A conserved in-frame stop codon acts as a multipotent defense mechanism in alphaviruses

Most alphaviruses maintain an in-frame opal stop codon that interrupts their non-structural polyprotein (nsP) ORF between nsP3 and nsP4 in both vertebrate and insect hosts. We show that the nsP3 opal stop codon confers a replicative advantage to Sindbis virus (SINV) in RNAi-competent mosquito cells and in Aedes aegypti mosquitoes, but not in cells or mosquitoes lacking RNAi. Mutation of the opal stop codon delays processing of the viral nsP polyprotein, disrupts viral replication spherule integrity, and renders viral RNA susceptible to Dicer 2 cleavage, resulting in higher antiviral siRNA responses against SINV. Similarly, these defects caused by opal codon mutations lead to increased viral RNA detection and enhanced immune signaling in vertebrate cells. Thus, a single stop codon in alphaviruses mediates a multipotent viral strategy to evade innate immune defenses across diverse hosts. TeaserA conserved ORF-interrupting stop codon helps alphaviruses avoid triggering innate antiviral immunity across diverse hosts.