How fitness tradeoffs of arboviruses selected for a single stop codon to become a multi-function biological sensor

Arboviruses are dual-host viruses that face fitness tradeoffs due to obligate transmission between divergent hosts, resulting in suboptimal fitness in either host. We explore these tradeoffs in a model arbovirus, Sindbis Virus (SINV), using experimental evolution and deep mutational scanning (DMS). Consistent with previous studies, we found strong evidence for fitness trade-offs after passages in human or mosquito cells. Intriguingly, most trade-offs can be attributed to temperature differences between mosquito (28ºC) and human cells (37ºC); there is no loss of fitness when SINV is forced to alternate between the two cells. We identified several convergent adaptive mutations in viral populations that affect RNA structure, structural proteins, and an opal stop codon within the non-structural polyprotein (nsP).

To better understand the nature of the trade-offs, we conducted a deeper analysis of the opal codon, which interrupts the non-structural polyprotein (nsP) open reading frame between the nsp3 and nsp4 genes in most alphaviruses. Through programmed readthrough (PRT) of the opal stop codon, the nsP genes of most alphaviruses encode two distinct polyproteins: nsP1-nsP2-nsP3 (P123) and (at a much lower rate) the longer nsP1-nsP2-nsP3-nsP4 polyprotein (P1234). Since nsP4 is the viral RNA-dependent RNA polymerase (RdRp), sufficient PRT is required for de novo viral RNA synthesis.

We showed that the nsP3 opal codon persists for two reasons. The first reason is temperature: the opal stop codon is the optimal solution to a trade-off between polymerase (nsP4) production via PRT and the polyprotein processing machinery, which is most severe at 37º but exacerbated at lower temperatures (28ºC), leading to recurrent loss of the opal codon in mosquito cells. The second reason is host immunity. Delays in nsP processing in the absence of the opal stop codon disrupt the integrity of replication spherules, which the virus constructs to ‘hide itself’ from innate immunity. As a result, loss of opal codon results in innate immune-driven antiviral responses in both mosquito and vertebrate cells. Thus, the opal codon is both a temperature sensor and an immune defense. We hypothesize that many such ‘inbuilt thermometers’ allow viruses to traverse their disparate hosts without losing fitness.