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

Natural Resistance-Associated Macrophage Protein Is a Cellular Receptor for Sindbis Virus in Both Insect and Mammalian Hosts

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

Natural Resistance-associated Macrophage Protein (NRAMP) is a cellular receptor for Sindbis virus in both insect and mammalian hosts

Alphaviruses, 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 for 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 non-permissive 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 multi-pass membrane proteins for infection

Weak Cross-Lineage Neutralization by Anti SARS-CoV-2 Spike Antibodies after Natural Infection or Vaccination Is Rescued by Repeated Immunological Stimulation

After over one year of evolution, through billions of infections in humans, SARS-CoV-2 has evolved into a score of slightly divergent lineages. A few different amino acids in the spike proteins of these lineages can hamper both natural immunity against reinfection, and vaccine efficacy. In this study, the in vitro neutralizing potency of sera from convalescent COVID-19 patients and vaccinated subjects was analyzed against six different SARS-CoV-2 lineages, including the latest B.1.617.2 (or Delta variant), in order to assess the cross-neutralization by anti-spike antibodies. After both single dose vaccination, or natural infection, the neutralizing activity was low and fully effective only against the original lineage, while a double dose or a single dose of vaccine, even one year after natural infection, boosted the cross-neutralizing activity against different lineages. Neither binding, nor the neutralizing activity of sera after vaccination, could predict vaccine failure, underlining the need for additional immunological markers. This study points at the importance of the anamnestic response and repeated vaccine stimulations to elicit a reasonable cross-lineage neutralizing antibody response

RUVBL1 and XPO1 restrict viral infection in mammalian cells.

<p><b>A–D.</b> Human U2OS cells were transfected with siRNAs against a control, hRuvBL1, or hXPO1 and challenged 3 days post transfection with WNV-KUN for 20 hours (<b>A–B</b>) or VSV for 12 hours (<b>C–D</b>). Cells were fixed, processed for microscopy and quantified in <b>A, C</b>. Mean ± SD of fold change compared to control for 3 independent experiments; * p<0.05, **p<0.01. Cells were processed for northern blots and quantified displaying the mean for 3 independent experiments with control set to 1; * p<0.05, **p<0.01 in <b>B, D</b>. <b>E</b>. 293T cells were transfected with siRNAs against control or two independent siRNAs against hTIP60 and challenged 3 days post transfection with WNV for 24 hours and processed by flow cytometry. Three independent experiments were quantified; Mean ± SD of the fold change in infection is shown and normalized to the control; **p<0.01. <b>F</b>. Primary neurons transduced with lentiviruses expressing the indicated shRNAs were infected with WNV for 24 hours and processed for viral yield by focus forming assays. Mean ± SD for 3 independent experiments; * p<0.05, **p<0.01. <b>G–H</b>. U2OS cells were treated with vehicle or LMB and infected with (<b>G</b>) WNV-KUN or (<b>H</b>) VSV. Mean ± SD of fold change in percent infection compared to control (vehicle) for 3 independent experiments; * p<0.05, ** p<0.01.</p

dRUVBL1 is a broadly antiviral gene.

<p><b>A.</b> Representative images of <i>Drosophila</i> cells treated with control (β-gal) or dRUVBL1 dsRNA, and infected with WNV, WNV-KUN, DEN, SIN, RVFV, or VSV (blue, nuclei; green, virus). <b>B</b>, Quantification of fold change in infection for dsRNA treated cells as in A. Mean ± SD for 3 independent experiments; * p<0.05, ** p<0.01. <b>C–D</b>. Viral RNA levels measured using qRT-PCR in <i>Drosophila</i> cells treated with β-gal (control) or dRUVBL1 dsRNA infected with WNV (<b>C</b>) or VSV (<b>D</b>) Mean ± SD of fold change for 3 independent experiments; ** p<0.01. <b>E–H</b>. Adult flies of the indicated genotypes were challenged with vehicle or WNV-KUN (<b>E–F</b>) or VSV (<b>G–H</b>). Mortality was monitored as a function of time post-infection (<b>E,G)</b> (log rank: * p<0.05, ** p<0.01). (<b>F,H)</b> Groups of 15 flies of the indicated genotypes were challenged, and viral titers were assessed by plaque assay in 4–7 independent experiments (shown as individual dots) with controls (set to 1) and fold change shown at day 6 post infection. Line represents mean.</p

dXPO1 has antiviral activity in insects.

<p><b>A.</b> Representative images of <i>Drosophila</i> cells treated with control (β-gal) or dXPO1 dsRNA, and infected with WNV, WNV-KUN, DEN, SIN, RVFV, or VSV (blue, nuclei; green, virus). <b>B</b>. Quantification of fold change in infection for dsRNA treated cells as in <b>A</b>. Mean ± SD for 3 independent experiments; * p<0.05, ** p<0.01. <b>C–D</b>. Viral RNA levels measured using RT-qPCR in Drosophila cells treated with β-gal (control) or dXPO1 dsRNA and infected with WNV (<b>C</b>) or VSV (<b>D</b>). Mean ± SD of fold change for 3 independent experiments; * p<0.05. <b>E–H</b>. Adult flies of the indicated genotypes were challenged with vehicle or WNV-KUN (<b>E, G</b>) or VSV (<b>F, H</b>) and mortality (<b>E, F</b>) was monitored as a function of time post-infection (** p<0.01 log rank). (<b>G, H</b>) Groups of 15 flies of the indicated genotypes were challenged, and viral titer was assessed by plaque assay in 3 or 4 independent experiments (shown as individual dots) with controls (set to 1) and fold change shown at day 6 post infection. Line represents mean. <b>I–J</b>. Aag2 cells were treated with the indicated dsRNA and then infected with (<b>I</b>) WNV-KUN or (<b>J</b>) VSV. Mean ± SD of fold change in percent infection compared to control (β-gal dsRNA) for 3 independent experiments; * p<0.05, ** p<0.01.</p

Tip60 complex has antiviral activity.

<p><b>A.</b> Table of RUVBL1-associated complexes, whether the complex is dependent on RUVBL2, and other genes in the complexes tested for antiviral activity. Genes in red were found to be antiviral against both WNV and VSV. <b>B–C</b>. DL1 cells were treated with the indicated dsRNA and then infected with (<b>B</b>) WNV or (<b>C</b>) VSV. Mean ± SD of fold change in percent infection compared to control (bgal dsRNA) for 3 independent experiments; * p<0.05, ** p<0.01. <b>D–E</b>. Aag2 cells were treated with the indicated dsRNA and then infected with (<b>D</b>) WNV-KUN or (<b>E</b>) VSV. Mean ± SD of fold change in percent infection compared to control (bgal dsRNA) for 3 independent experiments; * p<0.05, ** p<0.01.</p

Genome-wide RNAi screen in <i>Drosophila</i> for host factors involved in WNV infection.

<p><b>A.</b> Representative images of DL1 cells treated with the indicated dsRNAs and infected with WNV (nuclei, blue; WNV NS1, green). <b>B</b>. Quantification of fold change in infection for dsRNA treated cells as in A. Mean ± SD for 3 independent experiments; ** p<0.01. <b>C</b>. Schematic of screening pipeline including the scatter plot of Robust Z-scores for each gene assayed in duplicate. VSFs (376) and VRFs (161) are noted. <b>D</b>. Bioinformatics show fraction of candidate genes that have human or mosquito orthologs. Significant enrichment of conserved genes (p<0.0001) and under-enrichment of <i>Drosophila</i>-specific genes (p<0.0001) as analyzed by chi-squared test. <b>E</b>. Pie chart of candidate genes and validation results (50 VRF, 96 VSF, 71 not validated). <b>F–G</b>. Gene ontology enrichment of validated genes with five or more members displayed (p<0.001). <b>F</b>. VSF categories enriched. <b>G</b>. VRF categories enriched.</p

dXPO1 targets dALDOA and restricts viral infection.

<p><b>A.</b> Table of genes whose mRNA export is LMB-dependent, their level of induction by VSV infection, and whether they have been identified as antiviral previously. <b>B</b>. Schematic overview of glycolysis pathway (red, Aldolase (Ald); blue, enzymes tested; black, enzymes not tested; green, inhibitors). <b>C–D</b>. DL1 cells were treated with the indicated dsRNA and then infected with (<b>C</b>) WNV-KUN or (<b>D</b>) VSV. Data is presented as Mean ± SD of fold change in percent infection compared to control (β-gal dsRNA) for 3 independent experiments; * p<0.05.</p