A conserved opal termination codon optimizes a temperature-dependent trade-off between protein production and...

Most mosquito-transmitted alphaviruses encode a premature opal termination codon upstream of their viral polymerase. We show that the Sindbis virus (SINV) opal codon outperforms other stop codons in primate cells at 37°C due to optimal translational readthrough. However, increased readthrough of all stop codons reduces opal preference at 28°C in primate and mosquito cells. Opal also outperforms all sense codons because opal-to-sense substitutions lead to excess polyprotein production at 37°C, disrupting orderly polyprotein processing and production of viral genomic RNAs (gRNAs) required for virus production. Increased readthrough at 28°C dampens the fitness advantages of opal codons. Unexpectedly, we find that a naturally occurring SINV mutation restores sense-codon fitness by further delaying polyprotein processing, allowing adequate time to produce gRNAs. Similar temperature-dependent mechanisms occur in the distantly related dual-host alphavirus, Ross River virus. Our work highlights sophisticated strategies dual-host alphaviruses use to optimize replication in divergent temperatures through a single codon.