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RNA regulation during viral infection

Periodic Reporting for period 1 - VIRUSES AND RNA (RNA regulation during viral infection)

Reporting period: 2019-07-01 to 2020-12-31

Viral infections are responsible for significant morbidity and mortality and frequency and impact of epidemics are expected to increase. This is evidenced by the current SARS-CoV-2/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 application therefore is 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. However, cellular miRNAs increase translation and replication of certain viruses. Thus, hepatitis C virus (HCV) critically depends on the liver specific miR-122, which emerged as a therapeutic target. Further, HCV sequesters enough miR-122 to indirectly regulate cellular gene expression. I hypothesize that this RNA-based mechanism contributes to virus induced liver cancer, and aim to address this using our recently developed rodent model for HCV infection (Aim 1). Better understanding of viral RNA (vRNA) interactions could significantly contribute to basic infection biology and novel therapeutics. I therefore aim to systematically identify vRNA interactions with other cellular RNAs and proteins (Aim 2). I expect to identify interactions of value for functional regulation and therapeutic targeting. I finally hypothesize that translation of certain cellular mRNAs – similarly to viruses – increase upon miRNA binding, and aim to systematically screen for such virus-like alternative regulation, with potential to change understanding of post-transcriptional regulation (Aim 3).
In conclusion, this high-risk high-gain project has potential to shape novel dogmas for virus and RNA biology and to identify novel RNA-based therapeutic targets; a promising upcoming field of discovery.
The over-arching objective of this grant is to discover and understand RNA level virus-host interactions and related regulatory mechanisms of gene expression.
In Aim 1, we aim to characterize indirect cellular gene regulation through viral miRNA “sponging” 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 hypothesize that HCV sequesters or “sponges” miR-122 away from its normal targets. This is particularly interesting, given that miR-122 knock-out mice spontaneously develop liver diseases, including cancer, similarly to HCV patients. Thus, virus induced miR-122 sponging may be 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 employ a rat/mouse model of rodent hepacivirus (RHV); a virus that is similar to HCV. Here, we have performed a number of in vivo studies to improve characterization and understanding of the RHV model. We further have advanced towards development of miR-122 independent RHV, which will enable comparison of the role of miR-122 sponging on gene regulation and pathogenesis in vivo. In addition, we have developed in vitro systems for cultivation of RHV (Wolfisberg 2019 J Virol, and unpublished). These enable miRNA selection experiments in a more high-throughput manner; we performed proof-of-principle for this using the related virus, BVDV (Kokkonos 2020 Nucleic Acid Res). We also used RHV systems to confirm miR-122 dependency as a pre-requisite for using this model (Wolfisberg 2019 J Virol). 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, In Press, Hepatology).
In Aim 2, we aim to discover other types of cellular RNA interacting with viral RNA. Here, we have successfully established and improved several state-of-the-art RNA interaction assays in the lab. We have applied these to identify RNA interactors for HCV, SARS-CoV-2 (the cause of COVID-19), chikungunya virus, tick-borne encephalitis virus and yellow fever virus, and have among many other interactors confirmed HCV/miR-122. We are currently exploring the novel interactors identified.
In Aim 3, we have begun to explore the structural conditions of RNA molecules, which may govern how alternative miRNA regulation may happen in the cell. This will set the foundation for further understanding of the conditions underlaying such alternative miRNA induced gene regulation.
- 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, the closest relative to HCV, 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. Perhaps even in a more straight-forward bulk analysis instead of the more technically demanding single-cell analysis.
Graphical summary of the approach to discover and understand RNA level virus-host interactions.