Sequence Diversity in the 3′ Untranslated Region of Alphavirus Modulates IFIT2-Dependent Restriction in a Cell Type-Dependent Manner

Alphaviruses (family Togaviridae) are a diverse group of positive-sense RNA (+ssRNA) viruses that are transmitted by arthropods and are the causative agent of several significant human and veterinary diseases. Interferon (IFN)-induced proteins with tetratricopeptide repeats (IFITs) are a family of RNA-binding IFN-stimulated genes (ISGs) that are highly upregulated following viral infection and have been identified as potential restrictors of alphaviruses. The mechanism by which IFIT1 restricts RNA viruses is dependent on self and non-self-discrimination of RNA, and alphaviruses evade this recognition via their 5' untranslated region (UTR). However, the role of IFIT2 during alphavirus replication and the mechanism of viral replication inhibition is unclear. In this study, we identify IFIT2 as a restriction factor for Venezuelan equine encephalitis virus (VEEV) and show that IFIT2 binds the 3' 3'UTR of the virus. We investigated the potential role of variability in the 3'UTR of the virus affecting IFIT2 antiviral activity by studying infection with VEEV. Comparison of recombinant VEEV clones containing 3'UTR sequences derived from epizootic and enzootic isolates exhibited differential sensitivity to IFIT2 restriction <i>in vitro</i> infection studies, suggesting that the alphavirus 3'UTR sequence may function in part to evade IFIT2 restriction. <i>In vitro</i> binding assays demonstrate that IFIT2 binds to the VEEV 3'UTR; however, in contrast to previous studies, VEEV restriction did not appear to be dependent on the ability of IFIT2 to inhibit translation of viral RNA, suggesting a novel mechanism of IFIT2 restriction. Our study demonstrates that IFIT2 is a restriction factor for alphaviruses and variability in the 3'UTR of VEEV can modulate viral restriction by IFIT2. Ongoing studies are exploring the biological consequences of IFIT2-VEEV RNA interaction in viral pathogenesis and defining sequence and structural features of RNAs that regulate IFIT2 recognition.