Enterovirus Evolution Reveals the Mechanism of an RNA-Targeted Antiviral and Determinants of Viral Replication

Abstract

Selective pressures on positive-strand RNA viruses provide opportunities to establish target site specificity and mechanisms of action of antivirals. Here, Enterovirus-A71 revertant viruses with resistant mutations in the SLII IRES domain (SLIIresist) were selected at low doses of the antiviral DMA-135. The EV-A71 revertant viruses were resistant to DMA-135 at concentrations that robustly inhibit replication of wild-type virus. EV-A71 IRES structures harboring the suppressor mutations induced efficient expression of reporter Luciferase mRNA in the presence of non-cytotoxic doses of DMA-135 whereas DMA-135 dose-dependently inhibited Luciferase expression from the wild-type IRES element. NMR studies indicate that the resistant mutations change the structure of SLII at the bulge loop binding site of DMA-135 and at part of an extended surface recognized by host RNA-binding protein AUF1. Comparisons of biophysical analysis of complexes formed between AUF1, DMA-135, or either SLII or SLIIresist show that DMA-135 stabilizes a ternary complex with AUF1-SLII but not AUF1-SLIIresist. Further studies demonstrate that the hnRNP A1 protein retains binding affinity for SLIIresist, illustrating that DMA-135 inhibition and viral resistance do not perturb the SLII-hnRNP A1 arm of the regulatory axis. Taken together, this work demonstrates how viral evolution under selective pressures of small molecules can elucidate RNA binding site specificity, mechanisms of action, and provide additional insights into the viral pathways inhibited by the antiviral DMA-135.

Small Molecules Targeting Viral RNA

“…In parallel with the research on new targets, a focus must be as well the research of new molecules specifically acting on viral IRES in order not to risk targeting human IRES, which are involved in the translation of genes involved in stress and apoptosis [90], processes often ongoing in infected cells. Despite part of the compounds listed in Table 2 showing some aspecific activity, the results of prunin [7] and DMA-135 [13,74] are encouraging since a specific viral RNA targeted mechanism of action was identified thanks to the selection of resistance. These results prove that achieving specific viral inhibition by targeting the viral IRES is possible and the activity can be maintained up to in vivo [7].…”

“…The selection of resistance in vitro could reveal if the site targeted can be easily mutated, therefore hampering the further development of the molecule as an antiviral. However, the eventual selection of resistance is an important verification of the binding site that can be then re-engineered in dual luciferase assays or modeled to understand the change of the binding pocket of the molecule, as was previously done for inhibitors of the RNA of enterovirus 71 [7,13]. Importantly if the resistance mutation leads to the identification or validation of the binding site, it can also open the possibility of modifying the small molecule with a medicinal chemistry approach that could ameliorate the inhibition profile.…”

“…An important point to carefully monitor with the increase of RNA-targeting molecules will be the development of resistance. Despite the selection with some RNA-targeting antivirals targeting the IRES showed the possible insurgence of resistance [7,13], with others targeting the PRF, it was not possible to select resistant mutants and several artificial mutations were needed to observe a loss of efficacy [11,12]. Specific RNA targets might be, therefore, more resistant to mutations, particularly in coding regions since mutations in the ORF will result not only in a change of secondary or tertiary structure but also in a possible detrimental mutation in the encoded protein, with a consequent higher barrier to resistance.…”

The Repurposing of Cellular Proteins during Enterovirus A71 Infection

“…HnRNP A1 binds to a phylogenetically conserved bulge loop to change the structure of SLII, which in turn stimulates IRES activity. Mutations or deletions to the bulge loop abrogate hnRNP A1 binding, significantly attenuate IRES translation and inhibit EV-A1 replication by ~5 log 10 units [ 15 , 53 , 56 ]. Thus, the recruitment of hnRNP A1 to the EV-A71 5′-UTR is essential for efficient viral replication.…”

“…Further, this region has been successfully targeted by a small molecule antiviral DMA-135 (2-log and 5-log reduction of viral titers at 0.5 and 50 µM of DMA-135, respectively; IC 50 of 7.54 ± 0.0024 μM) where its mechanism of action is to allosterically increase the binding affinity of AUF1 to SLII, thus shifting the SLII regulatory axis towards translation repression [ 64 ]. Notably, EV-A71 can evolve drug resistance to DMA-135 by changing nucleotides that form part of the AUF1 binding site, while not perturbing the binding activity of hnRNP A1 [ 56 ]. These collective observations suggest that other small molecules with the capacity to selectively perturb specific 5′-UTR-ITAF interactions await discovery.…”