The flaviviruses dengue virus (DENV) and Zika virus (ZIKV) are severe health threats with rapidly expanding ranges. To identify the host cell dependencies of DENV and ZIKV, we completed orthologous functional genomic screens using RNAi and CRISPR/Cas9 approaches. The screens recovered the ZIKV entry factor AXL as well as multiple host factors involved in endocytosis (RAB5C and RABGEF), heparin sulfation (NDST1 and EXT1), and transmembrane protein processing and maturation, including the endoplasmic reticulum membrane complex (EMC). We find that both flaviviruses require the EMC for their early stages of infection. Together, these studies generate a high-confidence, systems-wide view of human-flavivirus interactions and provide insights into the role of the EMC in flavivirus replication.

Physiological Ca2+ signalling in T lymphocytes and other cells depends on the STIM-ORAI pathway of store-operated Ca2+ entry. STIM1 and STIM2 are Ca2+ sensors located in the endoplasmic reticulum (ER) membrane, with ER-luminal domains that monitor cellular Ca2+ stores and cytoplasmic domains that gate ORAI channels in the plasma membrane. The STIM ER-luminal domain dimerizes or oligomerizes upon dissociation of Ca2+, but the mechanism transmitting activation to the STIM cytoplasmic domain has not been defined. Here we demonstrate, using Tb3+–acceptor energy transfer, that dimerization of STIM1 ER-luminal domains can initiate an extensive conformational change in murine STIM1 cytoplasmic domains. The conformational change, triggered by apposition of the predicted coiled-coil 1 (CC1) regions, releases the ORAI-activating domains from their interaction with the CC1 regions and allows physical extension of the STIM1 cytoplasmic domain across the gap between ER and plasma membrane to communicate with ORAI channels.

SummaryClostridium difficile toxin B (TcdB) is a critical virulence factor causing diseases associated with C. difficile infection (CDI). Here we carried out CRISPR/Cas9-mediated genome-wide screens and identified the members of the Wnt receptor Frizzled (FZDs) family as TcdB receptors. TcdB binds to the conserved Wnt-binding site known as the cysteine-rich domain (CRD), with the highest affinity toward FZD1, 2, and 7. TcdB competes with Wnt for binding to FZDs, and its binding blocks Wnt signaling. FZD1/2/7 triple-knockout (KO) cells are highly resistant to TcdB, and recombinant FZD2-CRD prevented TcdB binding to the colonic epithelium. Colonic organoids cultured from FZD7 KO mice, combined with knock-down of FZD1 and 2, showed increased resistance to TcdB. The colonic epithelium in FZD7 KO mice was less susceptible to TcdB-induced tissue damage in vivo. These findings establish FZDs as physiologically relevant receptors for TcdB in the colonic epithelium.

Store-operated Ca 2+ entry through the plasma membrane Ca 2+ release-activated Ca 2+ (CRAC) channel in mammalian T cells and mast cells depends on the sensor protein stromal interaction molecule 1 (STIM1) and the channel subunit ORAI1. To study STIM1-ORAI1 signaling in vitro, we have expressed human ORAI1 in a sec6-4 strain of the yeast Saccharomyces cerevisiae and isolated sealed membrane vesicles carrying ORAI1 from the Golgi compartment to the plasma membrane. We show by in vitro Ca 2+ flux assays that bacterially expressed recombinant STIM1 opens wild-type ORAI1 channels but not channels assembled from the ORAI1 pore mutant E106Q or the ORAI1 severe combined immunodeficiency (SCID) mutant R91W. These experiments show that the STIM1-ORAI1 interaction is sufficient to gate recombinant human ORAI1 channels in the absence of other proteins of the human ORAI1 channel complex, and they set the stage for further biochemical and biophysical dissection of ORAI1 channel gating.Ca 2+ influx through the CRAC channel in mammalian T cells and mast cells is essential for transcriptional responses and other effector responses to physiological stimuli [1][2][3][4] . Gating of the CRAC channel is a classical instance of store-operated Ca 2+ entry, where an initial release of Ca 2+ from internal cellular stores, by depleting the stores, triggers sustained Ca 2+ signaling due to opening of plasma membrane Ca 2+ channels [5][6][7] . RNA interference (RNAi) screens and determination of the genetic basis of a human severe combined immunodeficiency (SCID) syndrome have identified two proteins required for CRAC channel function: STIM1, a protein anchored in the endoplasmic reticulum (ER) that senses depletion of ER Ca 2+ stores [8][9][10] , andCorrespondence should be addressed to P.G.H. (hogan@idi.harvard.edu). 4 These authors contributed equally to this work.Note: Supplementary information is available on the Nature Structural & Molecular Biology website. AUTHOR CONTRIBUTIONSP.G.H. set overall goals for the project and coordinated the work; Y.Z., P.M., A.R. and P.G.H. designed experiments and wrote the manuscript; Y.Z. prepared and characterized the STIM1 reagents, measured STIM-ORAI interactions and conducted the assays using mammalian cells; P.M. prepared and characterized sec6-4 ORAI1 vesicles; Y.Z. and P.M. conducted the in vitro Ca 2+ flux assays; D.M. and H.T.K. developed the P. pastoris membrane-flotation assay; M.O., with Y.Z., carried out reconstitution and Ca 2+ -imaging experiments using STIM1 −/− T cells; J.Z., with Y.Z., carried out SEC-MALLS analyses; Y.H., with Y.Z., contributed confocal microscopy; A.S. helped P.M. with construction of S. cerevisiae expression plasmids. COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/nsmb/.Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions/. NIH Public Access RESULTS Expression of recombinant ORAI1 and STIM...