Periodic Reporting for period 4 - ANTIViR (Molecular mechanisms of interferon-induced antiviral restriction and signalling)
Período documentado: 2022-06-01 hasta 2023-11-30
The current pandemic of coronavirus disease 2019 (COVID-19) has been a cruel reminder that emerging viruses can spread rapidly around the world, be deadly, and have a profound impact on our society. There is a dire need to better understand viral/host interactions in general, and, in particular, our natural defense mechanisms against viral infections, as this could pave the way for the development of innovative antiviral therapeutical strategies. In this regard, interferons (IFNs), which are signalling proteins produced by infected cells, are the first line of defence against viral infections. In infected and neighbouring cells, IFNs induce the expression of hundreds of IFN-stimulated genes (ISGs). The ISGs, in turn, induce in cells a potent antiviral state, capable of limiting replication of most viruses, including major human pathogens such as Human Immunodeficiency Virus type 1 (HIV-1), influenza A virus (IAV) or Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19 pandemic. Identifying the antiviral ISGs and understanding their mechanisms of action is therefore crucial to progress in the fight against viruses.
ISG-encoded proteins playing a role in the antiviral state have been identified, such as human MX1, a well-known antiviral factor able to restrict numerous viruses including IAV, and MX2, a potent HIV-1 inhibitor. Both proteins are thought to bind to viral components but their detailed mechanisms of action remain unclear. Moreover, our preliminary work had showed that additional anti-HIV-1 and anti-IAV ISGs remained to identify. The ANTIViR project, which was initially focused on pandemic HIV-1 and on IAV (which has a strong pandemic potential), was modified in 2020 to include SARS-CoV-2 when the COVID-19 pandemic started. Using notably the molecular scissors CRISPR at the whole-genome scale, we were able to identify several antiviral human genes essential in the fight against these viruses and to characterize in depth their molecular mechanisms of action.
Overall, our project provided a better understanding of the molecular mechanisms involved in the antiviral effect of IFN and also identified numerous intrinsically expressed genes regulating viral infections. In the future, our findings might help guiding the identification of novel therapeutic targets against major pathogenic viruses, and help to anticipate future pandemics.
ISG-encoded proteins playing a role in the antiviral state have been identified, such as human MX1, a well-known antiviral factor able to restrict numerous viruses including IAV, and MX2, a potent HIV-1 inhibitor. Both proteins are thought to bind to viral components but their detailed mechanisms of action remain unclear. Moreover, our preliminary work had showed that additional anti-HIV-1 and anti-IAV ISGs remained to identify. The ANTIViR project, which was initially focused on pandemic HIV-1 and on IAV (which has a strong pandemic potential), was modified in 2020 to include SARS-CoV-2 when the COVID-19 pandemic started. Using notably the molecular scissors CRISPR at the whole-genome scale, we were able to identify several antiviral human genes essential in the fight against these viruses and to characterize in depth their molecular mechanisms of action.
Overall, our project provided a better understanding of the molecular mechanisms involved in the antiviral effect of IFN and also identified numerous intrinsically expressed genes regulating viral infections. In the future, our findings might help guiding the identification of novel therapeutic targets against major pathogenic viruses, and help to anticipate future pandemics.
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.
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.
With the initial goal of revealing the ISG(s) responsible for the famous block to HIV-1 reverse transcription (characterized in Goujon et al. Journal of Virology 2010), the ANTIViR project identified DDX42, an intrinsic inhibitor of HIV-1 reverse transcription (and infection) and of many positive-strand RNA viruses and LINE-1 retroelements. This has opened novel avenues of research on this so far-poorly characterized DEAD-box RNA helicase.
The identification of NCOA7-AS as a novel ISG inhibiting pH-dependant entry of enveloped viruses (including IAV) (Doyle et al Nature Microb. 2018) and the study of its mode of action have brought to light novel ways of regulating vesicular pH (Arnaud-Arnould and Rebendenne et al. in preparation).
Moreover, the ANTIViR project have unravelled a novel and major block to IAV replication imposed by human MX1 protein and identified for the first time a cellular cofactor for MX1 (McKellar et al BioRxiv 2024).
Finally, bidirectional, genome-wide and secondary, CRISPR screens in multiple model cell lines revealed a high cell-line specificity of the genes identified as regulators of SARS-CoV-2 replication (Rebendenne et al. Nature Genetics 2022). The ANTIViR project hence clearly showed that an important variable of genetic screens is actually the chosen model cell line. This study also highlighted the fact that genetic screens performed with two cancer cell lines of similar origin could give completely different results. Hence, the ANTIViR project has clearly demonstrated the dire and urgent need to develop more pertinent, primary cellular models to perform CRISPR screens.
The identification of NCOA7-AS as a novel ISG inhibiting pH-dependant entry of enveloped viruses (including IAV) (Doyle et al Nature Microb. 2018) and the study of its mode of action have brought to light novel ways of regulating vesicular pH (Arnaud-Arnould and Rebendenne et al. in preparation).
Moreover, the ANTIViR project have unravelled a novel and major block to IAV replication imposed by human MX1 protein and identified for the first time a cellular cofactor for MX1 (McKellar et al BioRxiv 2024).
Finally, bidirectional, genome-wide and secondary, CRISPR screens in multiple model cell lines revealed a high cell-line specificity of the genes identified as regulators of SARS-CoV-2 replication (Rebendenne et al. Nature Genetics 2022). The ANTIViR project hence clearly showed that an important variable of genetic screens is actually the chosen model cell line. This study also highlighted the fact that genetic screens performed with two cancer cell lines of similar origin could give completely different results. Hence, the ANTIViR project has clearly demonstrated the dire and urgent need to develop more pertinent, primary cellular models to perform CRISPR screens.