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

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.


Supporting Figures
The spike trimer clearly labels more proteins than just ACE2, reflecting previously unknown host factors The same experiment was repeated with many other cell lines (A549 +/-ACE2, UH7, CACO-2, CALU-3, NULI-1 ACE2 S5 Figure S3: Quantitative proteomic analysis of enriched proteins using Ir-conjugated full-length spike trimer vs a free Ir catalyst control.
Calu-3 lung cells were incubated with free Ir catalyst or Ir-conjugated spike protein trimer for 1 hour at 4 ºC, followed by washing with PBS. Cell pellets were then reconstituted in 250 µM biotindiazirine and irradiated under blue light. Membrane lysates were then collected using the Mem-PER™ Plus Membrane Protein Extraction. After streptavidin enrichment, peptides were digested and then analyzed via quantitative proteomics. S6 Figure S4: Verification of pseudoviral entry in engineered cell lines expressing ACE2 and/or TMPRSS2.
SARS-CoV2pp (Wuhan strain) uptake of ACE2/TMPRSS2-expressing HEK293T and A549 cell lines. Cells were stably transduced with ACE2 and TMPRSS2 lentiviruses and then infected with SARS-CoV-2-Gluc or non-enveloped (NE) pseudoparticle (pp) and luciferase activity was ACE2 was detected using an anti-human ACE2 antibody combined with an AF594 secondary antibody. Lentiviral transduction was performed with a bicistronic construct against the zsGreen fluorescent protein, and entry factor candidate expression was determined by quantifying zsGreen positive cells.

ACE2 TMPRSS2
Factor X ---  A549 cells expressing various factors were infected with non-enveloped pseudoviral control particles encoding mScarlet. Cells were then stained with DAPI (blue) and visualized using immunofluorescence microscopy for both factor expression (szGreen) and viral uptake (mScarlet, red). Scale bar indicates 100 µm. S10 Figure S8: Binding of fluorophore-spike conjugate (Wuhan strain) to A549 cells expressing various factors.
A549 cells expressing various factors were incubated with increasing amounts of AlexaFluor 647conjugated recombinant spike protein (Wuhan strain) for 1 hour at 4 ºC. After washing twice with cold PBS, cells were constituted in PBS and then deposited into separate wells of a 96-well plate.
Fluorescence was then measured to quantify relative protein bound. All data are normalized to A549-ACE2 intensities and represent 3 independent trials. Error bars denote standard deviation.
Apparent dissociation constants (K'D) are denoted along with upper and lower limits for 95% confidence intervals. S11 Figure S9: Binding of fluorophore-spike conjugate (Delta variant) to A549 cells expressing various factors.
A549 cells expressing various factors were incubated with increasing amounts of AlexaFluor 647conjugated recombinant spike protein (Delta variant) for 1 hour at 4 ºC. After washing twice with cold PBS, cells were constituted in PBS and then deposited into separate wells of a 96-well plate.
Fluorescence was then measured to quantify relative protein bound. All data are normalized to A549-ACE2 intensities and represent 3 independent trials. Error bars denote standard deviation.
Apparent dissociation constants (K'D) are denoted along with upper and lower limits for 95% confidence intervals.

Synthetic methods
Organic solvents were purified according to the method of Grubbs i1 . Water was purified using a Millipore Milli-Q Integral Water Purification System. Organic solutions were concentrated under reduced pressure on a Büchi rotary evaporator using a water bath.

Cell Culture
Cell lines are cultured based on Key Resources Table.

Structures of reagents used for µMap experiments
S1 and S2 were prepared as described previously. 2,3 S3 and S4 are commercially available from BroadPharm.

Preparation of SARS-Cov-2 RBD and Spike Iridium Conjugates
To prepare free Iridium controls, a 5 mM solution of Ir-DBCO (S2) was prepared in DMSO. 20 µl of this solution was then reacted with 20 µl of a 5 mM solution of azido-PEG3-acid (BroadPharm) in DMSO according to the scheme below. After incubating in the dark for 1 hour, the material was used directly without further purification.
Scheme S1. Preparation of free Ir control.
To prepare RBD or spike Iridium conjugates,