{-}{-}{-}{-}{-}{-}{-}{-}RNA structures within Venezuelan equine encephalitis virus E1 alter macrophage replication fitness and contribute to viral emergence
Venezuelan equine encephalitis virus (VEEV) is a mosquito-borne +ssRNA virus belonging to the Togaviridae. VEEV is found throughout Central and South America and is responsible for periodic epidemic/epizootic outbreaks of febrile and encephalitic disease in equines and humans. Endemic/enzootic VEEV is transmitted between Culex mosquitoes and sylvatic rodents, whereas epidemic/epizootic VEEV is transmitted between mosquitoes and equids, which serve as amplification hosts during outbreaks. Epizootic VEEV emergence has been shown to arise from mutation of enzootic VEEV strains. Specifically, epizootic VEEV has been shown to acquire amino acid mutations in the E2 viral glycoprotein that facilitate viral entry and equine amplification. However, the abundance of synonymous mutations which accumulate across the epizootic VEEV genome suggests that other viral determinants such as RNA secondary structure may also play a role in VEEV emergence. In this study we identify novel RNA structures in the E1 gene which specifically alter replication fitness of epizootic VEEV in macrophages but not other cell types. We show that SNPs are conserved within epizootic lineages and that RNA structures are conserved across different lineages. We also identified several novel RNA-binding proteins that are necessary for altered macrophage replication. These results suggest that emergence of VEEV in nature requires multiple mutations across the viral genome, some of which alter cell-type specific replication fitness in an RNA structure-dependent manner. AUTHOR SUMMARYUnderstanding how viral pathogens emerge is critical for ongoing surveillance and outbreak preparedness. However, our understanding of the molecular mechanisms that drive viral emergence are still not completely understood. Emergence of the mosquito-borne virus Venezuelan equine encephalitis virus (VEEV) is known to require mutations in the viral attachment protein (E2), which drive viremia and transmission. We have observed that emergent strains (epizootic VEEV) also accumulate many silent mutations, suggesting that other determinants independent of protein sequence also contributes to emergence. In this study we identify novel RNA secondary structures associated with epizootic VEEV that alters viral replication in a cell-type dependent manner. We show that these RNA structures are conserved across epizootic viruses and identify host proteins that specifically bind these RNAs. These findings imply that viral emergence requires multiple mutations, a number of which likely alter viral structure in a manner that benefits viral replication and transmission.