A conserved in-frame stop codon acts as a multipotent defense mechanism in alphaviruses
- Tamanash Bhattacharya
- Tiia S. Freeman
- Eva M. Alleman
- Fang Wang
- Lyuba Chechik
- Michael Emerman
- Kevin M. Myles
- Harmit S. Malik
2026-06-17
Most alphaviruses maintain an in-frame opal stop codon that interrupts their nonstructural 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 Aedes cells and mosquitoes but not in RNAi-deficient cells or mosquitoes. 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.