SARS-CoV-2, the causative agent of COVID-19, uses the viral Spike (S) protein for host cell attachment and entry. The host protease furin cleaves the full-length precursor S glycoprotein into two associated polypeptides: S1 and S2. Cleavage of S generates a polybasic Arg-Arg-Ala-Arg C-terminal sequence on S1, which conforms to a C-end rule (CendR) motif that binds to cell surface Neuropilin-1 (NRP1) and Neuropilin-2 (NRP2) receptors. Here, we used X-ray crystallography and biochemical approaches to show that the S1 CendR motif directly bound NRP1. Blocking this interaction using RNAi or selective inhibitors reduced SARS-CoV-2 entry and infectivity in cell culture. NRP1 thus serves as a host factor for SARS-CoV-2 infection and may potentially provide a therapeutic target for COVID-19.

¶ These authors contributed equally. ∬ These authors contributed equally. § These authors jointly supervised this work and are joint corresponding authors. SARS-CoV-2 is the causative agent of COVID-19, a coronavirus disease thathas infected more than 6.6 million people and caused over 390,000 deaths worldwide 1,2 . The Spike (S) protein of the virus forms projections on the virion surface responsible for host cell attachment and penetration. This viral glycoprotein is synthesized as a precursor in infected cells and, to be active, must be cleaved to two associated polypeptides: S1 and S2 (3,4) . For SARS-CoV-2 the cleavage is catalysed by furin, a host cell protease, which cleaves the S protein precursor at a specific sequence motif that generates a polybasic Arg-Arg-Ala-Arg (RRAR) C-terminal sequence on S1. This sequence motif conforms to the C-end rule (CendR), which means that the C-terminal sequence may allow the protein to associate with cell surface neuropilin-1 (NRP1) and neuropilin-2 (NRP2) receptors 5 . Here we demonstrate using immunoprecipitation, site-specific mutagenesis, structural modelling, and antibody blockade that, in addition to engaging the known receptor ACE2, S1 can bind to NRP1 through the canonical CendR mechanism. This interaction enhances infection by SARS-CoV-2 in cell culture. NRP1 thus serves as a host factor for SARS-CoV-2 infection, and provides a therapeutic target for COVID- 19.A striking difference in the S protein of SARS-CoV-2 and SARS-CoV is the presence, in the former, of a polybasic sequence motif, RRAR, at the S1/S2 boundary. It provides a cleavage site for a proprotein convertase, furin, a membrane-bound host cell protease [3][4][5] (Figure 1A). The resulting two proteins, S1 and S2, remain noncovalently associated, with the serine protease TMPRSS2 further priming the S2 protein by proteolytic cleavage 6 . Several observations indicate that the furinmediated processing of the S protein increases the infection and affects the tropism of SARS-CoV-2 (3)(4)(5) . Proprotein convertase inhibitors that target furin robustly diminish SARS-CoV-2 entry into Calu-3 and HeLa cells exogenously expressing ACE2 (7) . Moreover, furin knockdown impairs S processing, and abrogation of the polybasic site in the S reduces syncytia formation in infected cells 4,7 .We noticed that the C-terminus of the S1 protein generated by furin has a polybasic amino acid sequence ( 682 RRAR 685 ), that conforms to a [R/K]XX[R/K] motif, termed the 'C-end rule' (CendR) (Figure 1B) 8 . CendR motifs bind to neuropilin-1 (NRP1) and NRP2, dimeric transmembrane receptors that regulate pleiotropic biological processes, including axon guidance, angiogenesis and vascular permeability 8-10 .To explore the possible association of the SARS-CoV-2 S1 protein with neuropilins we engineered a HEK293T cell line to stably express SARS-CoV-2 S protein. In this line we transiently expressed full length NRP1 tagged at the C-terminus with GFP were treated with the particle-mesh Ewald's method and a long-range dispersion co...

The membrane phospholipid phosphatidylinositol is the precursor of a family of lipid second-messengers, known as phosphoinositides, which differ in the phosphorylation status of their inositol group. A major advance in understanding phosphoinositide signalling has been the identification of a number of highly conserved modular protein domains whose function appears to be to bind various phosphoinositides. Such 'cut and paste' modules are found in a diverse array of multidomain proteins and recruit their host protein to specific regions in cells via interactions with phosphoinositides. Here, with particular reference to proteins involved in membrane traffic pathways, we discuss recent advances in our understanding of phosphoinositide-binding domains.

Autophagy, a lysosomal degradation pathway, plays an essential role in multiple aspects of immunity, including immune system development, regulation of innate and adaptive immune and inflammatory responses, selective degradation of intracellular microbes, and host protection against infectious diseases 1 , 2 . Unlike autophagy induction by stimuli such as nutrient deprivation and mTOR suppression, little is known about how autophagosomal biogenesis is initiated in mammalian cells in response to viral infection. We performed genome-wide siRNA screens and found that the endosomal protein sorting nexin 5 (SNX5) 3 , 4 is essential for virus-induced, but not for basal, stress- or endosome-induced, autophagy. We showed that SNX5 deletion increases cellular susceptibility to viral infection in vitro , and that Snx5 knockout in mice enhances lethality after infection with multiple human viruses. Mechanistically, SNX5 interacts with beclin 1 and ATG14-containing Class III phosphatidylinositol 3-kinase (PI3KC3) complex 1 (PI3KC3-C1), increases the lipid kinase activity of purified PI3KC3-C1, and is required for endosomal generation of PI3P and recruitment of the PI3P-binding protein WIPI2 to virion-containing endosomes. These findings identify a context- and organelle-specific mechanism – SNX5-dependent PI3KC3-C1 activation at endosomes – for autophagy initiation during viral infection.

Ras proteins are binary switches that, by cycling between inactive GDP-bound and active GTP-bound conformations, regulate multiple cellular signalling pathways including those that control cell growth, differentiation and survival. Approximately 30% of all human tumours express Ras-containing oncogenic mutations that lock the protein into a constitutively active conformation. The activation status of Ras is regulated by two groups of proteins: GEFs (guanine nucleotide-exchange factors) bind to Ras and enhance the exchange of GDP for GTP, thereby activating it, whereas GAPs (GTPase-activating proteins) inactivate Ras by binding to the GTP-bound form and enhancing the hydrolysis of the bound nucleotide back to GDP. In this review, we focus on a group of key regulators of Ras inactivation, the GAP1 family of Ras-GAPs. The members of this family are GAP1m, GAP1IP4BP, CAPRI (Ca2+-promoted Ras inactivator) and RASAL (Ras-GTPase-activating-like protein) and, as we will discuss, they are emerging as important modulators of Ras and small GTPase signalling that are subject to regulation by a diverse array of events and second messenger signals.

The concentration of essential micronutrients, such as copper (used here to describe both Cu+ and Cu2+), within the cell is tightly regulated to avoid their adverse deficiency and toxicity effects. Retromer-mediated sorting and recycling of nutrient transporters within the endo-lysosomal network is an essential process in regulating nutrient balance. Cellular copper homeostasis is regulated primarily by two transporters: the copper influx transporter copper transporter 1 (CTR1; also known as SLC31A1), which controls the uptake of copper, and the copper-extruding ATPase ATP7A, a recognised retromer cargo. Here, we show that in response to fluctuating extracellular copper, retromer controls the delivery of CTR1 to the cell surface. Following copper exposure, CTR1 is endocytosed to prevent excessive copper uptake. We reveal that internalised CTR1 localises on retromer-positive endosomes and, in response to decreased extracellular copper, retromer controls the recycling of CTR1 back to the cell surface to maintain copper homeostasis. In addition to copper, CTR1 plays a central role in the trafficking of platinum. The efficacy of platinum-based cancer drugs has been correlated with CTR1 expression. Consistent with this, we demonstrate that retromer-deficient cells show reduced sensitivity to the platinum-based drug cisplatin.

Centaurin-alpha is a 46 kDa in vitro binding protein for the lipid second messenger PtdIns(3,4,5)P3. In this report we have addressed whether centaurin-alpha1, a human homologue of centaurin-alpha, binds PtdIns(3,4,5)P3 in vivo and furthermore, identified a potential physiological function for centaurin-alpha1. Using confocal microscopy of live PC12 cells, transiently transfected with a chimera of green fluorescent protein (GFP) fused to the N-terminus of centaurin-alpha1 (GFP-centaurin-alpha1), we demonstrated the rapid plasma membrane recruitment of cytosolic GFP-centaurin-alpha1 following stimulation with either nerve growth factor or epidermal growth factor. This recruitment was dependent on the centaurin-alpha1 pleckstrin homology domains and was blocked by the PtdIns(4,5)P2 3-kinase (PI 3-kinase) inhibitors wortmannin (100 nM) and LY294002 (50 microM), and also by co-expression with a dominant negative p85. Functionally, we demonstrated that centaurin-alpha1 could complement a yeast strain deficient in the ADP-ribosylation factor (ARF) GTPase-activating protein Gcs1; a complementation that was blocked by mutagenesis of conserved cysteine residues within the ARF GTPase-activating protein analogous domain of centaurin-alpha1. Taken together, our data demonstrated that centaurin-alpha1 could potentially function as an ARF GTPase-activating protein that, on agonist stimulation, was recruited to the plasma membrane possibly through an ability to interact with PtdIns(3,4,5)P3.

Human retromer, a heterotrimer of VPS26 (VPS26A or VPS26B), VPS35 and VPS29, orchestrates the endosomal retrieval of internalised cargo and promotes their cell surface recycling, a prototypical cargo being the glucose transporter GLUT1 (also known as SLC2A1). The role of retromer in the retrograde sorting of the cation-independent mannose 6-phosphate receptor (CI-MPR, also known as IGF2R) from endosomes back to the trans -Golgi network remains controversial. Here, by applying knocksideways technology, we develop a method for acute retromer inactivation. While retromer knocksideways in HeLa and H4 human neuroglioma cells resulted in time-resolved defects in cell surface sorting of GLUT1, we failed to observe a quantifiable defect in CI-MPR sorting. In contrast, knocksideways of the ESCPE-1 complex – a key regulator of retrograde CI-MPR sorting – revealed time-resolved defects in CI-MPR sorting. Together, these data are consistent with a comparatively limited role for retromer in ESCPE-1-mediated CI-MPR retrograde sorting, and establish a methodology for acute retromer and ESCPE-1 inactivation that will aid the time-resolved dissection of their functional roles in endosomal cargo sorting.