Mutating novel interaction sites in NRP1 reduces SARS-CoV-2 spike protein internalization

The global pandemic of Coronavirus disease 2019 caused by severe acute respiratory syndrome coronavirus 2 has become a severe global health problem because of its rapid spread(1). Both angiotensin-converting enzyme 2 and neuropilin 1 provide initial viral binding sites for SARS-CoV-2 (2, 3). Here, we show that three cysteine residues located in a1/a2 and b1 domains of neuropilin 1 are necessary for SARS-CoV-2 spike protein internalization in human cells. Mutating cysteines C82, C104 and C147 altered neuropilin 1 stability and binding ability as well as cellular internalization and lysosomal translocation of the spike protein. This resulted in up to 4 times reduction in spike protein load in cells for the original, alpha and delta SARS-CoV-2 variants even in the presence of the endogenous angiotensin-converting enzyme 2 receptor. Transcriptome analysis of cells transfected with mutated NRP1 revealed significantly reduced expression of genes involved in viral infection and replication, including eight members of the ribosomal protein L, ten members of ribosomal protein S and five members of the proteasome {beta} subunit family proteins. We also observed higher expression of genes involved in suppression of inflammation and endoplasmic reticulum associated degradation. These observations suggest that these cysteines offer viable targets for therapies against COVID-19.