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SummaryAlphaviruses, including several emerging human pathogens, are a large family of mosquito-borne viruses with Sindbis virus being a prototypical member of the genus. The host factor requirements and receptors for entry of this class of viruses remain obscure. Using a Drosophila system, we identified the divalent metal ion transporter natural resistance-associated macrophage protein (NRAMP) as a host cell surface molecule required for Sindbis virus binding and entry into Drosophila cells. Consequently, flies mutant for dNRAMP were protected from virus infection. NRAMP2, the ubiquitously expressed vertebrate homolog, mediated binding and infection of Sindbis virus into mammalian cells, and murine cells deficient for NRAMP2 were nonpermissive to infection. Alphavirus glycoprotein chimeras demonstrated that the requirement for NRAMP2 is at the level of Sindbis virus entry. Given the conserved structure of alphavirus glycoproteins, and the widespread use of transporters for viral entry, other alphaviruses may use conserved multipass membrane proteins for infection.

Vector-borne viruses are an important class of emerging and re-emerging pathogens; thus, an improved understanding of the cellular factors that modulate infection in their respective vertebrate and insect hosts may aid control efforts. In particular, cell-intrinsic antiviral pathways restrict vector-borne viruses including the type I interferon response in vertebrates and the RNA interference (RNAi) pathway in insects. However, it is likely that additional cell-intrinsic mechanisms exist to limit these viruses. Since insects rely on innate immune mechanisms to inhibit virus infections, we used Drosophila as a model insect to identify cellular factors that restrict West Nile virus (WNV), a flavivirus with a broad and expanding geographical host range. Our genome-wide RNAi screen identified 50 genes that inhibited WNV infection. Further screening revealed that 17 of these genes were antiviral against additional flaviviruses, and seven of these were antiviral against other vector-borne viruses, expanding our knowledge of invertebrate cell-intrinsic immunity. Investigation of two newly identified factors that restrict diverse viruses, dXPO1 and dRUVBL1, in the Tip60 complex, demonstrated they contributed to antiviral defense at the organismal level in adult flies, in mosquito cells, and in mammalian cells. These data suggest the existence of broadly acting and functionally conserved antiviral genes and pathways that restrict virus infections in evolutionarily divergent hosts., Author Summary West Nile virus (WNV) is an insect-borne virus that has re-emerged globally and for which there are no specific therapeutics or vaccines. We set out to identify cellular factors that impact infection using Drosophila as a model insect. Using a genome-wide RNAi screen we identified a large number of genes that altered WNV infection. We focused on genes that restricted infection and validated 50 genes that were conserved from insects to humans that inhibited infection. Since WNV is a flavivirus, we tested whether additional flaviviruses were restricted by these genes and found that 17 also had antiviral activity against Dengue virus. There are additional families of insect-transmitted viruses that infect humans. Accordingly, we tested whether these genes also were antiviral against the bunyavirus Rift Valley Fever virus, the alphavirus Sindbis virus and the rhabdovirus Vesicular Stomatitis virus. From this analysis, we identified seven genes that are antiviral against all of these divergent arthropod-borne pathogens expanding our knowledge of cell-intrinsic immunity in insects. Lastly, we found that XPO1 and the Tip60 complex had antiviral activity in mammalian cells. These data demonstrate the existence of previously unknown antiviral genes that restrict infection of multiple viruses across divergent hosts.