The Researcher first did implement the proximal interactomics technologies (BioID and TurboID) in the host laboratory and then built stable inducible cell lines for the MONET project. During the COVID-19 lockdown, there was no mean to keep working on other subject than the SARS-CoV-2. The MONET project was then redirected using the molecular tools developed. This deviation was agreed upon with PO. Given the restriction, switching topic to SARS-CoV-2 was a mean to keep working in laboratory.
SARS-CoV-2 proximal interactome. We applied BioID to SARS-CoV-2 to delineate the first SARS-CoV-2 proximal interactome in human cells. The functional significance of our dataset is supported by the CRISPR-Cas screens identifying SARS-CoV-2 essential host factors. 432 protein-coding genes have been identified as essential in three or more independent studies. Our works identified 120 factors of them and provides molecular ties to at least one viral protein, more than any other study. We have applied our BioID pipeline to the 27 SARS-CoV-2 proteins, and performed the BioID experiments in un-stimulated cells or under poly(I:C) treatment to mimic viral infection. We thus have determined the first and most complete proteome proximal interactome of SARS-CoV-2, as well as the only virus-host proximal interactome upon immune stimulation.
Given that our interactome describes 5,626/10,185 PPIs not detected in any other study, we implemented N2H as an orthogonal approach to characterize these novel PPIs. We thus sampled 289 PPIs to identify the direct partners of viral proteins. We explored the binary, direct interactions between 156 high-confidence-proximal host factors and their cognate viral proteins. Binary virus-host interactions were measured using the N2H method, a Split-Nanoluciferase Complementation assay performed in human cells. A total of 85/289 BioID identified proximity interactions were detected at high confidence (p<0.01) of one or several viral proteins in the N2H assay (p<0.05). Thus, over 30% of the tested interactions are direct. We typically obtain 15-20% cross-validation when testing interactions identified by IP-MS technique, supposedly the gold standard to detect PPIs.
Our SARS-CoV-2 study identified NSP13-USP13 interaction by BioID, which was characterized as direct by N2H. When tested in an infectious context, a recently characterized UPS13 inhibitory compound, the Spautin-1, remarkably impaired SARS-CoV-2 replication, with an IC50 ~1µM, lower than gc376 (reference compound for in vitro SARS-CoV-2 life cycle inhibition), while no toxicity has been observed on cells treated at these doses. This molecule is under further characterization at our collaborator laboratory at Institut Pasteur, Paris.
MONET (initial plan). After lockdown, the initial MONET project resumed. TAs Interactomes assembly was achieved and analyzed to build the first functional hypothesis. Mass spectrometry-based experiments were extended to explore the changes induced in the proteome of cells expressing sT and LT at different times of induction. GST pull-down followed by mass spectrometry were done, using recombinant sT and LT fused to GST tag to identify which proximal interactors were able to directly bind the TAs. Given the epigenetic enrichment within the TAs proximal interactors, the TAs deregulate the cell transcriptome (thus proteome) through alterations the DNA binding proteins functions at the deregulated genes sites. Bioinformatic analysis are ongoing to identify which TAs proximal interactors are binding groups of genes encoding proteins identified as deregulated in our whole proteome analysis (ENCODE analysis). Results highlights are the finalization of the basal TAs proximal interactomes, the identification of EP400 as a new FBXW7 candidate target, the identification of PIN1 as the potential missing link between tLT-FBXW7 in oncogenesis, and the identification of a potential new addressing mechanism of PIN1 to FBXW7. Overall, although delayed and truncated, the MONET project has provided a wealth of data and opened several functional hypotheses which have been further explored.
