Effect of the G1896A precore mutation on drug sensitivity and replication yield of lamivudine‐resistant HBV in vitro

Hepatitis B e antigen (HBeAg) negative chronic hepatitis B (CHB) is frequently caused by a mutation (G1896A) in the hepatitis B virus (HBV) precore (PC) reading frame that creates a stop codon, causing premature termination of the PC protein. During lamivudine treatment, drug resistance develops at a similar rate in HBeAg positive and HBeAg negative CHB. Lamivudine‐resistant HBV mutants have been shown to replicate inefficiently in vitro in the absence of PC mutations, but it is unknown whether the presence of PC mutations affects replication efficiency or antiviral sensitivity. This study utilized the recombinant HBV baculovirus system to address these issues. HBV baculoviruses encoding the G1896A PC stop codon mutation were generated in wild‐type (WT) and lamivudine‐resistant (rtM204I and rtL180M + rtM204V) backgrounds, resulting in a panel of 6 related recombinant baculoviruses. In vitro assays were performed to compare the sensitivities of the PC mutant viruses with lamivudine and adefovir and to compare relative replication yields. The PC mutation did not significantly affect sensitivities to either adefovir or lamivudine. WT HBV and PC mutant HBV showed similar replication yields, whereas the replication yields of the lamivudine‐resistant mutants were greatly reduced in HBeAg positive HBVs, confirming previous observations. However, the presence of the PC mutation was found to compensate for the replication deficiency in each of the lamivudine‐resistant mutants, increasing the replication yields of each virus. In conclusion, the PC stop codon mutation appears to increase the replication efficacy of lamivudine‐resistant virus but does not affect in vitro drug sensitivity.

[1]  P. Marcellin,et al.  GS-98-437 a double-blind, randomized, placebo-controlled study of adefovir dipivoxil (ADV) for the treatment of patients with HBeAg+ chronic hepatitis B infection: 48 week results. , 2001 .

[2]  D. Vassilopoulos,et al.  Hepatitis B e antigen–negative chronic hepatitis B , 2001, Hepatology.

[3]  M. Manns,et al.  In Vitro Susceptibilities of Wild-Type or Drug-Resistant Hepatitis B Virus to (−)-β-d-2,6-Diaminopurine Dioxolane and 2′-Fluoro-5-Methyl-β-l-Arabinofuranosyluracil , 2001, Antimicrobial Agents and Chemotherapy.

[4]  D. Häussinger,et al.  Protein kinase C–dependent distribution of the multidrug resistance protein 2 from the canalicular to the basolateral membrane in human HepG2 cells , 2001, Hepatology.

[5]  N. Leung,et al.  Extended lamivudine treatment in patients with chronic hepatitis B enhances hepatitis B e antigen seroconversion rates: Results after 3 years of therapy , 2001, Hepatology.

[6]  M. Manns,et al.  Cross-Resistance Testing of Antihepadnaviral Compounds Using Novel Recombinant Baculoviruses Which Encode Drug-Resistant Strains of Hepatitis B Virus , 2001, Antimicrobial Agents and Chemotherapy.

[7]  C. Gibbs,et al.  Dynamics analyses provide insight into the influence of precore mutations on the mechanisms of viral CL , 2001 .

[8]  D. Richman,et al.  Nomenclature for antiviral‐resistant human hepatitis B virus mutations in the polymerase region , 2001, Hepatology.

[9]  R. Schinazi,et al.  The polymerase L528M mutation cooperates with nucleotide binding-site mutations, increasing hepatitis B virus replication and drug resistance. , 2001, The Journal of clinical investigation.

[10]  W. Delaney,et al.  Resistance of Hepatitis B Virus to Antiviral Drugs: Current Aspects and Directions for Future Investigation , 2001, Antiviral chemistry & chemotherapy.

[11]  L. Fu,et al.  Characterization of Novel Human Hepatoma Cell Lines with Stable Hepatitis B Virus Secretion for Evaluating New Compounds against Lamivudine- and Penciclovir-Resistant Virus , 2000, Antimicrobial Agents and Chemotherapy.

[12]  K. Hahm,et al.  Reversion from precore/core promoter mutants to wild‐type hepatitis B virus during the course of lamivudine therapy , 2000, Hepatology.

[13]  A. Lok,et al.  Evolution of hepatitis B virus polymerase gene mutations in hepatitis B e Antigen–negative patients receiving lamivudine therapy , 2000, Hepatology.

[14]  D. Richman The impact of drug resistance on the effectiveness of chemotherapy for chronic hepatitis B , 2000, Hepatology.

[15]  S. Hadziyannis,et al.  Efficacy of long‐term lamivudine monotherapy in patients with hepatitis B e antigen–negative chronic hepatitis B , 2000, Hepatology.

[16]  Y. Liaw,et al.  Effects of extended lamivudine therapy in Asian patients with chronic hepatitis B. Asia Hepatitis Lamivudine Study Group. , 2000, Gastroenterology.

[17]  S. Locarnini,et al.  In Vitro Antihepadnaviral Activities of Combinations of Penciclovir, Lamivudine, and Adefovir , 2000, Antimicrobial Agents and Chemotherapy.

[18]  T. Santantonio,et al.  Long-term follow-up of patients with anti-HBe/HBV DNA-positive chronic hepatitis B treated for 12 months with lamivudine. , 2000, Journal of hepatology.

[19]  A. Lok,et al.  Hepatitis B infection: pathogenesis and management. , 2000, Journal of hepatology.

[20]  C. Gibbs,et al.  Fulminant hepatic failure resulting from lamivudine-resistant hepatitis B virus in a renal transplant recipient: durable response after orthotopic liver transplantation on adefovir dipivoxil and hepatitis B immune globulin. , 1999, Transplantation.

[21]  C. Chu,et al.  Acute exacerbation and hepatitis B virus clearance after emergence of YMDD motif mutation during lamivudine therapy , 1999, Hepatology.

[22]  D. Faulds,et al.  Lamivudine , 1999, Drugs.

[23]  Y. Shiratori,et al.  YMDD motif in hepatitis B virus DNA polymerase influences on replication and lamivudine resistance: A study by in vitrofull‐length viral DNA transfection , 1999, Hepatology.

[24]  M. Buti,et al.  Efficacy of lamivudine in patients with hepatitis B e antigen-negative/hepatitis B virus DNA-positive (precore mutant) chronic hepatitis B. Lamivudine Precore Mutant Study Group. , 1999, Hepatology.

[25]  H. Isom,et al.  Hepatitis B virus replication in human HepG2 cells mediated by hepatitis B virus recombinant baculovirus , 1998, Hepatology.

[26]  R. D. de Man,et al.  Clinical impact of lamivudine resistance in chronic hepatitis B. , 1998, Journal of hepatology.

[27]  N. Leung,et al.  A one-year trial of lamivudine for chronic hepatitis B. Asia Hepatitis Lamivudine Study Group. , 1998, The New England journal of medicine.

[28]  Y. Cheng,et al.  Role of additional mutations outside the YMDD motif of hepatitis B virus polymerase in L(-)SddC (3TC) resistance. , 1998, Biochemical pharmacology.

[29]  J. Wands,et al.  Hepatitis B virus mutants associated with 3TC and famciclovir administration are replication defective , 1998, Hepatology.

[30]  J. Wands,et al.  Biologic properties of hepatitis B viral genomes with mutations in the precore promoter and precore open reading frame. , 1997, Virology.

[31]  R. D. de Man,et al.  Lamivudine resistance inimmunocompetent chronic hepatitis B , 1997 .

[32]  S. Locarnini,et al.  Synergistic inhibition of hepadnaviral replication by lamivudine in combination with penciclovir in vitro , 1997, Hepatology.

[33]  J. Wands,et al.  Posttranscriptional regulation of hepatitis B virus replication by the precore protein , 1997, Journal of virology.

[34]  D. O'reilly Use of baculovirus expression vectors. , 1997, Methods in molecular biology.

[35]  F. Chisari,et al.  The hepatitis B virus (HBV) precore protein inhibits HBV replication in transgenic mice , 1996, Journal of virology.

[36]  H. Schaller,et al.  Hepatitis B virus replication‐an update , 1996, Journal of viral hepatitis.

[37]  L. Boone,et al.  DNA polymerase activity of hepatitis B virus particles: differential inhibition by L-enantiomers of nucleotide analogs. , 1996, Antiviral research.

[38]  F. Boyce,et al.  Baculovirus-mediated gene transfer into mammalian cells. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[39]  U. Akarca,et al.  Mutations in the pre-core region of hepatitis B virus serve to enhance the stability of the secondary structure of the pre-genome encapsidation signal. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[40]  H. Zentgraf,et al.  Precore-mediated inhibition of hepatitis B virus progeny DNA synthesis , 1993, Journal of virology.

[41]  J. Pollack,et al.  An RNA stem-loop structure directs hepatitis B virus genomic RNA encapsidation , 1993, Journal of virology.

[42]  C. Trépo,et al.  Replication capacities of natural and artificial precore stop codon mutants of hepatitis B virus: relevance of pregenome encapsidation signal. , 1992, Virology.

[43]  R. Bartenschlager,et al.  Hepadnaviral assembly is initiated by polymerase binding to the encapsidation signal in the viral RNA genome. , 1992, The EMBO journal.

[44]  H. Will,et al.  Wild-type and e antigen-minus hepatitis B viruses and course of chronic hepatitis. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[45]  C. Guguen-Guillouzo,et al.  In vitro replication competence of a cloned hepatitis B virus variant with a nonsense mutation in the distal pre-C region. , 1991, Virology.

[46]  R. Bartenschlager,et al.  A short cis‐acting sequence is required for hepatitis B virus pregenome encapsidation and sufficient for packaging of foreign RNA. , 1990, The EMBO journal.

[47]  F Tsuda,et al.  Hepatitis B viruses with precore region defects prevail in persistently infected hosts along with seroconversion to the antibody against e antigen , 1990, Journal of virology.

[48]  H. Thomas,et al.  MUTATION PREVENTING FORMATION OF HEPATITIS B e ANTIGEN IN PATIENTS WITH CHRONIC HEPATITIS B INFECTION , 1989, The Lancet.

[49]  K. Koike,et al.  Hepatitis B virus (HBV) particles are produced in a cell culture system by transient expression of transfected HBV DNA. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[50]  B. Schwartz Letter: Ondine's curse. , 1976, Lancet.