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HLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options for restoring HLA-I antigen presentation are limited, we aimed to identify druggable HLA-I pathway targets. Using iterative genome-wide screens, we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augmented B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series GSLs. These GSLs sterically impeded antibody and receptor interactions with HLA-I and diminished CD8+ T cell activation. Furthermore, a disturbed SPPL3-B3GNT5 pathway in glioma correlated with decreased patient survival. We show that the immunomodulatory effect could be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that inhibits immune recognition and represents a potential therapeutic target in cancer, infection, and autoimmunity.

Cancer cells are able to evade immune surveillance through the expression of inhibitory ligands that bind their cognate receptors on immune effector cells. Expression of Programmed Death-Ligand 1 (PD-L1) in tumor micro-environments is a major immune checkpoint for tumor-specific T cell responses, by binding to Programmed Cell Death protein-1 (PD-1) on activated and dysfunctional T cells1. The activity of myeloid cells, such as macrophages and neutrophils, is likewise regulated by a balance between stimulatory and inhibitory signals. In particular, cell surface expression of the CD47 protein has been shown to form a “don’t eat me” signal on tumor cells, by binding to SIRPα expressed on myeloid cells2–5. Using a haploid genetic screen, we here identify glutaminyl-peptide cyclotransferase-like (QPCTL) as a major component of the CD47-SIRPα checkpoint. Biochemical analysis demonstrates that QPCTL is critical for pyroglutamate formation on CD47 at the SIRPα binding site shortly after biosynthesis. Both genetic and pharmacological interference with QPCTL activity enhances antibody-dependent cellular phagocytosis and cellular cytotoxicity of tumor cells. Furthermore, interference with QPCTL expression leads to a major increase in neutrophil-mediated tumor cell killing in vivo. These data identify QPCTL as a novel target to interfere with the CD47 pathway, and thereby augment antibody therapy of cancer.

Cellular antiviral programs can efficiently inhibit viral infection. These programs are often initiated through signaling cascades induced by secreted proteins, such as type I interferons, interleukin-6 (IL-6), or tumor necrosis factor alpha (TNF-α). In the present study, we generated an arrayed library of 756 human secreted proteins to perform a secretome screen focused on the discovery of novel modulators of viral entry and/or replication. The individual secreted proteins were tested for the capacity to inhibit infection by two replication-competent recombinant vesicular stomatitis viruses (VSVs) with distinct glycoproteins utilizing different entry pathways. Fibroblast growth factor 16 (FGF16) was identified and confirmed as the most prominent novel inhibitor of both VSVs and therefore of viral replication, not entry. Importantly, an antiviral interferon signature was completely absent in FGF16-treated cells. Nevertheless, the antiviral effect of FGF16 is broad, as it was evident on multiple cell types and also on infection by coxsackievirus. In addition, other members of the FGF family also inhibited viral infection. Thus, our unbiased secretome screen revealed a novel protein family capable of inducing a cellular antiviral state. This previously unappreciated role of the FGF family may have implications for the development of new antivirals and the efficacy of oncolytic virus therapy.IMPORTANCE Viruses infect human cells in order to replicate, while human cells aim to resist infection. Several cellular antiviral programs have therefore evolved to resist infection. Knowledge of these programs is essential for the design of antiviral therapeutics in the future. The induction of antiviral programs is often initiated by secreted proteins, such as interferons. We hypothesized that other secreted proteins may also promote resistance to viral infection. Thus, we tested 756 human secreted proteins for the capacity to inhibit two pseudotypes of vesicular stomatitis virus (VSV). In this secretome screen on viral infection, we identified fibroblast growth factor 16 (FGF16) as a novel antiviral against multiple VSV pseudotypes as well as coxsackievirus. Subsequent testing of other FGF family members revealed that FGF signaling generally inhibits viral infection. This finding may lead to the development of new antivirals and may also be applicable for enhancing oncolytic virus therapy.

The extraordinary virulence of the Ebola and Marburg filoviruses has spurred intensive research into the molecular mechanisms by which they multiply and cause disease. Carette et al. use a genome-wide genetic screen in human cells to identify factors required for entry of Ebola virus. The screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann–Pick C1 (NPC1). Cote et al. report the identification of a novel benzylpiperazine adamantane diamide-derived compound that inhibits EboV infection in cell culture, with NPC1 being the target. The unexpected role for the hereditary disease gene NPC1 in Ebola virus infection may facilitate the development of antifilovirus therapeutics. Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available1. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes2. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification3,4,5. Here we describe a genome-wide haploid genetic screen in human cells to identify host factors required for Ebola virus entry. Our screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes6, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann–Pick C1 (NPC1)7. Cells defective for the HOPS complex or NPC1 function, including primary fibroblasts derived from human Niemann–Pick type C1 disease patients, are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. We show that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. Our findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.

Viruses and Congenital Disorders Mutations in genes involved in α-dystroglycan O-linked glycosylation result in posttranslation modifications associated with the congenital disease Walker-Warburg syndrome (WWS). This cellular modification is also required for efficient Lassa virus infection of cells. Jae et al. (p. 479 , published online 21 March) screened for genes involved in O-glycosylation that affected Lassa virus infection and identified candidates involved in glycosylation. Individuals from different pedigrees exhibiting WWS had unique mutations among genes identified in the genetic screen. Thus, comprehensive forward genetic screens can be used to define the genetic architecture of a complex disease.