Acronimo: INTERROGA

Titolo: Uncovering novel principles of organelle biogenesis by untangling the functional INTERactome of SARS-Cov-2 proteins mediating the formation of the Replication OrGanelle and virion Assembly

Codice Progetto: 2022F2YJNK

Responsabile scientifico per il DMM: Prof. Gualtiero ALVISI - Università degli Studi di Padova
Coordinatore: Università degli Studi di Napoli Federico II - Prof.ssa Maria Antonietta DE MATTEIS

Partner-Unità di ricerca: Università degli Studi di Padova

Bando: PRIN 2022 - Decreto Direttoriale n. 104 del 02-02-2022
Durata: 28/09/2023 - 27/09/2025 (24 mesi)

Finanziamento progetto: € 251.788,00 – CUP C53D23003110006

 

Abstract del progetto

The COVID-19 pandemic has exposed our ignorance about several basic mechanisms underlying coronavirus–host interactions. The last few years have seen an enormous effort to make up for this lack of knowledge. Approaches that have been pursued range from “omics” (the interactome of viral proteins and CRISPR-based functional genetic screens in infected cells) to structural, biochemical, and functional studies of individually expressed viral proteins. These approaches have yielded an impressive wealth of data but they present important limitations. On the one hand, studies leading to the definition of protein interactomes by expressing individual viral proteins in uninfected cells must be interpreted with caution, as individually expressed viral proteins are likely to display artefactual subcellular localization and functionality, due to the lack of both physiological processing and interaction with other viral proteins. On the other hand, genome-wide functional screens performed so far have been based on cell survival, so genes essential for cell proliferation or involved in virion assembly or release have likely been missed. Indeed, a functional role in the viral life cycle has been validated for only a few host cell factors targeted by viral proteins, and little is known about the role of host factors in steps after viral entry that require the coordinated action of multiple viral proteins, such as viral replication, which occurs in virus-induced Replication Organelles (RO), and virion assembly and release. An approach that reduces the complexity and variability linked to viral infection while preserving the functional context of the different viral proteins consists in de-composing and reconstituting the different steps of the virus life cycle. Thanks to our expertise in virology and cell biology, we managed to reconstitute two key processes by expressing idoneous combinations of viral proteins: (1) biogenesis of the Replication Organelle (by expressing NSP3, NSP4, and NSP6) and (2) virion assembly and release (by expressing the N, E, and M proteins). Thus, we are in a strong position to study key protein-protein interactions in a context which is more physiological than the classical overexpression of a single viral protein, but also more controlled and reproducible than in the context of a wild-type virus infection. Defining the interactome of these key viral proteins co-expressed in functional modules is the overarching aim of INTERROGA, a project based on a close collaboration between a cell biologist (MADM) and a virologist (GA). Identified interactors will be functionally validated in infected cells using an approach that will distinguish between genes involved in entry/replication from genes involved in assembly/release. The expected outcome of INTERROGA is the identification of pro-viral host factors that could be targeted to develop broad-spectrum antivirals in preparation for possible future epidemics.