The project has progressed extremely well in the last period with important advances in the three main work packages. The results obtained are as followed:
WP1.
We have determined the RNA interactome of Sindbis virus (SINV), which is the pilot model proposed in the application. We have identified more than three hundred cellular proteins that engage with SINV RNA in infected cells. This include cellular RBPs with known roles in infection as well as more than hundred proteins with no prior connections to infection. Interestingly, many of these proteins are nuclear, despite the viral factories being in the cytoplasm of infected cells. We have validated these results using nuclear/cytoplasmic fractionation and single molecule fluorescence in situ hybridisation combined with immunofluorescence, demonstrating that several nuclear complexes are activated and recruited to the viral replication factories.
We have optimised viral RNA interactome (vRIC) for the other models proposed in the application, including Semliki Forest virus, Coxsackievirus B3, Zika virus and SARS-CoV-2. Therefore, we expect more progresses in this front in 2023. Note that due to the COVID19 pandemic and the biomedical relevance of coronaviruses, we are considering replacing the experiments proposed for ZIKV by SARS-CoV-2 .
In addition, we applied RNA interactome capture to cells treated with interferon and discovered that more than two hundreds RBPs exhibit differential association with RNA with no changes in protein abundance. These results show that interferon response is more complex than previously anticipated and does not only alter the abundance of interferon stimulated genes (ISGs) but also modulate the landscape of functionally active RBPs in the cell.
WP2
We have already tested 15 RBPs that bind to SINV RNA and are regulated by interferon (WP1). Interestingly, 6 out of 6 of the proteins tested that were upregulated functionally by interferon (with no changes in abundance) inhibited potently SINV gene expression, suggesting that they have strong antiviral activities. Conversely, most of the proteins tested whose activity was downregulated by interferon promoted SINV infection, suggesting that they are hijacked and are necessary for the viral life cycle. These results strongly support our initial hypothesis, in which we proposed that interferon would induce the engagement of antiviral proteins with viral RNA while causing the disengagement of proviral factors. We are currently extending these analyses to other viral models and trying to narrow down the steps in infection that these proteins regulate. The initial candidates that we have been able to test have produced consistent phenotypes in other viral models, but these results are still preliminary.
WP3
We have started to characterise two of the proteins causing strong phenotypes in a broad range of viruses. Our results have revealed that two of the proteins identified in WP2 are crucial supporting virus infection, we have identified their binding sites in SINV genome and have generated new insights into the complexes that they form in infection. We are currently exploring several working hypotheses and our results are very promissing. We expect to have more progress in this front in 2023.
WP4-5
We have contributed to science communication, both for academics and lay public. I have been invited to several conferences and top institutions to talk about our work. Also the members of the ERC team have participated in international conferences.
Invited talks
