Functional studies of viral and host cell factors involved in the regulation of coronaviruses replication and pathogenesis

• Main Author: Ng, Yan Ling
• Other Authors: James P Tam
• Format: Thesis-Doctor of Philosophy
• Language: English
• Published: Nanyang Technological University 2020
• Subjects: Science::Biological sciences::Microbiology::Virology
• Online Access: https://hdl.handle.net/10356/140136
• Institution: Nanyang Technological University

Upper respiratory tract infections can be caused by a variety of viruses, such as rhinoviruses, influenza viruses, and respiratory syncytial virus. Of these, coronavirus (CoV, Coronaviridae, Nidovirales) is an important upper respiratory tract pathogen that infects both humans and livestock. It is responsible for the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), Middle East Respiratory Syndrome coronavirus (MERS-CoV) and Coronavirus Disease 2019 (COVID-19) outbreaks that resulted in human cases with varying clinical outcomes, from minor upper respiratory tract infections to fatalities. Despite its medical importance, Coronavirus-host interactions that lead to increased mammalian infectivity and pathogenicity remain mostly enigmatic. In this study, two host factors identified from siRNA screening: RNA (Guanine-7-) Methyltransferase (RNMT) and Cleavage and Polyadenylation Factor I Subunit 1 (CLP1) are functionally characterized to better understand their contribution to Infectious Bronchitis Virus (IBV) replication and pathogenesis, through the generation of CRISPR/Cas9 mediated gene knockout (KO) cell lines. Here, we show that RNMT and CLP1 are enhancers in IBV infection. Total IBV genomic and subgenomic RNA expressed across multiple post-infection time points over 24 hours were under-expressed by at least 50% in RNMT-/- and CLP1-/- cells, compared to wildtype H1299 cells. Wild-type RNMT and CLP1-/- overexpression further confirmed its role as an enhancer in H1299 cells, with an average 2-fold increase in viral subgenomic RNA, protein expression, and viral titer within 24 hours (RNMT p-value: 0.0526; CLP1 p-value: 0.014). Continuous passage of wild-type IBV in RNMT- and CLP1- KO cells showed adaptation of the virus to the mutant cells. Sequencing of RNMT- and CLP1-adapted IBV revealed several point mutations in IBV non-structural proteins (NSPs) 3, 5, 15, and 16, with a unique tri-nucleotide deletion in the NSP5 of CLP1-adapted IBV. Our findings are a promising start to co-immunoprecipitation assays to determine the virus-host interface in CoV replication. These findings would shed light on the signaling pathways and immunoevasion mechanisms utilized by CoVs to interact with these host factors, which may initiate new research directions in understanding CoV-host interactions.