Amiloride inhibits the initiation of Coxsackievirus and poliovirus RNA replication by inhibiting VPg uridylylation

- Both CVB3 and PV1 RNA replication was inhibited by amiloride.
- Amiloride inhibited initiation by inhibiting VPgpUpU synthesis.
- Amiloride had no effect on polymerase elongation activity.
- Amiloride docks in the VPg binding site on the viral polymerase.
- A model is proposed in which amiloride competes with VPg for binding to polymerase.

The mechanism of amiloride inhibition of Coxsackievirus B3 (CVB3) and poliovirus type 1 (PV1) RNA replication was investigated using membrane-associated RNA replication complexes. Amiloride was shown to inhibit viral RNA replication and VPgpUpU synthesis. However, the drug had no effect on polymerase elongation activity during either (−) strand or (+) strand synthesis. These findings indicated that amiloride inhibited the initiation of RNA synthesis by inhibiting VPg uridylylation. In addition, in silico binding studies showed that amiloride docks in the VPg binding site on the back of the viral RNA polymerase, 3Dpol. Since VPg binding at this site on PV1 3Dpol was previously shown to be required for VPg uridylylation, our results suggest that amiloride inhibits VPg binding to 3Dpol. In summary, our findings are consistent with a model in which amiloride inhibits VPgpUpU synthesis and viral RNA replication by competing with VPg for binding to 3Dpol.