Type I interferon-stimulated genes (ISGs) have critical roles in inhibiting virus replication and dissemination. Despite advances in understanding of the molecular basis of restriction by ISGs, the antiviral mechanisms of many remain unclear. The 20 kDa ISG, ISG20, is a nuclear 3’-5’ exonuclease with preference for ssRNA, which has been implicated in the IFN-mediated restriction of several RNA viruses. While the exonuclease activity of ISG20 has been shown to degrade viral RNA in vitro, these findings have not been reconciled with proposed effects of ISG20 against RNA viruses that replicate in the cell cytoplasm. In the present study, we utilize a combination of an inducible, overexpression system for murine ISG20 and Isg20-/- mice to investigate mechanisms and consequences of ISG20-mediated restriction of alphaviruses. Overexpressed ISG20 primarily localized to Cajal bodies in the nucleus and potently restricted chikungunya virus and Venezuelan equine encephalitis virus replication by inhibiting the translation of infecting genomic RNA. However, degradation of viral RNAs was not observed. Translation inhibition was associated with an ISG20-induced upregulation of over 100 other gene products, many of which possess known antiviral activity. ISG20-responsive gene upregulation correlated with IRF3 activity among other transcription factors. Importantly, ISG20 modulated the production of IFIT1, an ISG that suppresses translation of RNAs possessing the type-0 5’ cap structure such as the alphavirus genome. Consistent with this, the replication and virulence of IFIT1-sensitive alphaviruses was significantly increased in Isg20-/- compared to congenic wild-type mice. Our findings establish an indirect role for ISG20 in the early restriction of RNA virus replication by altering regulation of other ISGs that inhibit virus translation and possibly other viral activities in the replication cycle
