Systematic analysis of small RNA function in respiratory virus infection
Item statusRestricted Access
Embargo end date31/07/2022
Respiratory syncytial virus (RSV) and parainfluenza virus (PIV) are among the most common causes of respiratory infections worldwide, causing morbidity and mortality especially in young children but also elderly or immunocompromised adults. Infections result in hundreds of thousands of hospitalizations each year, leading also to a significant global health and economic burden. RSV alone infects nearly all children by the age of 2 years and is estimated to cause a higher disease burden than influenza in the adult population. Despite intensive research, there are still no licenced vaccines or effective treatments against RSV and PIV infections. Most antiviral approaches to date directly target the virus, although many host factors are involved in the viral replication cycle. MicroRNAs (miRNAs) are a class of small regulatory RNAs that canonically supress gene expression by binding to the 3’ untranslated region (3’ UTR) of messenger RNAs (mRNAs). Several miRNAs have been reported to be altered upon viral infection and these alterations can suppress or boost host immune responses and the viral infection progress, depending on the targets of the miRNAs. However, there is no systematic analysis of miRNA and target regulation in respiratory virus infection. Previous functional screening from our group using miRNA mimics and inhibitors demonstrated that some miRNAs including miR-744 and miR-28 have antiviral properties against a wide range of viruses. However, the targets of these miRNAs and their mechanisms of action remain largely unknown. This thesis focuses on the global characterisation of host miRNA regulation in RSV and PIV-3 infections in the A549 epithelial cell line and then further investigates the targets of key de-regulated or antiviral miRNAs in RSV infections. Dynamic changes in miRNA expression over the time course of 96 h of RSV and PIV-3 infections were determined by small RNA sequencing. In general, RSV and PIV-3 induced similar de-regulation of miRNA levels, with miR-149 and miR-744 being down-regulated and the miR-34/miR-449 cluster being upregulated. Other miRNAs that we previously identified as antiviral (such as miR-28) were not differentially expressed. In addition, several viral small RNAs (vsRNAs) with a length of 22-30 nt were identified in the small RNA sequencing data for both RSV and PIV-3. The RSV vsRNAs were found to be present not only inside the cell but also in cell culture supernatant at early time points of infection. Small RNA sequencing from RSV-infected patients confirmed their presence in bronchial alveolar lavage fluid. Inhibition of one of these RNAs (vsRNA L-1) inhibited RSV infection and replication, making this RNA an interesting candidate for future therapeutic approaches. To investigate the mechanism of action of deregulated and antiviral miRNAs in RSV infection, miRNA targets were identified by immunoprecipitation of the RNA-induced silencing complex (RISC), followed by ligation of the miRNA to its target and sequencing of these chimeric RNAs. After initial optimisation, two protocols were tested side by side to identify optimal conditions for the A549 cell line. A variation of the CLEAR-CLIP (covalent ligation of endogenous Argonaute-bound RNAs– Crosslinking and immunoprecipitation) protocol resulted in 3-6% of miRNA-target interactions. Apart from canonical interactions with 3’ UTRs, high confidence targets were identified in coding sequences (CDS) and regulatory regions using a new bioinformatic approach developed by our laboratory. In general, miRNA expression correlated with the number of target counts. However, for a subset of miRNAs that were highly abundant, no high-confidence targets could be identified, including the miR-449 family. These data suggest that additional mechanisms, such as RISC association, regulate targeting of these miRNAs. In addition to the identification of host targets, this analysis also identified bindings sites in the viral genome for host miRNAs miR-26 and miR-27, which could represent a viral mechanism to suppress these host miRNAs and up-regulate host gene expression. High confidence targets were analysed in detail for a subset of lead candidate miRNAs to identify their mechanisms of action. Target network analysis suggested miR-26 and miR-27 as regulators of host genes important for viral processes. Too few targets were found to perform target network analysis for miR-28 or miR-744, but initial results confirm the Signal Peptidase Complex Subunit 3 (SPCS3) 3’ UTR as a target for miR-28. This gene was reported to be essential in viral infections and could explain the antiviral effect of miR-28 mimics. In summary, this thesis provides a global overview of miRNA regulation during the time course of respiratory virus infections and provides insight into new target interaction of de-regulated or antiviral miRNAs during RSV infection. Expanding our knowledge of miRNA-mediated gene regulations in the context of infection will contribute to a better understanding of pathways that could be targeted in new therapeutic strategies.