The Sindbis virus nsP3 opal codon protects viral RNA and fitness by maintaining replication spherule integrity

Most alphaviruses encode an in-frame opal stop codon between nsP3 and nsP4 in their non-structural ORF. This opal stop codon mediates a temperature-dependent balance between viral polymerase production and proteolytic processing in vertebrate hosts. Yet, why this opal codon is maintained in insect hosts is unknown. Here, we show that the nsP3 opal stop codon confers a replicative advantage to Sindbis virus (SINV) in RNAi-competent mosquito cells, but not in cells lacking RNAi. Through delays in nsP processing, the lack of opal stop codon disrupts viral replication spherule integrity and increases Dicer 2-dependent cleavage of viral RNA, resulting in higher antiviral siRNA responses to the virus. Moreover, in mammalian cells, the opal codon-mediated spherule integrity also blocks MDA5-dependent viral RNA detection and interferon signaling. Thus, the highly conserved alphavirus opal codon mediates a multipotent viral defensive strategy.