Enterovirus evolution reveals the mechanism of an RNA-targeted antiviral and determinants of viral replication.

Davila-Calderon, Jesse; Li, Mei-Ling; Penumutchu, Srinivasa R; Haddad, Christina; Malcolm, Linzy; King, Josephine; Hargrove, Amanda E; Brewer, Gary et al. · Sci Adv · 2024

basic_science · Level V

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Abstract

Selective pressures on viruses provide opportunities to establish target site specificity and mechanisms of antivirals. Enterovirus (EV)-A71 with resistant mutations in the stem loop (SL) II internal ribosome entry site (IRES) (SLII<sup>resist</sup>) were selected at low doses of the antiviral dimethylamiloride (DMA)-135. The EV-A71 mutants were resistant to DMA-135 at concentrations that inhibit replication of wild-type virus. EV-A71 IRES structures harboring resistant mutations induced efficient expression of Luciferase messenger RNA in the presence of noncytotoxic doses of DMA-135. Nuclear magnetic resonance indicates that the mutations change the structure of SLII at the binding site of DMA-135 and at the surface recognized by the host protein AU-rich element/poly(U)-binding/degradation factor 1 (AUF1). Biophysical studies of complexes formed between AUF1, DMA-135, and either SLII or SLII<sup>resist</sup> show that DMA-135 stabilizes a ternary complex with AUF1-SLII but not AUF1-SLII<sup>resist</sup>. This work demonstrates how viral evolution elucidates the (DMA-135)-RNA binding site specificity in cells and provides insights into the viral pathways inhibited by the antiviral.

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