Mutations in the nonstructural protein 3A confer resistance to the novel enterovirus replication inhibitor TTP-8307 - PubMed (original) (raw)

doi: 10.1128/AAC.00934-08. Epub 2009 Feb 23.

Hendrik Jan Thibaut, Lonneke van der Linden, Kjerstin Lanke, Ward Heggermont, Stephen Ireland, Robert Andrews, Murty Arimilli, Taleb H Al-Tel, Erik De Clercq, Frank van Kuppeveld, Johan Neyts

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Mutations in the nonstructural protein 3A confer resistance to the novel enterovirus replication inhibitor TTP-8307

Armando M De Palma et al. Antimicrob Agents Chemother. 2009 May.

Abstract

A novel compound, TTP-8307, was identified as a potent inhibitor of the replication of several rhino- and enteroviruses. TTP-8307 inhibits viral RNA synthesis in a dose-dependent manner, without affecting polyprotein synthesis and/or processing. Drug-resistant variants of coxsackievirus B3 were all shown to carry at least one amino acid mutation in the nonstructural protein 3A. In particular, three mutations located in a nonstructured region preceding the hydrophobic domain (V45A, I54F, and H57Y) appeared to contribute to the drug-resistant phenotype. This region has previously been identified as a hot sport for mutations that resulted in resistance to enviroxime, the sole 3A-targeting enterovirus inhibitor reported thus far. This was corroborated by the fact that TTP-8307 and enviroxime proved cross-resistant. It is hypothesized that TTP-8307 and enviroxime disrupt proper interactions of 3A(B) with other viral or cellular proteins that are required for efficient replication.

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Figures

FIG. 1.

FIG. 1.

Structural formulae of TTP-8307 (A) and enviroxime (B).

FIG. 2.

FIG. 2.

Dose-dependent inhibition of viral replication by TTP-8307. Vero cell cultures infected with CVB3 were treated with different concentrations of TTP-8307, and the effect on viral replication was monitored at day 3 postinfection. (A) Effect on virus-induced CPE formation (using an MTS-based cell protection assay). (B) Plaque reduction. The data represent averages ± the standard deviations (SD) from three independent experiments.

FIG. 3.

FIG. 3.

TTP-8307 inhibits accumulation of CVB3 viral RNA without affecting polyprotein processing. (A) Time of drug addition. TTP-8307 (25 μM) was added to CVB3-infected cell cultures at 1-h intervals, starting at 1 h before infection. At 8 h postinfection, the time needed for a single replication cycle, intra- and extracellular viral RNA was collected and quantified by means of real-time qRT-PCR. Values were standardized to the 0-h time point. The data represent averages ± the SD from two independent experiments. (B) Analysis of viral RNA accumulation with subgenomic replicon pCB53/T7-Luc. Accumulation of viral (+)RNA was monitored after transfection of BGM cells (in the presence or absence of TTP-8307) with RNA derived from a chimeric subgenomic replicon (pCB53/T7-Luc). At the indicated times posttransfection, the luciferase activity was quantified and expressed in (relative) light units [(R)LU]. The data represent averages ± the SD from three independent experiments. (C) Effect of TTP-8307 on polyprotein processing. BGM cells grown to confluence in 24-well plates were infected with CVB3. At 5h30 min postinfection, cells were pulse-labeled for 30 min with Met35[S] after starvation in methionine-free medium for 30 min, either in the presence (25 μM) or absence of TTP-8307. Cells were lysed and translation products were analyzed by SDS-polyacrylamide gel electrophoresis. The data represent averages ± the SD from three independent experiments.

FIG. 4.

FIG. 4.

Plaque phenotypes of wild-type CVB3 and recombinant clones.

FIG. 5.

FIG. 5.

Viral replication of wild-type and recombinant viruses in the presence of TTP-8307, enviroxime and TBZE-029. Each graph represents the number of PFU produced by wild-type CVB3 or by any of the four constructed 3A mutants—3A[I8T], 3A[V45A], 3A[I54F], and 3A[H57Y]—at a given compound concentration. Calculated EC90 values represent averages ± the SD from five independent experiments. **, P < 0.05.

FIG. 6.

FIG. 6.

(A) Sequence alignment of 3A proteins from CVB3, PV1, HRV2, and HRV14. The residues that are circled were earlier identified as amino acid mutations in enviroxime (or analog)-resistant variants (3, 22, 23). The arrows indicate mutations in TTP-8307 resistant CVB3, identified in the present study. The residues between brackets form a 22-residue hydrophobic region involved in membrane anchoring of 3A/3AB. The region underlined in green is suggested to be a “hot spot” for viral mutations when virus is selected in the presence of TTP-8307 or enviroxime. (B) Predicted solution structure of the N-terminal soluble domain of the PV 3A protein. The two cylinders represent two α-helices that form an α-helical hairpin. Adapted from Wessels et al. (46) with permission. (C) Location of the three predominant amino acid mutations involved in CVB3 resistance to TTP-8307 in a structural model of protein 3A.

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