Periodic Reporting for period 2 - vRNP-capture (Towards the discovery of cellular RNA-binding proteins with master regulatory roles in virus infection)
Periodo di rendicontazione: 2023-06-01 al 2024-11-30
RNA is a central molecule for RNA viruses as it functions not only as a blueprint for viral proteins in the form of messenger (m)RNA, but also as storage of genetic information as genome. Despite the central roles of RNA in virus infection, the interactions that this molecule establish in the host cell remain largely unknown. This project propose to use a novel approach to determine the viral RNA interactome in a comprehensive and systematic manner, and how it rearranges in permissive and repressive conditions (interferon α) for virus replication using different viral systems. We expect to discover critical host-virus interactions that will be characterised with molecular detail in this project.
The COVID-19 pandemic has revealed how important is to understand viruses and to repurpose and deploy the methods to study viruses to emerging threats. This project will generate the technical know-how to study host-virus interactions involving cellular RNA-binding proteins unbiasedly for any emergent virus.
Moreover, we have witnessed the limitations of rapid development of virus-targeting antivirals. This project aims to identify critical cellular proteins playing central roles in the infection of a wide range of viruses. Such host-virus interactions could be in the future targeted to generate new antiviral strategies with broad spectrum activity. Such antivirals could represent a first line of defence against emergent viruses.
The goals of this project are to discover the interactions that viral RNA establishes with host RNA-binding proteins (RBPs), how interferon alters them and what are their molecular roles in infection. These goals are separated into the following Work packages (WP):
1) Elucidating the core viral RNA interactome. We will employ viral RNA interactome capture (Kamel et al., Mol Cell 2021) in cells infected with a wide range of RNA viruses. We will also study if interferon α remodels the viral RNA interactome.
2) Discovering the RBPs that regulate virus infection. We will use a mid-throughput screening approach focusing on cellular proteins that interact with the RNA of different viruses and are regulated by interferon, to determine which cellular RBPs promote or restrict viral life cycle.
3) Determining the mechanism of action of regulatory cellular RBPs. We will use a wide range of molecular, cellular and structural biology approaches to elucidate the mechanism of action of cellular RBPs with critical roles in infection.
The COVID-19 pandemic has revealed how important is to understand viruses and to repurpose and deploy the methods to study viruses to emerging threats. This project will generate the technical know-how to study host-virus interactions involving cellular RNA-binding proteins unbiasedly for any emergent virus.
Moreover, we have witnessed the limitations of rapid development of virus-targeting antivirals. This project aims to identify critical cellular proteins playing central roles in the infection of a wide range of viruses. Such host-virus interactions could be in the future targeted to generate new antiviral strategies with broad spectrum activity. Such antivirals could represent a first line of defence against emergent viruses.
The goals of this project are to discover the interactions that viral RNA establishes with host RNA-binding proteins (RBPs), how interferon alters them and what are their molecular roles in infection. These goals are separated into the following Work packages (WP):
1) Elucidating the core viral RNA interactome. We will employ viral RNA interactome capture (Kamel et al., Mol Cell 2021) in cells infected with a wide range of RNA viruses. We will also study if interferon α remodels the viral RNA interactome.
2) Discovering the RBPs that regulate virus infection. We will use a mid-throughput screening approach focusing on cellular proteins that interact with the RNA of different viruses and are regulated by interferon, to determine which cellular RBPs promote or restrict viral life cycle.
3) Determining the mechanism of action of regulatory cellular RBPs. We will use a wide range of molecular, cellular and structural biology approaches to elucidate the mechanism of action of cellular RBPs with critical roles in infection.
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
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
The analysis of protein-RNA interactions is an emerging area of research since it was hindered by technical limitations. These limitations have recently been overcome because of advances of biochemical capture of viral ribonucleoproteins and the follow up proteomic analysis. I and my laboratory have been at the forefront of these developments and the results described above highlight the quality and robustness of our approaches. Although competition is growing in the field, we feel that we are in an optimal position to continue generating exciting novel results that will make the field move forward. This is illustrated by numerous invitations to prestigious international conferences and top institutions to talk about our research.
Based on our current progress, I think we are in a good position to achieve the proposed objetives and even more. Indeed, the fact that we had made progresses in the three work packages is already an outstanding outcome. I expect that we will be able to extend vRIC to other viral models soon and this will feed our screening to identify more master regulators of virus infection. I also expect to carry out important advances unveiling the mechanisms with our validated candidates.
Based on our current progress, I think we are in a good position to achieve the proposed objetives and even more. Indeed, the fact that we had made progresses in the three work packages is already an outstanding outcome. I expect that we will be able to extend vRIC to other viral models soon and this will feed our screening to identify more master regulators of virus infection. I also expect to carry out important advances unveiling the mechanisms with our validated candidates.