Virus-Host Interactions of Positive Sense RNA Viruses: Insights from SARS-CoV-2 and Bovine Viral Diarrhea Virus
| dc.contributor.advisor | Wilson, Joyce | |
| dc.contributor.committeeMember | Luo, Yu | |
| dc.contributor.committeeMember | van den Hurk, Sylvia | |
| dc.contributor.committeeMember | Kobryn, Kerri | |
| dc.contributor.committeeMember | Conn, Kristen | |
| dc.contributor.committeeMember | Banerjee, Arinjay | |
| dc.contributor.committeeMember | Colpitts, Che | |
| dc.creator | Khan, Juveriya Qamar | |
| dc.date.accessioned | 2024-08-12T14:24:16Z | |
| dc.date.available | 2024-08-12T14:24:16Z | |
| dc.date.copyright | 2024 | |
| dc.date.created | 2024-09 | |
| dc.date.issued | 2024-08-12 | |
| dc.date.submitted | September 2024 | |
| dc.date.updated | 2024-08-12T14:24:16Z | |
| dc.description.abstract | Early in the COVID-19 pandemic we initiated a collaboration to identify host dependency factors and consequently antiviral targets by using CRISPR knockout screens. To focus on human lung-relevant factors, we made NCI-H23 (lung adenocarcinoma) cells susceptible to SARS-CoV-2 by transduction of ACE2 receptor. CRISPR knockout screens done in this cell line identified >400 potential host dependency factors. To confirm impacts of host dependency factors on SARS-CoV-2 we used an siRNA screen and drugs that target specific genes and assessed their impact on SARS-CoV-2 replication. For rapid assessment of virus replication, we generated a NanoLuciferase reporter virus using reverse genetics for which NanoLuciferase expression correlates with virus replication. Of 165 genes targeted by the siRNAs, 55 showed an influence on virus replication. Of 21 drugs tested, four drugs targeting- HTR3E (Dihydroergocristine), KAT5 (Donepezil), and NRAS-MAPK pathway (Trametinib, Sorafenib) inhibited SARS-CoV-2 Wuhan, Delta, and Omicron variants. Furthermore, the drug and siRNA screens confirmed the NRAS-MAPK pathway as pro-viral for SARS-CoV-2 Wuhan and Delta variants. To investigate the mechanistic interplay between host factors and positive sense RNA viruses we studied the lifecycle promotion of bovine viral diarrhea virus (BVDV) by miR-17 and let-7, using a monocistronic subgenomic BVDV reporter replicon. Using DROSHA knockout cells (which lack miR-17 and let-7 expression) and miRNA supplementation we found that miR-17 is required for efficient replication whereas let-7 (a/b/i) is not. Using a set of perfectly complementary small RNAs that anneal to different locations on the 3’UTR, we mapped the boundaries in the 3’UTR to which small RNA annealing promotes virus replication, identified phenotypically distinct roles of two let-7 binding locations, and identified that translation enhancement by the miRNAs, more than genomic stabilization, plays a mechanistic role by which small RNA annealing promotes virus replication. Furthermore, we identified features of replication-promoting RNA structures of the 3’UTR induced by small RNA annealing using SHAPE analysis. In conclusion, our study identified host genes and four novel drugs that can be repurposed to treat SARS-CoV-2 and identified the NRAS-MAPK as an important pro-viral host pathway for SARS-CoV-2. We further defined the mechanism and RNA structure changes induced by small RNAs that promote BVDV lifecycle. | |
| dc.format.mimetype | application/pdf | |
| dc.identifier.uri | https://hdl.handle.net/10388/15895 | |
| dc.language.iso | en | |
| dc.subject | SARS-CoV-2 | |
| dc.subject | BVDV | |
| dc.subject | host-virus interactions | |
| dc.subject | CRISPR screen | |
| dc.subject | antivirals | |
| dc.subject | miRNAs | |
| dc.subject | RNA structures | |
| dc.title | Virus-Host Interactions of Positive Sense RNA Viruses: Insights from SARS-CoV-2 and Bovine Viral Diarrhea Virus | |
| dc.type | Thesis | |
| dc.type.material | text | |
| thesis.degree.department | Biochemistry, Microbiology and Immunology | |
| thesis.degree.discipline | Microbiology and Immunology | |
| thesis.degree.grantor | University of Saskatchewan | |
| thesis.degree.level | Doctoral | |
| thesis.degree.name | Doctor of Philosophy (Ph.D.) |