Neuropilin-1 is a host factor for SARS-CoV-2 infection

Abstract

¶ 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 D614G mutation in the SARS-CoV2 Spike protein increases infectivity in an ACE2 receptor dependent manner

“…The Spike protein is the largest structural protein of the SARS-CoV2 virus (2). This type I trimeric membrane protein mediates the viral entry into target cells through the binding of its primary receptor ACE2, and possibly also interaction with additional receptors or co-receptors (7). Activation of the Spike protein for receptor binding, requires proteolytic processing by serine proteases, such as furin, at the polybasic site during its secretion from the producer cells, or alternatively by the target cell plasma membrane TMPRSS2 protease or endosomal proteases such as cathepsins, into the S1 and S2 domains (4,8).…”

No evidence for basigin/CD147 as a direct SARS-CoV-2 spike binding receptor

“…Identifying the host receptors which a virus can recognize is an important step in mechanistically explaining viral infection, and can offer insight in a virus' cellular tropism and factors influencing susceptibility. Despite the importance of determining precisely which entry receptors SARS-CoV-2 uses to infect human cells, there remains considerable uncertainty amid multiple claimed viral receptors with variable qualities of data behind them 9,[32][33][34][35] . We investigated one of the most prominent claims among these, that human BSG acts as an alternate receptor for the virus to interact with, which has been the topic of several studies, news and review articles, and a clinical trial 8,14,15,[36][37][38][39] .…”

Simulations support the interaction of the SARS-CoV-2 spike protein with nicotinic acetylcholine receptors

“…In particular, the guanidinium group of R682 is the key anchoring point to the binding pocket, where it forms several interactions with the residues that form the aromatic box. Analysis of the structure and dynamics of the full-length glycosylated S protein shows that the Y674-R685 region protrudes outside the glycan shield, and is flexible, showing that it is accessible to bind to nAChRs (and to other receptors such as neuropilins 25 ). Modelling the interaction between the full-length S protein and nAChRs indicates that association is possible with the proteins in a non-parallel orientation to one another.…”