ACE2: Entry Receptor for SARS-CoV-2

An outbreak of SARS-CoV-2 infections has caused a pandemic associated with a severe acute pulmonary disease named COVID-19 (coronavirus induced disease 2019) (1). A related coronavirus, SARS-CoV, led to a much smaller outbreak in 2003, possibly due to infection occurring predominantly in the lower respiratory system, whereas SARS-CoV-2 spreads rapidly through active pharyngeal viral shedding (2). Despite these differences, uptake of both viruses is mediated by the identical cellular receptor, angiotensin-converting enzyme 2 (ACE2) (3–5). One attractive hypothesis to explain the enhanced spreading of SARS-CoV-2 is the presence of a polybasic furin-type cleavage site, RRAR^S, at the S1/S2 junction in the SARS-CoV-2 spike (S) protein that is absent in SARS-CoV (6). Similar sequences are found in the S proteins of many other pathogenic human viruses, including Ebola, HIV-1 and highly virulent strains of avian influenza (6, 7). The presence of the polybasic cleavage site in SARS-CoV-2 results in enhanced pathogenicity by priming the fusion activity (8) and could potentially create additional cell surface receptor binding sites. Proteolytic cleavage of RRAR^S by furin exposes a conserved carboxyterminal (C-terminal) motif RXXROH (where R is arginine and X is any amino acid; R can be substituted by lysine, K) in the S protein. Such C-terminal sequences that conform to the “C-end rule” (CendR) are known to bind to and activate neuropilin (NRP1 and NRP2) receptors at the cell surface (9). Recent cryo-electron microscopy structures of the SARS-CoV-2 S protein demonstrated that the S1/S2 junction is part of a solvent-exposed loop and therefore accessible for receptor interactions (10, 11).

To determine whether SARS-CoV-2 can use NRP1 for virus entry and infectivity, we generated lentiviral particles pseudotyped with the SARS-CoV-2 S protein. Pseudoviruses are well suited for virus entry assays, as they allow viral entry to be distinguished from other virus life cycle steps. HEK-293T cells, which have almost no detectable ACE2 and NRP1 transcripts (fig. S1), were transfected with plasmids encoding the two established host factors (4), human ACE2 and the transmembrane protease serine 2 (TMPRSS2), or NRP1. When expressed alone, ACE2 rendered cells susceptible to infection (Fig. 1A). While NRP1 only hardly promoted infection in HEK-293T cells, its co-expression with ACE2 and TMPRSS2 markedly enhanced infection (Fig. 1, A and B). NRP1 expression increased infection in Caco-2 cells, which endogenously express ACE2 (12) (Fig. 1C and fig. S1D), showing that NRP1 can potentiate infection in the presence of other host factors. To test the specificity of NRP1-dependent virus entry, we developed monoclonal antibodies (mAbs) that were designed to functionally block the extracellular b1b2 domain of NRP1, known to mediate the binding to CendR peptides (13). The mAb3 was observed to bind to the recombinant b1b2 domain of wild-type NRP1, but not to the triple mutant b1b2 domain (S346A, E348A and T349A in the CendR binding pocket) (fig. S2A). The potency of the mAbs in preventing cellular binding and internalization of NRP ligands was tested using 80 nm silver nanoparticles (AgNP) coated with the prototypic NRP1-binding CendR peptide RPARPAROH (9) (fig. S2B). mAb3 efficiently blocked AgNP-CendR binding (fig. S2C) and internalization (fig. S2, D and E), while another monoclonal antibody, mAb2, had no effect and was used as a control in further experiments. Treatment of HEK-293T with mAb3 significantly reduced infection by SARS-CoV-2 pseudoviruses in cells expressing ACE2/TMPRSS2/NRP1 (Fig. 1D), but not in cells expressing ACE2/TMPRSS2 only (fig. S2F). When SARS-CoV-2 pseudovirus was pre-incubated with recombinant, soluble extracellular b1b2 domain of NRP1, the wild-type significantly reduced infection compared to the triple mutant (Fig. 1E and fig. S2G).



Fig. 1 NRP1 facilitates the cellular entry of SARS-CoV-2 pseudotyped particles.
(A) Representative images and quantification of SARS-CoV-2 spike protein (SARS-2) (blue bars) and VSV-G pseudotype (gray bars) infectivity in HEK-293T cells transiently expressing control (ctrl) vector, ACE2, NRP1 or TMPRSS2 (TSS2). Data are normalized to the respective infectivity of SARS-2 and VSV-G pseudotype in ACE2-expressing cells. Two-way ANOVA with Tukey’s correction for multiple comparisons. (B) HEK-293T cells transiently expressing ACE2 and TMPRSS2 or NRP1, ACE2 and TMPRSS2 were inoculated with SARS-2 pseudotype. Data are normalized to SARS-2 infectivity in cells expressing ACE2 and TMPRSS2. One-way ANOVA with Tukey’s correction for multiple comparisons. (C) SARS-2 pseudotype infectivity in Caco-2 cells expressing NRP1 or control vector. Data are normalized to the respective infectivity of SARS-2 and VSV-G pseudotype in control cells. Two-way ANOVA with Sidak’s correction for multiple comparisons. (D and E) HEK-293T cells transiently expressing NRP1, ACE2 and TMPRSS2 were inoculated with SARS-2 pseudotype in the presence of mAb3 antibody against NRP1 (D, mAb3) or control mAb2 (D, ctrl Ab), and in the presence of soluble NRP1 wild-type b1b2 domain (E, wt b1b2) or the NRP1 mutant b1b2 domain (E, mut b1b2). Data in (E) are normalized to untreated cells expressing NRP1, ACE2 and TMPRSS2. Two-tailed unpaired Student’s t test. All data are represented as mean ± s.d. from three independent experiments (A to C) or three biological replicates (D and E). *p < 0.05, **p < 0.01, ****p < 0.0001. All images show GFP-positive, infected cells (magenta) and Hoechst (cyan). Scale bars, 100 μm.

Next, we explored the role of NRP1 using SARS-CoV-2 isolated from COVID-19 patients from the Helsinki University Hospital. We used wild-type SARS-CoV-2 and a cleavage-impaired SARS-CoV-2 mutant that was isolated from Vero-E6 which rapidly accumulate mutations at the furin cleavage site of the S protein over passaging (Fig. 2, A and B) (14). First, we confirmed that furin cleaved the wild-type, but not the mutant, SARS-CoV-2 S protein by analyzing S protein processing in CHO cells with functional (parental) or deficient (FD11) furin enzyme (fig. S3) (15). Next, we validated that exogenous ACE2 expression rendered HEK-293T cells susceptible to infection with SARS-CoV-2 (Fig. 2, C and D). NRP1 expression alone caused lower levels of infection, which were only detectable with increasing virus titer (Fig. 2, C and D). We then compared the ability of wild-type and mutant SARS-CoV-2 virus to infect HEK-293T stably expressing either ACE2, ACE2/TMPRSS2 or ACE2/TMPRSS2/NRP1. Infection of these cell lines by the wild-type, but not the mutant, virus increased in the presence of NRP1, providing further evidence that NRP1 requires a furin-cleaved substrate for its effects (Fig. 2, E and F). Next, we studied the effect of the NRP1-blocking antibody, mAb3, on infection of Caco-2 cells by wild-type and mutant SARS-CoV-2 virus, and found that pre-incubation with NRP1-blocking antibody reduced wild-type virus infection by ~40%, while the control mAb2 had no effect (Fig. 2, G and H). NRP1-blocking antibody had no effect on the infection by the mutated virus (Fig. 2, G and H).



Fig. 2 A blocking antibody against the b1b2 domain of NRP1 reduces infection by wild-type SARS-CoV-2 (SARS-2-wt), but not a mutant with a deletion at the furin-cleavage site (SARS-2-mut).
(A) Sequence analysis of viruses isolated at different passages (P) from different cell types. The first sequence is the reference from the Wuhan isolate (NC_045512.2). The sequence abundance in each virus population is indicated as a percentage (%). § = SARS-2-wt, §§ = SARS-2-mut, used in the experiments. (B) A deletion adjacent to the furin-cleavage site abrogates the enzymatic cleavage of the S protein. Immunoblot analysis of cell lysates from Vero-E6 cells infected for 16h with two viral populations (§ and §§). (C and D) Representative images and quantification of SARS-2-wt infectivity in HEK-293T cells stably expressing ACE2 (blue bars) or NRP1 (orange bars) compared to non-transfected cells (gray bars). Different virus titers were used. Data are normalized to the infectivity in ACE2-expressing cells at MOI 5. Two-way ANOVA with Tukey’s correction for multiple comparisons. (E and F) Representative images (E) and quantification (F) of HEK-293T cells stably expressing the indicated combinations of ACE2, TMPRSS2 (TSS2) and NRP1 following SARS-2-wt (wt, blue bars) or SARS-2-mut (mut, gray bars) inoculation. Data are normalized to the respective infectivity in ACE2-expressing cells. Two-way ANOVA with Tukey’s correction for multiple comparisons. (G and H) Caco-2 cell infection in the presence of control mAb2 (ctrl. Ab) or mAb3 blocking antibodies against NRP1 after SARS-2-wt (wt, blue bars) or SARS-2-mut (mut, gray bars) inoculation. Data are normalized to the respective vehicle control (PBS) sample. Two-way ANOVA with Tukey’s correction for multiple comparisons. Data are mean ± s.d. from three independent experiments, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. SARS-2-wt and SARS-2-mut infected cells (magenta), Hoechst (cyan). Scale bars, 50 μm.

Cleavage of SARS-CoV-2 S protein at the S1/S2 site generates the C-terminal end TQTNSPRRAROH. To determine whether this specific sequence can function as a substrate for NRP1, we used AgNPs coated with TQTNSPRRAROH peptide or different control peptides including one with a terminal amide group, which reduces NRP1 binding (TQTNSPRRARNH2) (9) (Fig. 3A). We found that AgNP-TQTNSPRRAROH, but not control AgNPs were efficiently taken up by HEK-293T cells expressing NRP1 (Fig. 3, B and C). Next, we determined whether AgNP-TQTNSPRRAROH particles were also internalized into cells in vivo. We chose to study nanoparticle entry in the mouse olfactory epithelium, owing to the known expression of NRP1 in the olfactory system (16) including olfactory neuronal cells of the epithelium (fig. S4). AgNPs-TQTNSPRRAROH and control AgNP-TQTNSPRRARNH2 were administered into the nose of anesthetized adult mice. 6 hours after administration, we observed a significantly larger uptake of AgNP-TQTNSPRRAROH compared to AgNP-TQTNSPRRARNH2 into the olfactory epithelium (Fig. 3, D and E), and unexpectedly also into neurons and blood vessels of the cortex (Fig. 3, F and G). Similar results were obtained for AgNPs coated with the prototypic NRP1-binding CendR peptide RPARPAROH (fig. S5).