Photochemical Identification of Auxiliary Severe Acute Respiratory Syndrome Coronavirus 2 Host Entry Factors Using μMap

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the infectious agent of the COVID-19 pandemic, remains a global medical problem. Angiotensin-converting enzyme 2 (ACE2) was identified as the primary viral entry receptor, and transmembrane serine protease 2 primes the spike protein for membrane fusion. However, ACE2 expression is generally low and variable across tissues, suggesting that auxiliary receptors facilitate viral entry. Identifying these factors is critical for understanding SARS-Cov-2 pathophysiology and developing new countermeasures. However, profiling host-virus interactomes involves extensive genetic screening or complex computational predictions. Here, we leverage the photocatalytic proximity labeling platform μMap to rapidly profile the spike interactome in human cells and identify eight novel candidate receptors. We systemically validate their functionality in SARS-CoV-2 pseudoviral uptake assays with both Wuhan and Delta spike variants and show that dual expression of ACE2 with either neuropilin-2, ephrin receptor A7, solute carrier family 6 member 15, or myelin and lymphocyte protein 2 significantly enhances viral uptake. Collectively, our data show that SARS-CoV-2 synergistically engages several host factors for cell entry and establishes μMap as a powerful tool for rapidly interrogating host-virus interactomes.

Figure 1.

a) SARS-Cov-2 utilizes ACE2 and TMPRSS2 for cell uptake, but additional entry factors are likely. b) μMap photoproximity labeling via iridium photocatalysts that activate nearby diazirines into reactive carbenes. c) Iridium conjugation with SARS-Cov-2 spike protein enables rapid receptor identification. d) Quantitative proteomics volcano plot of candidate cell-surface proteins after incubation of Ir-spike proteins with Calu-3 cells and photolabeling. Dataset compares full-length spike protein against receptor-binding domain (RBD). ACE2 interacts with both constructs and is thus near zero enrichment.

Figure 2.. SARS-CoV-2 uptake is enhanced in cells expressing candidate receptors and ACE2.

a) Schematic for generating pseudoviral particles and infection. b-e) Virus uptake into A549 cells expressing ACE2, TMPRSS2, and/or entry factor candidates. Entry was measured as relative luminescent signal (RLU) normalized against non-enveloped particle control. Datapoints represent mean (n = 6) and error bars denote standard deviation. Independent t-tests between ACE2 and expression lines are indicated * p < 0.05, ** p < 0.01., ***, p < 0.001.

Figure 3.. NRP2, MAL2, EPHA7, and SLC6A15 significantly enhance SARS-Cov-2 entry.

a) A549 cells expressing various factors were infected with SARS-CoV-2pp encoding mScarlet, stained with DAPI (blue), and visualized using fluorescence microscopy for factor expression (szGreen) and viral uptake (red). Scale bar indicates 100 μm, and insets display zoomed-in regions of each image. b) Entry quantification as percentage of red: (SARS positive)/(DAPI positive), green: percentage of (SARS positive)/(Factor positive). Datapoints represent mean from (n = 3), and error bars denote standard deviation. Independent t-tests of SARS+ values between ACE2 and ACE2–TMPRSS2 triple expression lines are indicated as significant (***, p < 0.001)

Figure 4.. Candidate receptors enhance Delta variant spike-mediated uptake.

a) Delta variant spike harbors 8 mutations compared to Wuhan strain. b) Delta pseudovirus uptake in cells expressing ACE2 and candidate factors. Entry was measured as relative luminescent signal (RLU) normalized against non-enveloped particle control. Datapoints represent mean (n = 3) and error bars denote standard deviation. Independent t-tests between ACE2 and respective lines are indicated as significant * p < 0.05, ** p < 0.01, ***, p < 0.001.

Figure 5.. Candidate receptors do not enhance SARS-Cov-2 spike binding on human cells.

a) Schematic for testing binding affinity of spike protein and candidate factors. b) Apparent dissociation constants (K’_d) for SARS-Cov-2 spike from Wuhan (blue) and Delta (green) strains towards cells expressing ACE2 and various cell-surface proteins. Datapoints represent mean (n = 3), and error bars denote 95% confidence intervals.