Beyond ACE2 and TMPRSS2: Neuropilin-1 Receptors Enhance Cellular Entry of SARS-CoV-2

Angiotensin-converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) have been established as the sites of entry for the SARS-CoV-2 virus into cells, but a new study published in Science demonstrates that another cell surface receptor, neuropilin-1 (NRP1), enhances the infectivity of the virus in vitro and in mice (Cantuti-Castelvetri et al., 2020). Of note, NRP1 is highly expressed in the olfactory system.

The theoretical basis of this study began with the identification of a polybasic furin-type cleavage site at the S1-S2 junction in the spike protein. This polybasic cleavage site can be cleaved by the enzyme furin, which exposes a C-terminal motif that can bind and activate neuropilin receptors. The authors note that similar sequences are found in other high pathogenic viruses such as Ebola, HIV-1, and avian influenza, but are absent in SARS-CoV. To test their hypothesis that neuropilin receptors are involved in SARS-CoV-2 entry, the authors transfected HEK293T cells with ACE2, TMPRSS2, and NRP1. NRP1 expression alone did not promote infection, but coexpression of all three markedly increased infection compared to ACE2 and TMPRSS2 alone, by approximately 2-3 fold. 

The authors then obtained both wild type SARS-CoV-2 virus and a mutant of the virus that lacked the furin cleavage site and found that the mutant had significantly reduced infectivity of HEK293T cells expressing ACE2, TMPRSS2, and NRP1. To really validate the role of NRP1 in reducing infectivity through binding of C-terminal motif of the cleaved spike protein, they then utilized a monoclonal antibody that blocks NRP1 and found that the antibody reduced the infectivity of the wild type virus, but not the mutant virus.

They then identified the specific C-terminal end sequence and found that it was efficiently taken up by HEK293T cells expressing NRP1. To confirm this finding in vivo, they administered nanoparticles containing this end sequence into the nose of anesthetized mice. Six hours after administration, they  observed “a significantly larger uptake of [C-terminal end peptide] than of [modified control peptide] into the olfactory epithelium and, unexpectedly, into neurons and blood vessels of the cortex.” 

The authors then examined  if NRP1 expression correlated with SARS-CoV-2 RNA presence in human COVID-19 patients samples. Using published single-cell transcriptomes obtained from cells isolated from the bronchoalveolar lavage fluid of COVID-19 patients, they found that SARS-CoV-2 infected cells were enriched with NRP1, FURIN, and TMPRSS11A compared to non-infected cells. They also found that both NRP1 and its homolog NRP2 “were abundantly expressed in almost all pulmonary and olfactory cells, with the highest levels of expression in endothelial cells” while ACE2 expression in these tissues was relatively low. Staining of COVID-19 autopsy samples revealed infection “of the olfactory epithelium in five of six” patients. Additionally, the infected epithelial cells highly expressed NRP1. 

This novel study reveals a new player in COVID-19 with NRP1. It also sheds a lot of light on why olfactory symptoms have been so prevalent with this virus, as NRP1 is so highly expressed in the olfactory epithelium and contributes to SARS-CoV-2 infectivity. Given their result that the C-terminal sequence nanoparticles were able to travel from the olfactory epithelium to the blood vessels and neurons of the cortex of mice, I am now interested to know if this end peptide could allow an intact virus to travel effectively into the brain and cause neurological symptoms. This also makes me wonder about other potential cell surface receptors that we may be ignoring when thinking about viral entry, especially in tissues that have relatively low expression of ACE2.