scholarly journals A Single Amino Acid in the Reverse Transcriptase Domain of Hepatitis B Virus Affects Virus Replication Efficiency

2001 ◽  
Vol 75 (23) ◽  
pp. 11827-11833 ◽  
Author(s):  
Xu Lin ◽  
Zheng-Hong Yuan ◽  
Li Wu ◽  
Jian-Ping Ding ◽  
Yu-Mei Wen
Keyword(s):  
Amino Acid ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcriptase ◽  
Single Amino Acid ◽  
Structural Basis ◽  
Replication Efficiency ◽  
P Gene ◽  
B Virus ◽  
The Difference

ABSTRACT To explore functional domains in the hepatitis B virus (HBV) polymerase, two naturally occurring HBV isolates (56 and 2-18) with 98.7% nucleic acid sequence homology but different replication efficiencies were studied. After transfection into HepG2 cells, HBV DNA isolated from intracellular virus core particles was much higher in 56-transfected cells than in cells transfected with 2-18. The structural basis for the difference in replication efficiency between these two isolates was studied by functional domain gene substitution. The complete polymerase (P) gene and its gene segments coding for the terminal protein (TP), spacer (SP), reverse transcriptase (RT), and RNase H in 2-18 were separately replaced with their counterparts from 56 to construct full-length chimeric genomes. Cell transfection analysis revealed that substitution of the complete P gene of 2-18 with the P gene from 56 slightly enhanced viral replication. The only chimeric genome that regained the high replication efficiency of the original 56 isolate was the one with substitution of the RT gene of 2-18 with that from 56. Within the RT region, amino acid differences between isolates 2-18 and 56 were located at positions 617 (methionine versus leucine), 652 (serine versus proline), and 682 (valine versus leucine). Point mutation identified amino acid 652 as being responsible for the difference in replication efficiency. Homologous modeling studies of the HBV RT domain suggest that the mutation of residue 652 from proline to serine might affect the conformation of HBV RT which interacts with the template-primer, leading to impaired polymerase activity.

scholarly journals Localization of a unique hepatitis B virus epitope sheds new light on the structure of hepatitis B virus surface antigen

1999 ◽  
Vol 80 (8) ◽  
pp. 2121-2126 ◽  
Author(s):  
W. P. Paulij ◽  
P. L. M. de Wit ◽  
C. M. G. Sünnen ◽  
M. H. van Roosmalen ◽  
A. Petersen-van Ettekoven ◽  
...  
Keyword(s):  
Amino Acid ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Surface Antigen ◽  
Single Amino Acid ◽  
Amino Acid Substitutions ◽  
Virus Surface ◽  
Virus Surface Antigen ◽  
B Virus ◽  
20 Nm

In a search for monoclonal antibodies (MAbs) that can bind hepatitis B virus surface antigen (HBsAg) with amino acid substitutions in the immune dominant ‘a’ region (escape mutants) we investigated the epitope recognition site of the human MAb 4-7B. Pepscan analysis and experiments with alanine substitution as well as substitutions known from nature pointed to residues 178–186 in the small S protein with the amino acid sequence PFVQWFVGL (key amino acids in bold) as the minimal epitope. Single amino acid substitutions at positions 122(R/K)(d/y), 134(Y/F), 145(G/R), 148(T/A) and 160(K/R)(w/r), representing ‘a’ region variants in recombinant HBsAg COS-I cells, did not influence binding of MAb 4-7B. Synthetic peptides (residues 175–189) including the 4-7B epitope sequence were able to evoke an anti-HBs response in rabbits. According to established polypeptide models, the 4-7B epitope region is located in the lipid layer of 20 nm HBsAg particles. The present results, however, suggest that residues 178–186 are exposed on the surface of the 20 nm particle. This may change our view of the structure of HBsAg.

Generation of duck hepatitis B virus polymerase mutants through site-directed mutagenesis which demonstrate resistance to lamivudine [(--)-beta-L-2', 3'-dideoxy-3'-thiacytidine] in vitro.

1996 ◽  
Vol 40 (8) ◽  
pp. 1957-1960 ◽  
Author(s):  
K P Fischer ◽  
D L Tyrrell
Keyword(s):  
Amino Acid ◽  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Amino Acid Change ◽  
Single Amino Acid ◽  
Wild Type Virus ◽  
Duck Hepatitis B Virus ◽  
Duck Hepatitis ◽  
Single Amino Acid Change ◽  
B Virus

Hepatitis B virus replication is very sensitive to lamivudine. A single amino acid change in human immunodeficiency virus reverse transcriptase is responsible for high-level resistance to this compound. Duck hepatitis B virus mutants were created bearing the analogous amino acid change in the duck hepatitis B virus polymerase. Viral DNA production was reduced 92% for the wild-type virus at 2 micrograms of lamivudine per ml, while the mutants required 40 micrograms of lamivudine per ml to inhibit replication by greater than 80%.

scholarly journals The Hepatitis B Virus Polymerase Mutation rtV173L Is Selected during Lamivudine Therapy and Enhances Viral Replication In Vitro

2003 ◽  
Vol 77 (21) ◽  
pp. 11833-11841 ◽  
Author(s):  
William E. Delaney ◽  
Huiling Yang ◽  
Christopher E. Westland ◽  
Kalyan Das ◽  
Eddy Arnold ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Nucleic Acid ◽  
Hepatitis B ◽  
Viral Replication ◽  
Reverse Transcriptase ◽  
Polymerization Reaction ◽  
Replication Efficiency ◽  
Template Strand ◽  
B Virus

ABSTRACT Therapy of chronic hepatitis B virus (HBV) infection with the polymerase inhibitor lamivudine frequently is associated with the emergence of viral resistance. Genotypic changes in the YMDD motif (reverse transcriptase [rt] mutations rtM204V/I) conferred resistance to lamivudine as well as reducing the in vitro replication efficiency of HBV. A second mutation, rtL180M, was previously reported to partially restore replication fitness as well as to augment drug resistance in vitro. Here we report the functional characterization of a third polymerase mutation (rtV173L) associated with resistance to lamivudine and famciclovir. rtV173L was observed at baseline in 9 to 22% of patients who entered clinical trials of adefovir dipivoxil for the treatment of lamivudine-resistant HBV. In these patients, rtV173L was invariably found as a third mutation in conjunction with rtL180M and rtM204V. In vitro analyses indicated that rtV173L did not alter the sensitivity of wild-type or lamivudine-resistant HBV to lamivudine, penciclovir, or adefovir but instead enhanced viral replication efficiency. A molecular model of HBV polymerase indicated that residue rtV173 is located beneath the template strand of HBV nucleic acid near the active site of the reverse transcriptase. Substitution of leucine for valine at this residue may enhance polymerization either by repositioning the template strand of nucleic acid or by affecting other residues involved in the polymerization reaction. Together, these results suggest that rtV173L is a compensatory mutation that is selected in lamivudine-resistant patients due to an enhanced replication phenotype.

scholarly journals The L80I Substitution in the Reverse Transcriptase Domain of the Hepatitis B Virus Polymerase Is Associated with Lamivudine Resistance and Enhanced Viral Replication In Vitro

2007 ◽  
Vol 51 (7) ◽  
pp. 2285-2292 ◽  
Author(s):  
Nadia Warner ◽  
Stephen Locarnini ◽  
Michael Kuiper ◽  
Angeline Bartholomeusz ◽  
Anna Ayres ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Reverse Transcriptase ◽  
Resistance Mutations ◽  
Hbv Genotype ◽  
Lamivudine Resistance ◽  
Replication Efficiency ◽  
B Virus ◽  
High Level

ABSTRACT Long-term lamivudine (LMV) treatment of chronic hepatitis B almost inevitably engenders viral resistance. Mutations that result in the replacement of the methionine at position 204 of the deoxynucleoside triphosphate-binding site of the hepatitis B virus (HBV) reverse transcriptase (rt) by isoleucine, valine, or (rarely) serine (rtM204I/V/S) confer high-level resistance to LMV but reduce replication efficiency. The subsequent selection or coselection of secondary mutations that partially restore replication efficiency is common and may influence drug resistance. Genotyping has shown that LMV treatment can select for HBV rtL80V/I mutants, but their prevalence and phenotype have not been documented. Analysis of a large sequence database revealed that rtL80V/I occurred almost exclusively in association with LMV resistance, and 85% of these isolates encoded rtL80I. Coselection of rtL80V/I occurred in 46% of isolates in which LMV resistance was attributable to rtM204I but only 9% of those in which resistance was attributable to rtM204V. Moreover, rtL80V/I did not occur in HBV genotype A isolates but occurred at similar frequencies in genotype B, C, and D isolates. In vitro phenotyping showed that although the rtL80I mutant by itself replicated less efficiently and was hypersensitive to LMV compared to the replication efficiency and sensitivity of its wild-type parent, the presence of rtL80I enhanced the replication efficiency of rt204I/V mutants without significantly affecting LMV resistance. Molecular modeling revealed that rt80 does not interact directly with the enzyme's substrates. Collectively, these results suggest that coselection of rtL80V/I and rtM204I/V occurs because the former compensates for the loss of replication efficiency associated with the acquisition of LMV resistance, particularly in the case of rtM204I.

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