Date of Award

2021

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

Susan R. Weiss

Abstract

In the last decade, two novel coronaviruses have emerged from zoonotic sources to humans. Middle East Respiratory Syndrome Coronavirus (MERS-CoV) emerged in 2012 causing several outbreaks of severe respiratory illness with a high case fatality ratio of 35%. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerged in late 2019 causing a devastating pandemic that has caused over 2.7 million deaths in over 124 million cases as of March 2021. Coronaviruses are positive sense single-stranded RNA viruses and are adept at delaying or suppressing activation of innate immune responses in their hosts, despite detectable double-stranded (ds)RNA production during infection. Our goal was to understand how the highly pathogenic human coronaviruses, MERS-CoV and SARS-CoV-2, interact with and evade the dsRNA-induced innate immune pathways: type I and III interferon (IFN) production and signaling, protein kinase R (PKR), and oligo adenylate synthetase ribonuclease L (OAS/RNase L). We found that MERS-CoV evades activation of IFN, PKR and OAS/RNase L due to the activities of three different proteins. We show during authentic MERS-CoV infection that dsRNA-binding accessory protein NS4a blocks PKR activation and IFN mRNA expression, and that accessory protein NS4b blocks IFN induction through its phosphodiesterase (PDE) activity and nuclear localization. Furthermore, we examined the role of conserved coronavirus protein nsp15 endoribonuclease (EndoU) catalytic activity during MERS-CoV infection. We found that inactivation of EndoU during MERS-CoV infection had mild effects on the dsRNA-induced innate immune pathways. However, inactivation of EndoU in combination with loss of expression of accessory protein NS4a or inactivation of PDE activity of NS4b caused defects in infectious virus production and robust activation of the innate immune pathways in MERS-CoV infected A549DPP4 cells. This highlighted redundant functions of EndoU, NS4a, and NS4b that together lead to strong suppression and evasion of dsRNA induced innate immunity during MERS-CoV infection. We also investigated the interactions of SARS-CoV-2 with the dsRNA-induced pathways. We found that SARS-CoV-2, unlike MERS-CoV, induced mild IFN expression and moderately activated PKR and OAS/RNase L in lung derived cell lines. Our findings fill some of the gaps in knowledge of how highly pathogenic human coronaviruses interact with the innate immune system.

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Virology Commons

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