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

Bhattacharya, Tamanash; Freeman, Tiia S; Alleman, Eva M; Wang, Fang; Chechik, Lyuba; Emerman, Michael; Myles, Kevin M; Malik, Harmit S · Sci Adv · 2026

basic_science · Level V

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Abstract

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 <i>Aedes</i> 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.

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