerases for the natural dNTP substrates and physiological intracellular concentrations of dNTPs that are limiting for the replication of YMDD-mutant HBV strains.

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Pharmacology of β-l-Thymidine and β-l-2′-Deoxycytidine in HepG2 Cells and Primary Human Hepatocytes: Relevance to Chemotherapeutic Efficacy against Hepatitis B Virus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.6.1728-1733.2002 ◽

2002 ◽

Vol 46 (6) ◽

pp. 1728-1733 ◽

Cited By ~ 41

Author(s):

B. Hernandez-Santiago ◽

L. Placidi ◽

E. Cretton-Scott ◽

A. Faraj ◽

E. G. Bridges ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Hepg2 Cells ◽

Hepatitis Virus ◽

Parent Compound ◽

Human Hepatocytes ◽

Primary Human Hepatocytes ◽

B Virus ◽

Intracellular Concentrations

ABSTRACT β-l-Thymidine (l-dT) and β-l-2′-deoxycytidine (l-dC) are potent and highly specific inhibitors of hepatitis B virus (HBV) replication both in vivo and in vitro (50% effective concentrations, 0.19 to 0.24 μM in 2.2.15 cells). The intracellular metabolisms of l-dT and l-dC were investigated in HepG2 cells and primary cultured human hepatocytes. l-dT and l-dC were extensively phosphorylated in both cell types, with the 5′-triphosphate derivative being the predominant metabolite. In HepG2 cells, the 5′-triphosphate levels were 27.7 ± 12.1 and 72.4 ± 1.8 pmol/106 cells for l-dT and l-dC, respectively. In primary human hepatocytes, the 5′-triphosphate levels were 16.5 ± 9.8 and 90.1 ± 36.4 pmol/106 cells for l-dT and l-dC, respectively. Furthermore, a choline derivative of l-dCDP was detected at concentrations of 15.8 ± 1.8 and 25.6 ± 0.1 pmol/106 cells in human hepatocytes and HepG2 cells, respectively. In HepG2 cells exposed to l-dC, the 5′-monophosphate and 5′-triphosphate derivatives of β-l-2′-deoxyuridine (l-dUMP and l-dUTP, respectively) were also observed, reaching intracellular concentrations of 6.7 ± 0.4 and 18.2 ± 1.0 pmol/106 cells, respectively. In human hepatocytes, l-dUMP and l-dUTP were detected at concentrations of 5.7 ± 2.4 and 43.5 ± 26.8 pmol/106 cells, respectively. It is likely that deamination of l-dCMP by deoxycytidylate deaminase leads to the formation of l-dUMP, as the parent compound, l-dC, was not a substrate for deoxycytidine deaminase. The intracellular half-lives of l-dTTP, l-dCTP, and l-dUTP were at least 15 h, with intracellular concentrations of each metabolite remaining above their respective 50% inhibitory concentrations for the woodchuck hepatitis virus DNA polymerase for as long as 24 h after removal of the drug from cell cultures. Exposure of HepG2 cells to l-dT in combination with l-dC led to concentrations of the activated metabolites similar to those achieved with either agent alone. These results suggest that the potent anti-HBV activities of l-dT and l-dC are associated with their extensive phosphorylation.

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IL-33 Inhibits Hepatitis B Virus through Its Receptor ST2 in Hydrodynamic HBV Mouse Model

Mediators of Inflammation ◽

10.1155/2020/1403163 ◽

2020 ◽

Vol 2020 ◽

pp. 1-9

Author(s):

Xiuzhu Gao ◽

Xiumei Chi ◽

Xiaomei Wang ◽

Ruihong Wu ◽

Hongqin Xu ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Mouse Model ◽

Hepg2 Cells ◽

Hbv Dna ◽

Dependent Manner ◽

Wild Type ◽

Antiviral Effects ◽

B Virus

Interleukin-33 has been demonstrated to be associated with liver damage. However, its potential value in hepatitis B virus (HBV) infection remains unknown. This study was designed to investigate the role of IL-33 in hydrodynamic HBV mouse model. Different doses of IL-33 were used to treat HBV wild-type, ST2 knockout, CD8+ T depletion, NK depletion C57BL/6 mice and C.B-17 SCID and nod SCID mouse, respectively. The concentrations of HBV DNA, HBsAg, HBeAg, and molecules related to liver function were detected in the collected serum at different time points from model mice. Intrahepatic HBcAg was visualized by immunohistochemical staining of liver tissues. In vitro, hepG2 cells were transfected with pAAV-HBV 1.2, then treated with IL-33. The results showed that IL-33 significantly reduced HBV DNA and HBsAg in a dose-dependent manner in HBV wild-type mice. However, in the IL-33 specific receptor ST2 knockout mice, their antiviral effects could not be exerted. Through immunodeficient animal models and in vivo immune cell depletion mouse model, we found that IL-33 could not play antiviral effects without NK cells. Moreover, IL-33 could reduce the levels of HBsAg and HBeAg in the supernatant of HBV-transfected hepG2 cells in vitro. Our study revealed that IL-33 could inhibit HBV through ST2 receptor in the HBV mouse model, and this effect can be impaired without NK cell. Additionally, IL-33 had the direct anti-HBV effect in vitro, indicating that IL-33 could be a potent inducer of HBV clearance and a promising drug candidate.

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Suppression of hepatitis B virus antigen production and replication by wild-type HBV dependently replicating HBV shRNA vectors in vitro and in vivo

Antiviral Research ◽

10.1016/j.antiviral.2016.08.007 ◽

2016 ◽

Vol 134 ◽

pp. 117-129 ◽

Cited By ~ 7

Author(s):

Baosheng Li ◽

Shuo Sun ◽

Minran Li ◽

Xin Cheng ◽

Haijun Li ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Virus Antigen ◽

Wild Type ◽

Antigen Production ◽

B Virus

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Efficacies of Entecavir against Lamivudine-Resistant Hepatitis B Virus Replication and Recombinant Polymerases In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.8.2525-2532.2002 ◽

2002 ◽

Vol 46 (8) ◽

pp. 2525-2532 ◽

Cited By ~ 145

Author(s):

S. Levine ◽

D. Hernandez ◽

G. Yamanaka ◽

S. Zhang ◽

R. Rose ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Cross Resistance ◽

Wild Type ◽

Resistance Mutations ◽

Hbv Replication ◽

B Virus ◽

Inhibition Studies

ABSTRACT Entecavir (ETV) is a potent and selective inhibitor of hepatitis B virus (HBV) replication in vitro and in vivo that is currently in clinical trials for the treatment of chronic HBV infections. A major limitation of the current HBV antiviral therapy, lamivudine (3TC), is the emergence of drug-resistant HBV in a majority of treated patients due to specific mutations in the nucleotide binding site of HBV DNA polymerase (HBV Pol). To determine the effects of 3TC resistance mutations on inhibition by ETV triphosphate (ETV-TP), a series of in vitro studies were performed. The inhibition of wild-type and 3TC-resistant HBV Pol by ETV-TP was measured using recombinant HBV nucleocapsids, and compared to that of 3TC-TP. These enzyme inhibition studies demonstrated that ETV-TP is a highly potent inhibitor of wild-type HBV Pol and is 100- to 300-fold more potent than 3TC-TP against 3TC-resistant HBV Pol. Cell culture assays were used to gauge the potential for antiviral cross-resistance of 3TC-resistant mutants to ETV. Results demonstrated that ETV inhibited the replication of 3TC-resistant HBV, but 20- to 30-fold higher concentrations were required. To gain further perspective regarding the potential therapeutic use of ETV, its phosphorylation was examined in hepatoma cells treated with extracellular concentrations representative of drug levels in plasma in ETV-treated patients. At these concentrations, intracellular ETV-TP accumulated to levels expected to inhibit the enzyme activity of both wild-type and 3TC-resistant HBV Pol. These findings are predictive of potent antiviral activity of ETV against both wild-type and 3TC-resistant HBV.

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IL-17 Activates the IL-6/STAT3 Signal Pathway in the Proliferation of Hepatitis B Virus-Related Hepatocellular Carcinoma

Cellular Physiology and Biochemistry ◽

10.1159/000484390 ◽

2017 ◽

Vol 43 (6) ◽

pp. 2379-2390 ◽

Cited By ~ 14

Author(s):

Zongqiang Hu ◽

Ding Luo ◽

Dongdong Wang ◽

Linjie Ma ◽

Yingpeng Zhao ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Knockout Mice ◽

Hepg2 Cells ◽

Wild Type ◽

B Virus ◽

And Migration ◽

Proliferation And Migration

Background/Aims: We performed this study to determine the role of IL-17 in the immune microenvironment of hepatitis B virus- (HBV-) related hepatocellular carcinoma (HCC). Methods: HepG2 cells were treated with IL-17, STAT3 inhibitor S31-201 or IL-6 neutralizing monoclonal antibody (IL-6 mAb). Cell proliferation and migration were compared using the Cell Counting kit-8 (CCK-8) and Transwell assays, respectively. Real-time quantitative PCR (RT-qPCR), Western Blot, ELISA, immunofluorescence and histological staining were used for determining the expression levels of IL-17, IL-6, MCP-1, CCL5, VEGF, STAT3 and p-STAT3. HCC xenograft models were constructed in wild type and IL-17 knockout mice to clarify the effects of IL-17 on HCC in vivo. Results: Exogenous IL-17 enhanced the proliferation and migration of HepG2 cells, and it activated the phosphorylation of STAT3. RT-qPCR and ELISA showed that IL-17 promoted the expression of IL-6. The CCK-8 and Transwell assays showed that S31-201 or IL-6 mAb remarkably reversed the promotion effects of proliferation and migration by exogenous IL-17 in HepG2 cells. Additionally, IL-6 could promote the phosphorylation of STAT3, while IL-6 mAb acted as an inhibitor, and exogenous IL-17 could neutralize the inhibitory effects of IL-6 mAb. In vivo, compared to the wild type mice, the tumor volume, weight, density and size were decreased in IL-17 knockout mice. Additionally, the expression levels of p-STAT3, IL-6, MCP-1, CCL5 and VEGF decreased in IL-17 knockout mice. Conclusions: IL-17 can enhance the proliferation of HepG2 cells in vitro and in vivo via activating the IL-6/STAT3 pathway. Therefore, the IL-17/IL-6/STAT3 signaling pathway is a potential therapeutic target for HBV-related HCC.

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Identification of Disubstituted Sulfonamide Compounds as Specific Inhibitors of Hepatitis B Virus Covalently Closed Circular DNA Formation

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00473-12 ◽

2012 ◽

Vol 56 (8) ◽

pp. 4277-4288 ◽

Cited By ~ 140

Author(s):

Dawei Cai ◽

Courtney Mills ◽

Wenquan Yu ◽

Ran Yan ◽

Carol E. Aldrich ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Polymerase Activity ◽

Circular Dna ◽

Drug Candidates ◽

Covalently Closed Circular Dna ◽

Hbv Cccdna ◽

B Virus

ABSTRACTHepatitis B virus (HBV) covalently closed circular DNA (cccDNA) plays a central role in viral infection and persistence and is the basis for viral rebound after the cessation of therapy, as well as the elusiveness of a cure even after extended treatment. 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 HBV e antigen 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 low micromolar 50% effective concentrations (EC50s) 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 thein vitroendogenous 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 interfere primarily with rcDNA conversion into cccDNA. In addition, we demonstrated that CCC-0975 was able to reduce cccDNA biosynthesis in duck HBV-infected primary duck hepatocytes. This is the first attempt, to our knowledge, to identify 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.

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Phenylpropenamide Derivatives AT-61 and AT-130 Inhibit Replication of Wild-Type and Lamivudine-Resistant Strains of Hepatitis B Virus In Vitro

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.9.3057-3060.2002 ◽

2002 ◽

Vol 46 (9) ◽

pp. 3057-3060 ◽

Cited By ~ 108

Author(s):

William E. Delaney ◽

Ros Edwards ◽

Danni Colledge ◽

Tim Shaw ◽

Phil Furman ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Nucleoside Analogue ◽

Wild Type ◽

Hbv Replication ◽

B Virus ◽

Resistant Strains

ABSTRACT The phenylpropenamide derivatives AT-61 and AT-130 are nonnucleoside analogue inhibitors of hepatitis B virus (HBV) replication. They inhibited the replication of wild-type HBV with 50% inhibitory concentrations of 21.2 ± 9.5 and 2.40 ± 0.92 μM, respectively, compared to 0.064 ± 0.020 μM lamivudine. There were no significant differences in sensitivity between wild-type and nucleoside analogue-resistant (rtL180M, rtM204I, and rtL180M + rtM204V) HBV.

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Two-Year Assessment of Entecavir Resistance in Lamivudine-Refractory Hepatitis B Virus Patients Reveals Different Clinical Outcomes Depending on the Resistance Substitutions Present

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00833-06 ◽

2006 ◽

Vol 51 (3) ◽

pp. 902-911 ◽

Cited By ~ 166

Author(s):

Daniel J. Tenney ◽

Ronald E. Rose ◽

Carl J. Baldick ◽

Steven M. Levine ◽

Kevin A. Pokornowski ◽

...

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Reverse Transcriptase ◽

Clinical Outcomes ◽

Chronic Infection ◽

Hbv Dna ◽

Wild Type ◽

Pcr Assay ◽

B Virus

ABSTRACT Entecavir (ETV) is a deoxyguanosine analog approved for use for the treatment of chronic infection with wild-type and lamivudine-resistant (LVDr) hepatitis B virus (HBV). In LVD-refractory patients, 1.0 mg ETV suppressed HBV DNA levels to below the level of detection by PCR (<300 copies/ml) in 21% and 34% of patients by Weeks 48 and 96, respectively. Prior studies showed that virologic rebound due to ETV resistance (ETVr) required preexisting LVDr HBV reverse transcriptase substitutions M204V and L180M plus additional changes at T184, S202, or M250. To monitor for resistance, available isolates from 192 ETV-treated patients were sequenced, with phenotyping performed for all isolates with all emerging substitutions, in addition to isolates from all patients experiencing virologic rebounds. The T184, S202, or M250 substitution was found in LVDr HBV at baseline in 6% of patients and emerged in isolates from another 11/187 (6%) and 12/151 (8%) ETV-treated patients by Weeks 48 and 96, respectively. However, use of a more sensitive PCR assay detected many of the emerging changes at baseline, suggesting that they originated during LVD therapy. Only a subset of the changes in ETVr isolates altered their susceptibilities, and virtually all isolates were significantly replication impaired in vitro. Consequently, only 2/187 (1%) patients experienced ETVr rebounds in year 1, with an additional 14/151 (9%) patients experiencing ETVr rebounds in year 2. Isolates from all 16 patients with rebounds were LVDr and harbored the T184 and/or S202 change. Seventeen other novel substitutions emerged during ETV therapy, but none reduced the susceptibility to ETV or resulted in a rebound. In summary, ETV was effective in LVD-refractory patients, with resistant sequences arising from a subset of patients harboring preexisting LVDr/ETVr variants and with approximately half of the patients experiencing a virologic rebound.

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N-Linked Glycosylation Is Not Essential for Sodium Taurocholate Cotransporting Polypeptide To Mediate Hepatitis B Virus Infection In Vitro

Journal of Virology ◽

10.1128/jvi.00732-18 ◽

2018 ◽

Vol 92 (15) ◽

Cited By ~ 5

Author(s):

Jiwon Lee ◽

Li Zong ◽

Alexander Krotow ◽

Yanli Qin ◽

Lucy Jia ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Surface Antigen ◽

Hepg2 Cells ◽

Hepatitis B Surface Antigen ◽

Sodium Taurocholate ◽

Wild Type ◽

Heparg Cells ◽

B Virus

ABSTRACT Sodium taurocholate cotransporting polypeptide (NTCP) has been identified as a hepatitis B virus (HBV) receptor, and its overexpression in HepG2 cell lines leads to efficient secretion of hepatitis B e antigen (HBeAg) following challenge with a large dose of cell culture-derived HBV (cHBV) particles. However, NTCP-reconstituted HepG2 cells are inefficiently infected by patient serum-derived HBV (sHBV) and release very little hepatitis B surface antigen (HBsAg) following cHBV infection, unlike differentiated HepaRG cells, which are naturally susceptible to both cHBV and sHBV particles. Here, we investigated whether NTCP could explain the different behaviors of the two cell types. Endogenous NTCP protein from differentiated HepaRG cells was unglycosylated despite wild-type coding sequence. HepaRG cells stably transfected with an epitope-tagged NTCP expression construct displayed higher sHBV but not cHBV susceptibility than cells transfected with the null mutant. Tagged NTCP introduced to both HepG2 and HepaRG cells was glycosylated, with N5 and N11 being sites of N-linked glycosylation. Mutating N5, N11, or both did not alter cell surface availability of NTCP or its subcellular localization, with both the singly glycosylated and nonglycosylated forms still capable of mediating cHBV infection in HepG2 cells. In conclusion, nonglycosylated NTCP is expressed by differentiated HepaRG cells and capable of mediating cHBV infection in HepG2 cells, but it cannot explain differential susceptibility of HepaRG and HepG2/NTCP cells to cHBV versus sHBV infection and different HBsAg/HBeAg ratios following cHBV infection. The responsible host factor(s) remains to be identified. IMPORTANCE HBV can infect differentiated HepaRG cells and also HepG2 cells overexpressing NTCP, the currently accepted HBV receptor. However, HepG2/NTCP cells remain poorly susceptible to patient serum-derived HBV particles and release very little hepatitis B surface antigen following infection by cell culture-derived HBV. We found differentiated HepaRG cells expressed nonglycosylated NTCP despite a wild-type coding sequence. NTCP introduced to HepG2 cells was glycosylated at two N-linked glycosylation sites, but mutating either or both sites failed to prevent infection by cell culture-derived HBV or to confer susceptibility to serum-derived HBV. Overexpressing NTCP in HepRG cells did not increase infection by cell culture-derived HBV or distort the ratio between the two viral antigens. These findings suggest that host factors unique to HepaRG cells are required for efficient infection by serum-derived HBV, and factors other than NTCP contribute to balanced viral antigen production following infection by cell culture-derived HBV.

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