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Resistance mutations of hepatitis B virus in entecavir-refractory patients.

ABSTRACT: The emergence of resistance mutations in the reverse transcriptase gene of hepatitis B virus (HBV) is associated with treatment failure. Entecavir (ETV) is one of the most potent anti-HBV reagents; it has a very low resistance rate and is used as the first-line treatment for chronic hepatitis B. In this study, we isolated HBVs in 4 ETV-refractory patients (2 with viral breakthrough, 1 with partial virological response, and 1 with flare-up) and assessed ETV resistance using replication-competent 1.38-fold HBV genome-length molecular clones. The full genome sequences of infected HBVs in ETV-refractory patients were determined. The HBV molecular clones were generated with the patient-derived sequences. After transfection of these molecular clones into HepG2 cells, viral replications and ETV susceptibilities were evaluated by measuring the amount of intracellular core-particle-associated HBV DNA using Southern blotting and real-time polymerase chain reaction. Among these cases, ETV-resistant variants were detected in 2 patients with viral breakthrough and responsible amino acid mutations in reverse transcriptase were successfully identified in these variants. No ETV-resistant mutation was detected in the other cases. The identified ETV-resistant mutations did not confer resistance to tenofovir disoproxil fumarate. Conclusion: The HBV replication model with patient-derived sequences is useful for assessing replication efficiency, susceptibility to anti-HBV reagents, and responsible resistance mutations and can aid in choosing the appropriate treatment strategy for treatment-failure cases of chronic hepatitis B. (Hepatology Communications 2017;1:110-121).

SUBMITTER: Yamada N

PROVIDER: S-EPMC5721430 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Resistance mutations of hepatitis B virus in entecavir-refractory patients.

Yamada Norie N Sugiyama Ryuichi R Nitta Sayuri S Murayama Asako A Kobayashi Minoru M Okuse Chiaki C Suzuki Michihiro M Yasuda Kiyomi K Yotsuyanagi Hiroshi H Moriya Kyoji K Koike Kazuhiko K Wakita Takaji T Kato Takanobu T

Hepatology communications 20170309 2

The emergence of resistance mutations in the reverse transcriptase gene of hepatitis B virus (HBV) is associated with treatment failure. Entecavir (ETV) is one of the most potent anti-HBV reagents; it has a very low resistance rate and is used as the first-line treatment for chronic hepatitis B. In this study, we isolated HBVs in 4 ETV-refractory patients (2 with viral breakthrough, 1 with partial virological response, and 1 with flare-up) and assessed ETV resistance using replication-competent ...[more]

PMID: 29404449

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Project description:BACKGROUND: Entecavir (ETV) is a deoxyguanosine analog competitive inhibitor of hepatitis B virus (HBV) polymerase that exhibits delayed chain termination of HBV DNA. A high barrier to entecavir-resistance (ETVr) is observed clinically, likely due to its potency and a requirement for multiple resistance changes to overcome suppression. Changes in the HBV polymerase reverse-transcriptase (RT) domain involve lamivudine-resistance (LVDr) substitutions in the conserved YMDD motif (M204V/I +/- L180M), plus an additional ETV-specific change at residues T184, S202 or M250. These substitutions surround the putative dNTP binding site or primer grip regions of the HBV RT. METHODS/PRINCIPAL FINDINGS: To determine the mechanistic basis for ETVr, wildtype, lamivudine-resistant (M204V, L180M) and ETVr HBVs were studied using in vitro RT enzyme and cell culture assays, as well as molecular modeling. Resistance substitutions significantly reduced ETV incorporation and chain termination in HBV DNA and increased the ETV-TP inhibition constant (K(i)) for HBV RT. Resistant HBVs exhibited impaired replication in culture and reduced enzyme activity (k(cat)) in vitro. Molecular modeling of the HBV RT suggested that ETVr residue T184 was adjacent to and stabilized S202 within the LVDr YMDD loop. ETVr arose through steric changes at T184 or S202 or by disruption of hydrogen-bonding between the two, both of which repositioned the loop and reduced the ETV-triphosphate (ETV-TP) binding pocket. In contrast to T184 and S202 changes, ETVr at primer grip residue M250 was observed during RNA-directed DNA synthesis only. Experimentally, M250 changes also impacted the dNTP-binding site. Modeling suggested a novel mechanism for M250 resistance, whereby repositioning of the primer-template component of the dNTP-binding site shifted the ETV-TP binding pocket. No structural data are available to confirm the HBV RT modeling, however, results were consistent with phenotypic analysis of comprehensive substitutions of each ETVr position. CONCLUSIONS: Altogether, ETVr occurred through exclusion of ETV-TP from the dNTP-binding site, through different, novel mechanisms that involved lamivudine-resistance, ETV-specific substitutions, and the primer-template.

Project description:BACKGROUND:It is unclear whether immune escape-associated mutations in the major hydrophilic region of hepatitis B virus surface antigen (HBsAg) are associated with nucleoside/nucleotide analog resistance. AIM:To evaluate the association between immune escape-associated mutations and nucleoside/nucleotide analog resistance mutations. METHODS:In total, 19440 patients with chronic hepatitis B virus infection, who underwent resistance testing at the Fifth Medical Center of Chinese PLA General Hospital between July 2007 and December 2017, were enrolled. As determined by sequence analysis, 6982 patients harbored a virus with resistance mutations and 12458 harbored a virus lacking resistance mutations. Phenotypic analyses were performed to evaluate HBsAg production, replication capacity, and drug-induced viral inhibition of patient-derived drug-resistant mutants with or without the coexistence of sA159V. RESULTS:The rate of immune escape-associated mutation was significantly higher in 9 of the 39 analyzed mutation sites in patients with resistance mutations than in patients without resistance mutations. In particular, these mutations were sQ101H/K/R, sS114A/L/T, sT118A/K/M/R/S/V, sP120A/L/Q/S/T, sT/I126A/N/P/S, sM133I/L/T, sC137W/Y, sG145A/R, and sA159G/V. Among these, sA159V was detected in 1.95% (136/6982) of patients with resistance mutations and 1.08% (134/12,458) of patients lacking resistance mutations (P < 0.05). The coexistence of sA159V with lamivudine (LAM) and entecavir (ETV)-resistance mutations in the same viral genome was identified during follow-up in some patients with drug resistance. HBsAg production was significantly lower and the replication capacity was significantly higher, without a significant difference in LAM/ETV susceptibility, in sA159V-containing LAM/ETV-resistant mutants than in their sA159V-lacking counterparts. CONCLUSION:In summary, we observed a close link between the increase in certain immune escape-associated mutations and the development of resistance mutations. sA159V might increase the fitness of LAM/ETV-resistant mutants under environmental pressure in some cases.

Project description:Entecavir (ETV) is a widely used anti-hepatitis B virus (HBV) drug. However, the emergence of resistant mutations in HBV reverse transcriptase (RT) results in treatment failure. To understand the mechanism underlying the development of ETV resistance by HBV RT, we analyzed the L180M, M204V, and L180M/M204V mutants using a combination of biochemical and structural techniques. ETV-triphosphate (ETV-TP) exhibited competitive inhibition with dGTP in both wild-type (wt) RT and M204V RT, as observed using Lineweaver-Burk plots. In contrast, RT L180M or L180M/M204V did not fit either competitive, uncompetitive, noncompetitive, or typical mixed inhibition, although ETV-TP was a competitive inhibitor of dGTP. Crystallography of HIV RTY115F/F116Y/Q151M/F160M/M184V, mimicking HBV RT L180M/M204V, showed that the F115 bulge (F88 in HBV RT) caused by the F160M mutation induced deviated binding of dCTP from its normal tight binding position. Modeling of ETV-TP on the deviated dCTP indicated that a steric clash could occur between ETV-TP methylene and the 3'-end nucleoside ribose. ETV-TP is likely to interact primarily with HBV RT M171 prior to final accommodation at the deoxynucleoside triphosphate (dNTP) binding site (Y. Yasutake, S. Hattori, H. Hayashi, K. Matsuda, et al., Sci Rep 8:1624, 2018, https://doi.org/10.1038/s41598-018-19602-9). Therefore, in HBV RT L180M/M204V, ETV-TP may be stuck at M171, a residue that is conserved in almost all HBV isolates, leading to the strange inhibition pattern observed in the kinetic analysis. Collectively, our results provide novel insights into the mechanism of ETV resistance of HBV RT caused by L180M and M204V mutations. IMPORTANCE HBV infects 257 million people in the world, who suffer from elevated risks of liver cirrhosis and cancer. ETV is one of the most potent anti-HBV drugs, and ETV resistance mutations in HBV RT have been extensively studied. Nevertheless, the mechanisms underlying ETV resistance have remained elusive. We propose an attractive hypothesis to explain ETV resistance and effectiveness using a combination of kinetic and structural analyses. ETV is likely to have an additional interaction site, M171, beside the dNTP pocket of HBV RT; this finding indicates that nucleos(t)ide analogues (NAs) recognizing multiple interaction sites within RT may effectively inhibit the enzyme. Modification of ETV may render it more effective and enable the rational design of efficient NA inhibitors.

Project description:Background and aimsEntecavir (ETV) resistance of hepatitis B virus (HBV) conventionally requires rt184, 202, or 250 mutations plus lamivudine-resistance mutation (rtM204V/I ± L180M). This study aimed to clarify whether rtL180M+A181C+M204V mutations may contribute to HBV ETV resistance.MethodsSerum samples were collected from 22,009 patients who underwent resistance testing in Beijing 302 Hospital from 2007 to 2016. HBV reverse transcriptase (RT) gene was screened by direct sequencing and verified by clonal sequencing. Phenotypic analysis was performed for evaluating replication capacity and drug susceptibility.ResultsClassical ETV-resistance mutations of HBV were detected in 1252 patients who were receiving ETV therapy. The rtA181C mutation was detected with rtL180M+M204V mutations in 18 lamivudine-experienced ETV-treated patients, and the emergence of the mutations was associated with virological breakthrough or inadequate virological response to ETV. Patient-derived representative rtA181C-containing mutants, rtL180M+A181C+M204V, rtL180M+A181C+M204V+M250V, and rtL180M+A181C+S202G+M204V, exhibited 45.7%, 25.9%, and 25.0% replication capacity and 85.6-, 356.1-, and 307.1-fold decreased susceptibility to ETV respectively compared to the wild-type strain, while the three mutants remained sensitive to tenofovir (TDF). Artificial elimination of rtA181C largely restored the rtL180M+A181C+M204V mutant's sensitivity to ETV. Molecular modelling of viral RT binding to ETV showed that the rtL180M+A181C+M204V mutant had a less stable conformation compared to rtL180M+M204V mutant. In clinical practice, undetectable serum HBV DNA was achieved in two of five longitudinally followed rtA181C-positive patients who received switching-to TDF therapy, but not in the other three who received add-on adefovir therapy during observation.ConclusionsBoth clinical and experimental data support rtL180M+A181C+M204V as a novel non-classical ETV-resistance mutation pattern.

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Resistance mutations of hepatitis B virus in entecavir-refractory patients.

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Resistance mutations of hepatitis B virus in entecavir-refractory patients.

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