ESCPE-1 mediates retrograde endosomal sorting of the SARS-CoV-2 host factor Neuropilin-1

Significance To facilitate internalization into the host cell’s endosomal network, viruses recognize cell surface receptors and host factors. Upon network entry, the dynamic process of endosomal sorting underpins infection by regulating the intracellular trafficking and turnover of host factors and associated pathogenic proteins. Here, we identify Neuropilin-1, a host factor for infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Epstein-Barr virus (EBV), and human T cell lymphotropic virus type 1 (HTLV-1), as a direct cargo sorted by the endosomal SNX-BAR sorting complex promoting exit 1 (ESCPE-1). Disruption of this process perturbs NRP1-dependent endosomal trafficking of the SARS-CoV-2 spike protein, thus revealing an intracellular pathway that may contribute to the mechanism of SARS-CoV-2 infection and a number of other pathogenic viruses.

3 under these short simulation conditions. This conformation of the NRP1-SNX5 complex became one of the models proposed for experimental exploration.
for the identification of the HRP-TGN46-labelled proteome. Scale bars = 20 µm. (F) Volcano plot of proteins identified following streptavidin affinity isolation displayed as a ratio of heavy (HeLa + BP + H2O2) over light (HRP-TGN46 -BP + H2O2) abundance. Proteins significantly enriched in the heavy condition (p < 0.05, Log2 fold change >2) were filtered out of subsequent analysis. Asterisks represent significantly enriched proteins with known endogenous biotin-binding affinity.

Movie S1 (separate file).
HeLa cells were transfected with GFP-Nrp1. 24 hours after transfection, cells were live imaged using a confocal laser-scanning microscope at 37ºC and incidences of GFP-Nrp1 (greyscale) localisation on tubular structures were observed. Representative frames are displayed in Fig. S3D.
Representative frames are displayed in Figure 3D.
Representative frames are displayed in Fig. 3E.

Dataset S1 (separate file).
Complete list of proteins identified by SILAC-based proteomics following proximity labelling by HRP-TGN46. Untransfected HeLa cells were labelled in light (R0K0) SILAC media and treated with BP + H2O2, HRP-TGN46-expressing HeLa cells were labelled in medium (R6K4) SILAC media and treated with BP + H2O2, and HRP-TGN46-expressing HeLa cells were treated with BP in the absence of H2O2. N = 5 independent replicates. 10 proteins that were significantly enriched in the heavy (HRP-TGN46 expressing cells incubated with BP in the absence of H2O2) relative to untransfected HeLa cells, some of which were endogenous biotin-binding proteins, are highlighted in red and were removed from subsequent analyses.