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).
