Substitution of proline 306 in the reverse transcriptase domain of hepatitis B virus regulates replication

The proline residue at position 306 in hepatitis B virus (HBV) reverse transcriptase (rtP306) has been suggested to constrain the conformation of the α-helices in the thumb subdomain that interacts with the viral DNA template-primer. To study the impact of residue rt306 in HBV replication further, 11 site-directed mutants were constructed that substituted rtP306 with different amino acids. The replicative competencies of these mutants were assayed by HepG2 cell transfection and real-time PCR. When rtP306 was substituted with glycine or threonine, the replication competency of these mutants was drastically reduced to 1·96 and 4·51 % of the wild-type HBV level, respectively. When rtP306 was substituted with glutamic acid, the replicative competency of the mutant increased up to 9·4-fold compared with wild-type virus. The results also showed that changes in the replicative competency of these constructed mutants were not associated with functional changes of HBV enhancer I. These results indicate the importance of amino acid(s) at the interface between the thumb and palm subdomains in modulating the replicative competency of HBV isolates. This regulatory residue(s) could serve as a new target for the development of anti-HBV drugs.

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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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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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Complementation of recombinant baculoviruses by coinfection with wild-type virus facilitates production in insect larvae of antigenic proteins of hepatitis B virus and influenza virus.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.86.5.1453 ◽

1989 ◽

Vol 86 (5) ◽

pp. 1453-1456 ◽

Cited By ~ 19

Author(s):

P. M. Price ◽

C. F. Reichelderfer ◽

B. E. Johansson ◽

E. D. Kilbourne ◽

G. Acs

Keyword(s):

Influenza Virus ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Recombinant Baculoviruses ◽

Wild Type Virus ◽

Type Virus ◽

Wild Type ◽

Insect Larvae ◽

Antigenic Proteins ◽

B Virus

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The Hepatitis B Virus X Protein Modulates Hepatocyte Proliferation Pathways To Stimulate Viral Replication

Journal of Virology ◽

10.1128/jvi.02196-09 ◽

2010 ◽

Vol 84 (6) ◽

pp. 2675-2686 ◽

Cited By ~ 69

Author(s):

Tricia L. Gearhart ◽

Michael J. Bouchard

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Hepatocyte Proliferation ◽

X Protein ◽

Experimental Conditions ◽

Hbv Replication ◽

B Virus ◽

The Impact

ABSTRACT Worldwide, there are over 350 million people who are chronically infected with the human hepatitis B virus (HBV); chronic HBV infections are associated with the development of hepatocellular carcinoma (HCC). The results of various studies suggest that the HBV X protein (HBx) has a role in the development of HBV-associated HCC. HBx can regulate numerous cellular signal transduction pathways, including those that modulate cell proliferation. Many previous studies that analyzed the impact of HBx on cell proliferation pathways were conducted using established or immortalized cell lines, and when HBx was expressed in the absence of HBV replication, and the precise effect of HBx on these pathways has often differed depending on experimental conditions. We have studied the effect of HBx on cell proliferation in cultured primary rat hepatocytes, a biologically relevant system. We demonstrate that HBx, both by itself and in the context of HBV replication, affected the levels and activities of various cell cycle-regulatory proteins to induce normally quiescent hepatocytes to enter the G1 phase of the cell cycle but not to proceed to S phase. We linked HBx regulation of cell proliferation to cytosolic calcium signaling and HBx stimulation of HBV replication. Cumulatively, our studies suggest that HBx induces normally quiescent hepatocytes to enter the G1 phase of the cell cycle and that this calcium-dependent HBx activity is required for HBV replication. These studies identify an essential function of HBx during HBV replication and a mechanism that may connect HBV infections to the development of HCC.

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Inhibition of Hepatitis B Virus (HBV) Replication by Pyrimidines Bearing an Acyclic Moiety: Effect on Wild-Type and Mutant HBV

Journal of Medicinal Chemistry ◽

10.1021/jm058271d ◽

2006 ◽

Vol 49 (6) ◽

pp. 2049-2054 ◽

Cited By ~ 19

Author(s):

Wassila Semaine ◽

Monika Johar ◽

D. Lorne J. Tyrrell ◽

Rakesh Kumar ◽

B. Agrawal

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Wild Type ◽

Hbv Replication ◽

B Virus

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Acute hepatitis C virus (HCV) infection in chronic carriers of hepatitis B virus (HBV): the impact of underlying active HBV replication on persistence of HCV infection and antibody responses to HCV

Gut ◽

10.1136/gut.51.1.95 ◽

2002 ◽

Vol 51 (1) ◽

pp. 95-99 ◽

Cited By ~ 21

Author(s):

C-M Chu ◽

C T Yeh ◽

I S Sheen ◽

Y F Liaw

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Hepatitis B Virus ◽

Hepatitis B ◽

Hcv Infection ◽

Antibody Responses ◽

Acute Hepatitis C ◽

Hbv Replication ◽

B Virus ◽

The Impact

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Enhancement of Hepatitis B Virus Replication by the Regulatory X Protein In Vitro and In Vivo

Journal of Virology ◽

10.1128/jvi.02020-06 ◽

2006 ◽

Vol 81 (6) ◽

pp. 2656-2662 ◽

Cited By ~ 112

Author(s):

Victor V. Keasler ◽

Amanda J. Hodgson ◽

Charles R. Madden ◽

Betty L. Slagle

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Virus Replication ◽

Hepg2 Cells ◽

Wild Type ◽

Tail Vein ◽

Hbv Replication ◽

Tail Vein Injection ◽

B Virus

ABSTRACT The 3.2-kb hepatitis B virus (HBV) genome encodes a single regulatory protein termed HBx. While multiple functions have been identified for HBx in cell culture, its role in virus replication remains undefined. In the present study, we combined an HBV plasmid-based replication assay with the hydrodynamic tail vein injection model to investigate the function(s) of HBx in vivo. Using a greater-than-unit-length HBV plasmid DNA construct (payw1.2) and a similar construct with a stop codon at position 7 of the HBx open reading frame (payw1.2*7), we showed that HBV replication in transfected HepG2 cells was reduced 65% in the absence of HBx. These plasmids were next introduced into the livers of outbred ICR mice via hydrodynamic tail vein injection. At the peak of virus replication, at 4 days postinjection, intrahepatic markers of HBV replication were reduced 72% to 83% in mice injected with HBx-deficient payw1.2*7 compared to those measured in mice receiving wild-type payw1.2. A second plasmid encoding HBx was able to restore virus replication from payw1.2*7 to wild-type levels. Finally, viremia was monitored over the course of acute virus replication, and at 4 days postinjection, it was reduced by nearly 2 logs in the absence of HBx. These studies establish that the role for HBx in virus replication previously shown in transfected HepG2 cells is also apparent in the mouse liver within the context of acute hepatitis. Importantly, the function of HBx can now be studied in an in vivo setting that more closely approximates the cellular environment for HBV replication.

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Presence of Replicating Virus in Recombinant Hepadnavirus Stocks Results from Recombination and Can Be Eliminated by the Use of a Packaging Cell Line

Journal of Virology ◽

10.1128/jvi.77.5.2873-2881.2003 ◽

2003 ◽

Vol 77 (5) ◽

pp. 2873-2881 ◽

Cited By ~ 2

Author(s):

Uta Klöcker ◽

Heike Oberwinkler ◽

Timo Kürschner ◽

Ulrike Protzer

Keyword(s):

Hepatitis B Virus ◽

Hepatitis B ◽

Wild Type Virus ◽

Recombinant Hepatitis ◽

Type Virus ◽

Wild Type ◽

Viral Genomes ◽

B Virus ◽

Viral Sequences

ABSTRACT Mutant hepatitis B viruses are useful tools to study the viral life cycle and viral pathogenesis. Furthermore, recombinant hepatitis B viruses are candidate vectors for liver-directed gene therapy. Because wild-type viruses present in recombinant or mutant virus stocks may falsify experimental results and are detrimental for a viral vector, we investigated whether and to what extent wild-type virus is present in recombinant virus stocks and where it originates from. We took advantage of the duck model of hepatitis B virus infection which allows very sensitive detection of replication-competent viruses by infection of primary duck hepatocytes or of ducklings in vivo. Recombinant hepatitis B virus stocks contained significant amounts of wild-type viruses, which were most probably generated by homologous recombination between plasmids containing homologous viral sequences. In addition, replication-competent viral genomes were reconstituted from plasmids which contained replication-deficient but redundant viral sequences. Using a stable cell line for packaging of deficient viral genomes, no wild-type virus was detected, neither by infection of primary hepatocytes nor in vivo.

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