Mechanistic characterization and molecular modeling of hepatitis B virus polymerase resistance to entecavir

Abstract

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.

Figure 1. Incorporation of [³H]-ETV into HBV nucleocapsid DNA in culture.

[³H]-ETV was added to cultures of HepG2 cells transfected with an HBV expression construct, as detailed under Materials and Methods. HBV nucleocapsids were isolated from cell lysates, as detailed under Materials and Methods, and radiolabeled HBV DNA was quantified through scintillation counting. The levels of nucleocapsid-associated [³H] from cells grown in [³H]-ETV are presented as percent wildtype control values ± standard deviation. Yields of HBV nucleocapsid DNA were standardized according to real-time PCR quantification of HBV DNA within isolated nucleocapsids, as detailed under Materials and Methods. Similar results were obtained by standardizing total HBV nucleocapsid DNA levels with nucleocapsids from parallel cultures metabolically labeled with [³H]-thymidine (data not shown). WT, wildtype nucleocapsids; LVDr, M204V+L180M substituted HBV nucleocapsids, LVDr+M250V, M204V+L180M+M250V substituted nucleocapsids; LVDr+T184G+S202I, M204V+L180M+T184G+S202I substituted nucleocapsids. HBVs were tested independently 3 to 4 times, except the LVDr+M250V, which was tested twice.

Figure 2. Molecular homology model of resistant HBV RTs.

The ETV-TP binding pocket of HBV RT. . (A) HBV RT with LVDr substitutions, M204V+L180M, (B) HBV RT with LVDr + S202G. HBV RT, ETV-TP and primer-template DNAs are labeled. The residues lining the pocket are orange, changes from LVDr are red, from ETVr are blue, and the M250, S202, and T184 residue positions (panel B) are pink. Panel A was essential reproduced from with permission.

Figure 3. The T184-S202-204 hydrogen bonding network stabilizes the YMDD loop.

The YMDD loop is shown with residues M204V+L180M and the H-bonding between residues T184, S202 and M204V are shown as dotted white lines.

Figure 4. Position of residue M250 in the HBV RT/DNA molecular model.

A) The relative location of the M250, Y203, M204V, and other resistance residues are shown with primer-template DNA and ETV-TP in the dNTP binding site. The proximity of the M250 residue to both primer DNA and the Y203 residue are shown, indicating possible mechanisms for effects of M250 substitutions on the dNTP binding site, as well as the influence different side chains could have on primer positioning. B) M250 is packed against L66 and N65 forms hydrogen bonds (white dotted lines) to the hydroxyl groups on the backbone of the RNA template strand. C) The smaller side chain of the M250V no longer packs against L66. As indicated by the arrows (protein:green; DNA:gray; M250V:purple), the resulting conformational change in the protein to close the resultant hole repositions the RNA/DNA slightly over the NTP binding site. The slight modification to the NTP site enhances LVD binding while decreasing the binding affinity of ETV.

Figure 5. Analysis of ETVr through strand-specific DNA synthesis in culture.

Cell culture ETV EC₅₀ determinations were made for wildtype and various resistant HBVs. The levels of HBV DNA synthesized in the presence of ETV was determined by HBV-specific probe hybridization to HBV nucleocapsid DNA from the cultures. The probes used were the typical double-stranded DNA probe, or strand-specific riboprobes which hybridized to a single HBV DNA strand. Comparison of strand-specific EC₅₀ versus double stranded EC₅₀ for wildtype polymerase (WT) or LVDr M204V+L180M HBV, or the LVDr substitutions with ETVr substitutions (+T184, +S202, +M250) as indicated. Values at 4000 nM indicate that 50% inhibition was not observed at the highest ETV concentration tested.