Beth Gold

Virus recognition by Toll-7 activates antiviral autophagy in Drosophila

Innate immunity is highly conserved and relies on pattern recognition receptors (PRRs) such as Toll-like receptors (identified through their homology to Drosophila Toll) for pathogen recognition. Although Drosophila Toll is vital for immune recognition and defense, roles for the other eight Drosophila Tolls in immunity have remained elusive. Here we have shown that Toll-7 is a PRR both in vitro and in adult flies; loss of Toll-7 led to increased vesicular stomatitis virus (VSV) replication and mortality. Toll-7, along with additional uncharacterized Drosophila Tolls, was transcriptionally induced by VSV infection. Furthermore, Toll-7 interacted with VSV at the plasma membrane and induced antiviral autophagy independently of the canonical Toll signaling pathway. These data uncover an evolutionarily conserved role for a second Drosophila Toll receptor that links viral recognition to autophagy and defense and suggest that other Drosophila Tolls may restrict specific as yet untested pathogens, perhaps via noncanonical signaling pathways.

Efficient RNA virus control in Drosophila requires the RNA methyltransferase Dnmt2.

Drosophila use small‐interfering RNA mechanisms to limit the amplification of viral genomes. However, it is unclear how small RNA interference components recognize and separate viral from cellular RNA. Dnmt2 enzymes are highly conserved RNA methyltransferases with substrate specificity towards cellular tRNAs. We report here that Dnmt2 is required for efficient innate immune responses in Drosophila. Dnmt2 mutant flies accumulate increasing levels of Drosophila C virus and show activated innate immune responses. Binding of Dnmt2 to DCV RNA suggests that Dnmt2 contributes to virus control directly, possibly by RNA methylation. These observations demonstrate a role for Dnmt2 in antiviral defence.

Genome-wide RNAi screen identifies broadly-acting host factors that inhibit arbovirus 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.

Stem loop recognition by DDX17 facilitates miRNA processing and antiviral defense

DEAD-box helicases play essential roles in RNA metabolism across species, but emerging data suggest that they have additional functions in immunity. Through RNAi screening, we identify an evolutionarily conserved and interferon-independent role for the DEAD-box helicase DDX17 in restricting Rift Valley fever virus (RVFV), a mosquito-transmitted virus in the bunyavirus family that causes severe morbidity and mortality in humans and livestock. Loss of Drosophila DDX17 (Rm62) in cells and flies enhanced RVFV infection. Similarly, depletion of DDX17 but not the related helicase DDX5 increased RVFV replication in human cells. Using crosslinking immunoprecipitation high-throughput sequencing (CLIP-seq), we show that DDX17 binds the stem loops of host pri-miRNA to facilitate their processing and also an essential stem loop in bunyaviral RNA to restrict infection. Thus, DDX17 has dual roles in the recognition of stem loops: in the nucleus for endogenous microRNA (miRNA) biogenesis and in the cytoplasm for surveillance against structured non-self-elements.

Microbiota-Dependent Priming of Antiviral Intestinal Immunity in Drosophila

Enteric pathogens must overcome intestinal defenses to establish infection. In Drosophila, the ERK signaling pathway inhibits enteric virus infection. The intestinal microflora also impacts immunity but its role in enteric viral infection is unknown. Here we show that two signals are required to activate antiviral ERK signaling in the intestinal epithelium. One signal depends on recognition of peptidoglycan from the microbiota, particularly from the commensal Acetobacter pomorum, which primes the NF-kB-dependent induction of a secreted factor, Pvf2. However, the microbiota is not sufficient to induce this pathway; a second virus-initiated signaling event involving release of transcriptional paused genes mediated by the kinase Cdk9 is also required for Pvf2 production. Pvf2 stimulates antiviral immunity by binding to the receptor tyrosine kinase PVR, which is necessary and sufficient for intestinal ERK responses. These findings demonstrate that sensing of specific commensals primes inflammatory signaling required for epithelial responses that restrict enteric viral infections.

Genome-wide RNAi screen identifies SEC61A and VCP as conserved regulators of Sindbis virus entry

Alphaviruses are a large class of insect-borne human pathogens and little is known about the host-factor requirements for infection. To identify such factors, we performed a genome-wide RNAi screen using model Drosophila cells and validated 94 genes that impacted infection of Sindbis virus (SINV), the prototypical alphavirus. We identified a conserved role for SEC61A and valosin-containing protein (VCP) in facilitating SINV entry in insects and mammals. SEC61A and VCP selectively regulate trafficking of the entry receptor NRAMP2, and loss or pharmacological inhibition of these proteins leads to altered NRAMP2 trafficking to lysosomal compartments and proteolytic digestion within lysosomes. NRAMP2 is the major iron transporter in cells, and loss of NRAMP2 attenuates intracellular iron transport. Thus, this study reveals genes and pathways involved in both infection and iron homeostasis that may serve as targets for antiviral therapeutics or for iron-imbalance disorders.

Nup98 promotes antiviral gene expression to restrict RNA viral infection in Drosophila

Significance The innate immune system is a highly conserved mode of defense that induces gene expression programs to restrict microbial infections. However, much remains unknown about how the target genes are poised for their rapid induction. Using a Drosophila model, we demonstrate that Nup98 plays an essential antiviral role in insects against human insect-borne viruses. Although Nup98 is known for its role in nuclear-cytoplasmic transport, our data suggest that this antiviral function is not at the nuclear pore, rather at promoters controlling expression of a subset of virus-induced genes. Our findings suggest that the Nup98 primes virus-stimulated genes by regulating the occupancy of active RNA polymerase at these promoters poising them for rapid induction, thereby coordinating a robust and complex antiviral response. In response to infection, the innate immune system rapidly activates an elaborate and tightly orchestrated gene expression program to induce critical antimicrobial genes. While many key players in this program have been identified in disparate biological systems, it is clear that there are additional uncharacterized mechanisms at play. Our previous studies revealed that a rapidly-induced antiviral gene expression program is active against disparate human arthropod-borne viruses in Drosophila. Moreover, one-half of this program is regulated at the level of transcriptional pausing. Here we found that Nup98, a virus-induced gene, was antiviral against a panel of viruses both in cells and adult flies since its depletion significantly enhanced viral infection. Mechanistically, we found that Nup98 promotes antiviral gene expression in Drosophila at the level of transcription. Expression profiling revealed that the virus-induced activation of 36 genes was abrogated upon loss of Nup98; and we found that a subset of these Nup98-dependent genes were antiviral. These Nup98-dependent virus-induced genes are Cdk9-dependent and translation-independent suggesting that these are rapidly induced primary response genes. Biochemically, we demonstrate that Nup98 is directly bound to the promoters of virus-induced genes, and that it promotes occupancy of the initiating form of RNA polymerase II at these promoters, which are rapidly induced on viral infection to restrict human arboviruses in insects.

The Transcription Factor FoxK Participates with Nup98 To Regulate Antiviral Gene Expression

ABSTRACT 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. IMPORTANCE Innate immunity is characterized by rapid gene expression programs, from insects to mammals. Furthermore, we find that Nup98, known for its roles in the nuclear pore, plays a noncanonical role in binding the promoters and poising a subset of loci for rapid antiviral gene induction. It was unclear how Nup98 accesses these specific genes, and we here demonstrate that Nup98 cooperates with the transcription factor FoxK to regulate this gene expression program. Depletion of FoxK specifically reduces the induction of Nup98-dependent genes. Further, we find that the antiviral function of FoxK is conserved, as the human ortholog FOXK1 is also antiviral and regulates gene expression from virus-induced promoters. Although other forkhead transcription factors have been implicated in immunity, a role for FoxK in antiviral defense was previously unappreciated. Our findings reveal a conserved and novel role for FoxK in coordinating with Nup98 to promote a robust and complex antiviral transcriptional response. Innate immunity is characterized by rapid gene expression programs, from insects to mammals. Furthermore, we find that Nup98, known for its roles in the nuclear pore, plays a noncanonical role in binding the promoters and poising a subset of loci for rapid antiviral gene induction. It was unclear how Nup98 accesses these specific genes, and we here demonstrate that Nup98 cooperates with the transcription factor FoxK to regulate this gene expression program. Depletion of FoxK specifically reduces the induction of Nup98-dependent genes. Further, we find that the antiviral function of FoxK is conserved, as the human ortholog FOXK1 is also antiviral and regulates gene expression from virus-induced promoters. Although other forkhead transcription factors have been implicated in immunity, a role for FoxK in antiviral defense was previously unappreciated. Our findings reveal a conserved and novel role for FoxK in coordinating with Nup98 to promote a robust and complex antiviral transcriptional response.

Aging-associated dysbiosis increases susceptibility to enteric viral infection in Drosophila

Age is associated with increased susceptibility to enteric infections, but the molecular mechanisms are unclear. We find that aged Drosophila are more susceptible to enteric viral infections and that this increase in susceptibility is due to the aged microbiota, since depletion of the microbiota or reconstitution with a young microbiome suppressed infection. Metagenomic analysis of the aged microbiome revealed dysbiosis with an increased abundance in reactive oxygen species (ROS) producing pathways. This aged microbiota drives intestinal ROS production and we could restore immune function in old flies by reducing ROS genetically or pharmacologically. Moreover, we found that reconstitution of old flies with a cocktail of commensals, including L. fructivorans and heat-killed A. pomorum, could fully restore immunity. Altogether, these findings provide a mechanistic link between age-dependent dysbiosis and antiviral immunity and show that we can restore innate protection in aged animals, suggesting that this is a treatable and reversible state.