Periodic Reporting for period 4 - VIRUSES AND RNA (RNA regulation during viral infection)
Periodo di rendicontazione: 2024-01-01 al 2024-06-30
Viral infections are responsible for significant morbidity and mortality, evidenced by the COVID-19 pandemic. Thorough understanding of basic virology is critical for informed development of prevention and control. Most systematic studies of virus-host interactions have focused on proteins, however, with recent methodological advances the intersecting fields of viral infection and RNA biology hold great promise for basic and therapeutic exploration. The goal of this project therefore was to discover and dissect RNA-based virus-host interactions and related regulatory mechanisms of gene expression.
Micro-RNAs (miRNAs) fine-tune gene expression by repressing messenger RNA (mRNA) targets. Interestingly, certain viruses exploit this system. For example, miR-122 increases translation and replication of hepatitis C virus (HCV) to the extent that the virus critically depends on this miRNA, which has also emerged as a therapeutic target. Further, HCV sequesters enough miR-122 to indirectly regulate cellular gene expression, at least in vitro.
Given that miR-122 is a tumor-suppressor, we here hypothesized that this RNA-based mechanism contributes to virus induced liver cancer. Since all clinical HCV isolates bind miR-122, the causal relationship cannot be investigated in patients. In Aim 1, we therefore first established in vivo and in vitro systems of the related Norway rat hepacivirus (NrHV), and observed HCV-related liver pathology. Using evolution-in-a-dish experiments, we re-directed the miRNA tropism of NrHV, allowing for comparative studies in vivo of infection with and without miR-122 sequestration.
Better understanding of viral RNA interactions could significantly contribute to basic infection biology and novel therapeutics. In Aim 2, we therefore aimed to systematically identify viral RNA interactions with cellular RNAs and proteins. In this context, we characterized several cases of virus-miRNA interaction landscapes. We developed methods to characterize the RNA-RNA interaction landscape more broadly during viral infection, and also applied methods to study the regulation of RNA-binding proteins during infection.
In Aim 3, we set out to explore interactions between cellular RNA and RNA-binding proteins, which may govern how viruses or cellular genes can take advantage of alternative miRNA regulation. We have screened for novel RNA binding proteins (RBPs) and have validated a number of such candidates.
In conclusion, this project has made major progress towards understanding novel RNA-based mechanisms of disease, mapped virus-host RNA interactions during a number of viral infections, and uncovered regulatory mechanisms of RNA-binding proteins.
Micro-RNAs (miRNAs) fine-tune gene expression by repressing messenger RNA (mRNA) targets. Interestingly, certain viruses exploit this system. For example, miR-122 increases translation and replication of hepatitis C virus (HCV) to the extent that the virus critically depends on this miRNA, which has also emerged as a therapeutic target. Further, HCV sequesters enough miR-122 to indirectly regulate cellular gene expression, at least in vitro.
Given that miR-122 is a tumor-suppressor, we here hypothesized that this RNA-based mechanism contributes to virus induced liver cancer. Since all clinical HCV isolates bind miR-122, the causal relationship cannot be investigated in patients. In Aim 1, we therefore first established in vivo and in vitro systems of the related Norway rat hepacivirus (NrHV), and observed HCV-related liver pathology. Using evolution-in-a-dish experiments, we re-directed the miRNA tropism of NrHV, allowing for comparative studies in vivo of infection with and without miR-122 sequestration.
Better understanding of viral RNA interactions could significantly contribute to basic infection biology and novel therapeutics. In Aim 2, we therefore aimed to systematically identify viral RNA interactions with cellular RNAs and proteins. In this context, we characterized several cases of virus-miRNA interaction landscapes. We developed methods to characterize the RNA-RNA interaction landscape more broadly during viral infection, and also applied methods to study the regulation of RNA-binding proteins during infection.
In Aim 3, we set out to explore interactions between cellular RNA and RNA-binding proteins, which may govern how viruses or cellular genes can take advantage of alternative miRNA regulation. We have screened for novel RNA binding proteins (RBPs) and have validated a number of such candidates.
In conclusion, this project has made major progress towards understanding novel RNA-based mechanisms of disease, mapped virus-host RNA interactions during a number of viral infections, and uncovered regulatory mechanisms of RNA-binding proteins.
The over-arching objective of this project was to discover and understand RNA level virus-host interactions and related regulatory mechanisms of gene expression.
In Aim 1, we aimed to characterize indirect cellular gene regulation through viral miRNA sequestration in vivo. Hepatitis C virus (HCV) directly binds, and critically depends on, the cellular micro-RNA, miR-122. In addition to its direct use of this host factor during viral replication, we hypothesized that HCV sequesters miR-122 away from its normal targets. This is particularly interesting, given that miR-122 is a tumor-suppressor, and that virus induced miR-122 sponging therefore may serve as an RNA-based mechanism providing an environment fertile for liver cancer. However, this is not easily addressed in patients, since all HCV isolates bind miR-122. To enable studies of causative links, we therefore employed a rat/mouse model of Norway rat hepacivirus (NrHV); a virus that is related to HCV. Here, we have developed in vitro and in vivo models for NrHV and characterized the model, including its liver pathology Wolfisberg J Virol 2019, Wolfisberg Hepatology 2022, Wolfisberg J Virol 2023 and Brown Hepatology 2023). We further have used our in vitro systems to develop miR-122 independent NrHV, which enabled direct comparison of the role of miR-122 sequestration on gene regulation and pathogenesis in vivo. These studies are currently ongoing. Finally, we have used experimental infection with equine hepacivirus in horses to demonstrate that a miR-122 sponge effect indeed is observable by RNA-seq of liver biopsies in vivo (Tomlinson Hepatology 2021).
In Aim 2, we aimed to discover novel virus-host interactions at the RNA level. This led to a thorough characterization of another case of viral dependency on host miRNAs; the case of bovine viral diarrhea virus (BVDV), an important veterinary pathogen, and its interaction with miR-17 (Kokkonos 2020 Nucl Acids Res). For SARS-CoV-2, we globally mapped miRNA interactions across the viral genome and identified six major miRNA binding sites. We further mapped specific cellular miRNA regulation upon SARS-CoV-2 infection and how this translated to cellular gene regulation (Fossat 2023 Cell Rep). Finally, we successfully established and improved several state-of-the-art RNA interaction assays in the lab, and applied these to map and characterized cellular RNA and protein interactors of viral RNA. These studies are currently being finalized for publication.
In Aim 3, we have been exploring interactions between cellular RNA and RNA-binding proteins, including the structural conditions of RNA molecules, which may govern how viruses or cellular genes can take advantage of alternative miRNA regulation. This has led us to identify a completely novel type of cap structure on RNA, which could serve as protection for the RNA molecule, and which may just be the beginning of a new understanding of RNA cap structures and of viral protection against host responses (Sherwood, Rivera-Rangel 2023 Nature). We in addition have screened for novel RNA binding proteins (RBPs) and have validated a number of such candidates. We specifically studied the role of Rbm5 in regulation of mRNA splicing, including its potential role in Huntington’s disease (Mullari, Fossat 2023 Nat Comms).
In Aim 1, we aimed to characterize indirect cellular gene regulation through viral miRNA sequestration in vivo. Hepatitis C virus (HCV) directly binds, and critically depends on, the cellular micro-RNA, miR-122. In addition to its direct use of this host factor during viral replication, we hypothesized that HCV sequesters miR-122 away from its normal targets. This is particularly interesting, given that miR-122 is a tumor-suppressor, and that virus induced miR-122 sponging therefore may serve as an RNA-based mechanism providing an environment fertile for liver cancer. However, this is not easily addressed in patients, since all HCV isolates bind miR-122. To enable studies of causative links, we therefore employed a rat/mouse model of Norway rat hepacivirus (NrHV); a virus that is related to HCV. Here, we have developed in vitro and in vivo models for NrHV and characterized the model, including its liver pathology Wolfisberg J Virol 2019, Wolfisberg Hepatology 2022, Wolfisberg J Virol 2023 and Brown Hepatology 2023). We further have used our in vitro systems to develop miR-122 independent NrHV, which enabled direct comparison of the role of miR-122 sequestration on gene regulation and pathogenesis in vivo. These studies are currently ongoing. Finally, we have used experimental infection with equine hepacivirus in horses to demonstrate that a miR-122 sponge effect indeed is observable by RNA-seq of liver biopsies in vivo (Tomlinson Hepatology 2021).
In Aim 2, we aimed to discover novel virus-host interactions at the RNA level. This led to a thorough characterization of another case of viral dependency on host miRNAs; the case of bovine viral diarrhea virus (BVDV), an important veterinary pathogen, and its interaction with miR-17 (Kokkonos 2020 Nucl Acids Res). For SARS-CoV-2, we globally mapped miRNA interactions across the viral genome and identified six major miRNA binding sites. We further mapped specific cellular miRNA regulation upon SARS-CoV-2 infection and how this translated to cellular gene regulation (Fossat 2023 Cell Rep). Finally, we successfully established and improved several state-of-the-art RNA interaction assays in the lab, and applied these to map and characterized cellular RNA and protein interactors of viral RNA. These studies are currently being finalized for publication.
In Aim 3, we have been exploring interactions between cellular RNA and RNA-binding proteins, including the structural conditions of RNA molecules, which may govern how viruses or cellular genes can take advantage of alternative miRNA regulation. This has led us to identify a completely novel type of cap structure on RNA, which could serve as protection for the RNA molecule, and which may just be the beginning of a new understanding of RNA cap structures and of viral protection against host responses (Sherwood, Rivera-Rangel 2023 Nature). We in addition have screened for novel RNA binding proteins (RBPs) and have validated a number of such candidates. We specifically studied the role of Rbm5 in regulation of mRNA splicing, including its potential role in Huntington’s disease (Mullari, Fossat 2023 Nat Comms).
- Given the current COVID-19 pandemic, we have included the causative virus, SARS-CoV-2, in our studies. We have already successfully mapped its miRNA interactions and are currently also mapping other types of RNA interactions for this virus. This has the potential to reveal novel RNA-based therapeutic avenues for COVID-19 disease.
- Studies of experimental equine hepacivirus (EqHV) infection in horses support our hypothesis of virus induced miRNA sponging. In gene expression analysis performed on liver biopsies during infection, we see that cellular genes normally specifically repressed by miR-122 are now upregulated. This points to functional action of a virus induced miR-122 sponge effect. Although the equine model was not originally thought to contribute to Aim 1, this finding lends strong support to our idea and therefore strengthen our belief that this concept can be explored using the rodent model.
- Studies of the RNA termini led us to identify a completely novel type of cap structure on RNA, which could serve as protection for the RNA molecule, and which may just be the beginning of a new understanding of RNA cap structures and of viral protection against host responses.
- Studies of experimental equine hepacivirus (EqHV) infection in horses support our hypothesis of virus induced miRNA sponging. In gene expression analysis performed on liver biopsies during infection, we see that cellular genes normally specifically repressed by miR-122 are now upregulated. This points to functional action of a virus induced miR-122 sponge effect. Although the equine model was not originally thought to contribute to Aim 1, this finding lends strong support to our idea and therefore strengthen our belief that this concept can be explored using the rodent model.
- Studies of the RNA termini led us to identify a completely novel type of cap structure on RNA, which could serve as protection for the RNA molecule, and which may just be the beginning of a new understanding of RNA cap structures and of viral protection against host responses.