The experiment was performed twice with representative images shown. species and insect vectors separated by hundreds of millions of years of evolutionary history. Entry into evolutionarily divergent host cells can be accomplished by recognition GNE-207 of different cellular receptors in different species, or by binding to receptors that are highly conserved across species. Although multiple alphavirus receptors have been described1C3, most are not shared among vertebrate and invertebrate hosts. Here we identify the very low-density lipoprotein receptor (VLDLR) as a receptor for the prototypic alphavirus Semliki forest virus. We show that the E2 and E1 glycoproteins (E2CE1) of Semliki forest virus, eastern equine encephalitis virus and Sindbis virus interact with the ligand-binding domains (LBDs) of VLDLR and apolipoprotein E receptor 2 (ApoER2), two closely related receptors. Ectopic expression of either protein facilitates cellular attachment, and internalization of virus-like particles, a VLDLR LBDCFc fusion protein or a ligand-binding antagonist block Semliki GNE-207 forest virus E2CE1-mediated infection of human and mouse neurons in culture. The administration of a VLDLR GNE-207 LBDCFc fusion protein has protective activity against rapidly fatal Semliki forest virus infection in mouse neonates. We further show that invertebrate receptor orthologues from mosquitoes and worms can serve as functional alphavirus receptors. We propose that the ability of some alphaviruses to infect a wide range of hosts is a result of their engagement of evolutionarily conserved lipoprotein receptors and contributes to their pathogenesis. as the top candidate (Fig. ?(Fig.1a,1a, Supplementary Table 2). VLDLR is a part of the low-density Rabbit polyclonal to ALX3 lipoprotein receptor (LDLR) family and mediates endocytosis of lipoproteins and other ligands8. Open in a separate window Fig. 1 A CRISPRCCas9 screen identifies VLDLR as a host factor for SFV E2CE1-mediated infection.a, Results of MAGeCK49 analysis for the screen performed with SFV RVPs in HEK 293T-Cas9 cells?showing enriched genes on the basis of top?robust rank aggregation (RRA) scores. b, Wild-type?(WT) cells, VLDLR-knockout (KO) cells and VLDLR-knockout cells transiently transfected with cDNA encoding VLDLR with an N-terminal Flag tag (VLDLRCFlag) were infected with SFV single-cycle RVPs expressing GFP, and infection was measured by fluorescence-activated cell sorting (FACS). VLDLR cell surface expression was monitored by immunostaining (Extended Data Fig. ?Fig.2b).2b). c, Infection of HEK 293T cells with single-cycle SFV RVPs in the presence of an antibody against VLDLR or a control antibody against human leukocyte antigen (HLA), measured by FACS. d, Infection of Vero cells with SFV or CHIKV single-cycle RVPs expressing GFP in the presence of the indicated antibodies. Cells were imaged by fluorescence microscopy. Scale bar, 100?m. The experiment was performed twice with representative images shown. e, Infection of Vero cells with replication-competent SINV chimeras expressing GFP and the structural proteins of SFV (SINV-SFV) or CHIKV (SINV-CHIKV) at a multiplicity of infection (MOI) of 1 1 in the presence of the indicated antibodies. GFP expression was measured by FACS 24?h after infection. f, Infection of HEK 293T cells with GFP-expressing single-cycle RVPs in the presence of receptor-associated protein (RAP) or transferrin (Tf) control, measured by FACS. Data are mean??s.d. from two experiments (or is included as a reporter downstream of the capsid (C) after a 2A peptide sequence. The arrow indicates a subgenomic promoter. b, SDS-PAGE gel of purified RVPs imaged with a stain-free imaging system. The experiment was performed twice independently, and a representative gel image is shown. c, Screening strategy. HEK?293T-Cas9 cells are first transduced with the guide (sgRNA) library using vesicular stomatitis virus (VSV) glycoprotein G pseudotyped lentiviruses and are then infected with RVPs expressing CD20. Infected cells are depleted using magnetic beads against CD20. Selection is repeated iteratively to improve the signal-to-noise ratio of the screen. noninfected, CD20 negative cells are sequenced GNE-207 using next.