Flaviviruses infect hundreds of millions of people annually, with no antiviral therapy available1,2. We performed a genome-wide CRISPR/Cas9-based screen to identify host genes that when edited resulted in reduced flavivirus infection. We validated nine human genes required for flavivirus infectivity, and these were associated with endoplasmic reticulum (ER) functions including translocation, protein degradation, and N-linked glycosylation. In particular, a subset of ER-associated signal peptidase complex (SPCS) proteins was necessary for the proper cleavage of the flavivirus structural proteins (prM and E) and secretion of viral particles. Loss of SPCS1 expression resulted in markedly reduced yield of all Flaviviridae family members tested (West Nile, Dengue, Zika, yellow fever, Japanese encephalitis, and hepatitis C viruses), yet had little impact on alphavirus, bunyavirus, or rhabdovirus infection or the surface expression or secretion of diverse host proteins. We found that SPCS1 dependence could be bypassed by replacing the native prM protein leader sequences with a class I MHC antigen leader sequence. Thus, SPCS1, either directly or indirectly via its interactions with unknown host proteins, preferentially promotes the processing of specific protein cargo, and Flaviviridae have a unique dependence on this signal peptide processing pathway. SPCS1 and other signal processing pathway members could represent pharmacological targets for inhibiting infection of the expanding number of flaviviruses of medical concern.

Zika virus is an emerging arthropod-borne flavivirus for which there are no vaccines or specific therapeutics. We screened a library of 2000 ‘bioactive’ compounds for their ability to block Zika virus infection in three distinct cell-types with two different strains of Zika virus. Using a microscopy-based assay, we validated 38 drugs that inhibited Zika virus infection, including FDA approved nucleoside analogs. Cells expressing high levels of the attachment factor AXL can be protected from infection with receptor tyrosine kinase inhibitors, while placental-derived cells that lack AXL expression are insensitive to this inhibition. Importantly, we identified nanchangmycin as a potent inhibitor of Zika virus entry across all cell types tested including physiologically relevant primary cells. Nanchanmycin was also active against other medically relevant viruses including West Nile, dengue, and chikungunya virus that use a similar route of entry. This study provides a resource of small molecules to study Zika virus pathogenesis.

RNA degradation is tightly regulated to selectively target aberrant RNAs, including viral RNA, but this regulation is incompletely understood. Through RNAi screening in Drosophila cells, we identified the 3 ′ -to-5 ′ RNA exosome and two components of the exosome cofactor TRAMP (Trf4/5-Air1/2-Mtr4 polyadenylation) complex, dMtr4 and dZcchc7, as antiviral against a panel of RNA viruses. We extended our studies to human orthologs and found that the exosome as well as TRAMP components hMTR4 and hZCCHC7 are antiviral. While hMTR4 and hZCCHC7 are normally nuclear, infection by cytoplasmic RNA viruses induces their export, forming a cytoplasmic complex that specifically recognizes and induces degradation of viral mRNAs. Furthermore, the 3 ′ untranslated region (UTR) of bunyaviral mRNA is sufficient to confer virus-induced exosomal degradation. Altogether, our results reveal that signals from viral infection repurpose TRAMP components to a cytoplasmic surveillance role where they selectively engage viral RNAs for degradation to restrict a broad range of viruses.

Despite the cytopathic nature of influenza A virus (IAV) replication, we recently reported that a subset of lung epithelial club cells is able to intrinsically clear virus and survive infection. However, the mechanisms that drive cell survival during a normally lytic infection remained unclear. Using a loss-of-function screening approach, we discovered that the DNA mismatch repair (MMR) pathway is essential for club cell survival of IAV infection. Repair of virallyinduced oxidative damage by the DNA MMR pathway not only allowed cell survival of infection but also facilitated host gene transcription, including the expression of antiviral and stress response genes. Enhanced viral suppression of the DNA MMR pathway prevented club cell survival and increased the severity of viral disease in vivo. Altogether, these results identify previously unappreciated roles for DNA MMR as a central modulator of cellular fate and a contributor to the innate antiviral response, which together, control influenza viral disease severity.

Upon infection, pathogen recognition leads to a rapidly activated gene expression program that induces antimicrobial effectors to clear the invader. We recently found that Nup98 regulates the expression of a subset of rapidly activated antiviral genes to restrict disparate RNA virus infections in Drosophila by promoting RNA polymerase occupancy at the promoters of these antiviral genes. How Nup98 specifically targets these loci was unclear; however, it is known that Nup98 participates with transcription factors to regulate developmental-gene activation. We reasoned that additional transcription factors may facilitate the Nup98-dependent expression of antiviral genes. In a genome-wide RNA interference (RNAi) screen, we identified a relatively understudied forkhead transcription factor, FoxK, as active against Sindbis virus (SINV) in Drosophila. Here we find that FoxK is active against the panel of viruses that are restricted by Nup98, including SINV and vesicular stomatitis virus (VSV). Mechanistically, we show that FoxK coordinately regulates the Nup98-dependent expression of antiviral genes. Depletion of FoxK significantly reduces Nup98-dependent induction of antiviral genes and reduces the expression of a forkhead response element-containing luciferase reporter. Together, these data show that FoxK-mediated activation of gene expression is Nup98 dependent. We extended our studies to mammalian cells and found that the mammalian ortholog FOXK1 is antiviral against two disparate RNA viruses, SINV and VSV, in human cells. Interestingly, FOXK1 also plays a role in the expression of antiviral genes in mammals: depletion of FOXK1 attenuates virus-inducible interferon-stimulated response element (ISRE) reporter expression. Overall, our results demonstrate a novel role for FOXK1 in regulating the expression of antiviral genes, from insects to humans.