First, ANTIViR shed light on the modes of action of the broadly-acting antiviral restriction factors MX1. Chimeric proteins, deletion and point mutants revealed a motif, conserved in human, murine and bat MX1 proteins, that is essential for broad antiviral activity (McKellar et al. JBC 2023). Moreover, Oxford Nanopore long read sequencing and imaging approaches revealed a novel block to IAV replication imposed by human MX1, which impedes IAV ribonucleoprotein complex (vRNP) transport towards the plasma membrane. MX1 transiently associates with the vRNPs and induces their dynein-dependant retrograde transport towards the MTOC where they remained sequestrated. Dynein inhibition rescues IAV infectious particle production, identifying dynein as the first cellular cofactor for MX1 (McKellar et al, bioRXiv 2024).
Next, ANTIViR took advantage of the hostile environment induced by IFN to reveal novel HIV-1 inhibitors through genome-wide CRISPR KO screening. This led to the identification of the DEAD-box RNA helicase DDX42 as a potent intrinsic inhibitor of HIV-1 and other retroviruses, as well as LINE-1 retrotransposons and various positive-strand RNA viruses, including coronaviruses and alphaviruses (Bonaventure et al. EMBO reports 2022, Bonaventure and Goujon, J Gen Virol 2022). DDX42 was found in the close proximity to viral elements upon infection and interacted with LINE-1 and viral RNAs, suggesting a direct mode of action. Concerning IAV, we identified, in collaboration with my former lab at King’s College London, a novel ISG participating in the IFN-induced restriction against this virus: NCOA7-AS (Doyle et al. Nature Microbiol. 2018). We showed that NCOA7-AS interacted with the vacuolar ATPase, increasing its activity and thereby decreasing the endolysosomal pH, which seemed detrimental to IAV entry. We then solved the structure of an essential domain of NCOA7-AS and identified essential partners for its antiviral activity, and characterized in depth NCOA7-AS mode of action (Arnaud-Arnould et al. 2021, Arnaud-Arnould and Rebendenne et al, in preparation). We also performed genome-wide screens to identify human genes regulating IAV replication. This led us to (re-)identify genes essential to replication; the validation work is still in progress (Chaves Valadão and García de Gracia et al, in preparation).
Finally, ANTIViR characterized the cell host responses to SARS-CoV-2 replication, using both mode cell lines and primary airway epithelia. We showed really strong, but somewhat late, type 1 and type 3 IFN responses by infected cells (both in model cell lines and primary airway epithelia), due to MDA-5 mediated sensing (Rebendenne et al, Journal of Virology 2021, Jouvenet, Goujon and Banerjee, Trends in Immunol. 2021). In parallel, we performed bidirectional, genome-wide and secondary CRISPR screens (activation and KO screens) in multiple model cell lines in order to reveal both the cellular dependency factors and inhibitors of SARS-CoV-2. These screens revealed a number of human genes regulating positively and negatively SARS-CoV-2 replication, including numerous intrinsic inhibitors and some ISGs (e.g. mucins, LY6E, IL6R, CD44 etc) (Rebendenne et al. Nature Genetics 2022).
Overall, the ANTIViR project has provided a better understanding of the molecular mechanisms involved in the innate and intrinsic innate immune defences of our cells against pathogenic viruses.
