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.

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Adenovirus (Ad) enters target cells by receptor-mediated endocytosis, escapes to the cytosol, and then delivers its DNA genome into the nucleus. Here we analyzed the trafficking of fluorophore-tagged viruses in HeLa and TC7 cells by time-lapse microscopy. Our results show that native or taxol-stabilized microtubules (MTs) support alternating minus- and plus end–directed movements of cytosolic virus with elementary speeds up to 2.6 μm/s. No directed movement was observed in nocodazole-treated cells. Switching between plus- and minus end–directed elementary speeds at frequencies up to 1 Hz was observed in the periphery and near the MT organizing center (MTOC) after recovery from nocodazole treatment. MT-dependent motilities allowed virus accumulation near the MTOC at population speeds of 1–10 μm/min, depending on the cell type. Overexpression of p50/dynamitin, which is known to affect dynein-dependent minus end–directed vesicular transport, significantly reduced the extent and the frequency of minus end–directed migration of cytosolic virus, and increased the frequency, but not the extent of plus end–directed motility. The data imply that a single cytosolic Ad particle engages with two types of MT-dependent motor activities, the minus end– directed cytoplasmic dynein and an unknown plus end– directed activity.

Adenovirus type 2 (Ad2) binds the coxsackie B virus Ad receptor and is endocytosed upon activation of the αv integrin coreceptors. Here, we demonstrate that expression of dominant negative clathrin hub, eps15, or K44A-dynamin (dyn) inhibited Ad2 uptake into epithelial cells, indicating clathrin-dependent viral endocytosis. Surprisingly, Ad strongly stimulated the endocytic uptake of fluid phase tracers, coincident with virus internalization but without affecting receptor-mediated transferrin uptake. A large amount of the stimulated endocytic activity was macropinocytosis. Macropinocytosis depended on αv integrins, PKC, F-actin, and the amiloride-sensitive Na+/H+ exchanger, which are all required for Ad escape from endosomes and infection. Macropinocytosis stimulation was not a consequence of viral escape, since it occurred in K44A-dyn–expressing cells. Surprisingly, 30–50% of the endosomal contents were released into the cytosol of control and also K44A-dyn–expressing cells, and the number of fluid phase–positive endosomes dropped below the levels of noninfected cells, indicating macropinosomal lysis. The release of macropinosomal contents was Ad dose dependent, but the presence of Ad particles on macropinosomal membranes was not sufficient for contents release. We conclude that Ad signaling from the cell surface controls the induction of macropinosome formation and leakage, and this correlates with viral exit to the cytosol and infection.

¶ 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...