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Photocatalyst-based ViraMap platform maps SARS-CoV-2 spike protein interactions on the cell surface

Nicholas by Nicholas
September 7, 2022
in Health
0

In a recent study posted to the bioRxiv* preprint server, researchers performed the photocatalytic mapping of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein-host cell membrane interactions.

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Study: High resolution photocatalytic mapping of SARS-CoV-2 Spike protein-host cell membrane interactions. Image Credit: Kjpargeter/Shutterstock

Understanding viral entrance and pathogenicity can be improved by identifying protein habitats at the virus-host cell interface. The virus responsible for the ongoing coronavirus disease 2019 (COVID-19) pandemic, SARS-CoV-2, uses the angiotensin-converting enzyme-2 (ACE2) protein as a primary receptor. However, the role of other cellular proteins in the entrance process is uncertain.

About the study

In the present study, the team developed a viral-host protein microenvironment mapping technology (ViraMap) using iridium photocatalysts (IrPC) conjugated to SARS-CoV-2 spike protein for visible-light-driven proximity labeling on host cells.

The team generated spike protein photocatalyst conjugates for targeted labeling on ACE2-expressing cells to analyze the interactions in the SARS-CoV-2 spike-host cell surface microenvironment using ViraMap. The SARS-CoV-2 spike protein trimer and its associated variants, including the SARS-CoV-1 spike and SARS-CoV-2 D614G spike, were chosen for conjugation with IrPC.

Further, the researchers performed targeted labeling on the cells by exposing them to spike-IrPC conjugates in the presence of a biotin-diazirine probe. This was followed by blue light irradiation and subsequent monitoring with flow cytometry and confocal imaging analysis. To selectively label host cell surface contacts and avoid cellular internalization of spike-IrPC conjugates, HEK293T+ACE2 cells were treated with spike-IrPC at 4 °C.

The team assessed cell surface binding in the presence of free ACE2 protein to establish that the spike-IrPC compound preserved its affinity for ACE2 (ACE2-Fc). For cell surface proximity labeling on HEK293T+ACE2 cells, three spike-IrPC variants such as SARS-CoV-1, SARS-CoV-2, and SARS-CoV-2 D614G spike proteins, were employed.

Furthermore, the team used a primary-secondary antibody combination comprising an anti-ACE2 primary antibody and an antibody-photocatalyst conjugate for targeted labeling. After extraction from the cell membrane fraction and streptavidin enrichment, the biotinylated host proteins from these targeted labeling studies were quantitatively characterized using a tandem mass tag (TMT)-based liquid chromatography–mass spectrometry (LC-MS)/MS quantification.

Results

When compared to a non-binding antibody immunoglobulin G (IgG) control conjugated with IrPC, flow cytometry analysis revealed a clear change in the biotinylation signal for spike-IrPC variant conjugates-treated cells. Compared to the mouse-IgG-IrPC control, confocal imaging of spike-IrPC induced labeling revealed biotin localisation to the host cell surface environment. Furthermore, because the recombinant spike construct had a poly-His-tag, cellular biotinylation might be achieved through targeted labeling with an anti-His antibody IrPC conjugate.

IrPC-conjugated SARS-CoV-1, SARS-CoV-2, SARS-CoV-2 D614G spike proteins, as well as anti-ACE2 primary/secondary antibody systems resulted in statistically significant enrichment of distinct groups of cellular proteins compared to the IrPC isotype conjugate as a negative control. Using the SAINTexpress and mass spectrometry interaction statistics (MiST) scoring algorithms, the team identified 96 high-confidence enriched proteins across all spike-IrPC labeling studies.

Almost 23 of the 96 high-confidence enriched proteins were shared by all three spike variants, whereas the remaining two, 25, and 46 enriched proteins were unique to the SARS-CoV-1, SARS-CoV-2, and SARS-CoV-2 D614G spike-IrPC spikes, respectively. The gene ontology enrichment analysis of the 23 enriched proteins found a significant relationship with viral entrance, followed by immune cell differentiation and co-stimulation activities. Furthermore, 70% of the enriched proteins had a high abundance in most human tissues, including the lung, kidney, gastrointestinal tract, fat, cardiac muscle, soft tissues, reproductive tissues, and brain.

Out of 23, a total of 10 high-confidence enriched proteins could be divided into two groups based on whether or not they showed co-enrichment with the anti-ACE2 primary/secondary antibody system. The first class of cell surface receptors included ACE2, neuropilin 1 (NRP1), prostaglandin F2 receptor negative (PTGFRN), and neurogenic locus notch homolog protein 2 (NOTCH-2). Evidence of PTGFRN protein and messenger ribonucleic acid (mRNA) expression in a wide range of tissue cells, particularly cardiomyocytes and lung fibroblasts, implied that these cell types are highly susceptible to SARS-CoV-2 via various entry points. Galectins were the second set of host proteins found in all three spike-IrPC labeling assays.

The results of the targeted labeling experiments point to putative cell surface co-receptors involved in viral entry and antiviral immunity. As a result, functional experiments were performed to assess which of the identified host proteins are involved in SARS-CoV-2 entrance into cells. For functional validation, the study focused on the ten overlapping proteins shared by all three spike-IrPC variants. The clustered regularly interspaced short palindromic repeats /Cas9-mediated knockdown (KD) only reduced ACE2 abundance by 30%. Although there was a 40% reduction in protein expression relative to controls, there was no significant change in pseudoparticle entrance following NRP1 KD.

Overall, the study findings showed that the ViraMap technology successfully facilitated the assessment of known as well as unknown virus-host protein interactions that occur on the outer cell membrane of the host cell using cell surface recognition targeting modalities.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Tags: ACE2AngiotensinAntibodyCellCell MembraneCoronavirusCoronavirus Disease COVID-19covid-19CytometryEnzymeFlow CytometryImagingMass SpectrometryMembranePandemicProteinReceptorRespiratorySARSSARS-CoV-2Secondary AntibodySevere Acute RespiratorySevere Acute Respiratory SyndromeSpectrometrySpike ProteinSyndromeVirus

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