Investigation on the host determinants and underlying mechanisms for the pathogenesis of MERS-CoV and SARS-CoV-2

Abstract

Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a highly pathogenic beta coronavirus, which was first reported in 2012 in Saudi Arabia. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that cause Coronavirus Disease 2019 (COVID-19) since 2019. This study investigates the pathogenesis of SARS-CoV-2 and MERS-CoV.

The thesis first investigated the binding process of coronavirus spike proteins to the host cell receptors during virus entry. Carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) was identified as a novel cell surface binding target of MERS-CoV using the viral overlay protein binding assay (VOPBA). This finding was validated using multiple experimental assays including overexpression, siRNA knockdown, antibody blocking assay, and recombinant protein blocking assay. The identification of CEACAM5 as an attachment factor of MERS-CoV that assisted MERS-CoV infection by augmenting the attachment of the virus to the host cell surface promoted our understanding on the infection mechanism of MERS-CoV.

Next, we further investigated on the pathogenic mechanism of MERS-CoV infection and revealed that MERS-CoV infection triggered necroptosis in the infected cells. Necroptosis is a newly identified form of regulated cell death that is mechanistically distinct from apoptosis. We first confirmed the involvement of necroptosis in MERS-CoV-infected cells. Next, we showed that RIPK1, RIPK3, and MLKL contributed in MERS-CoV-induced necroptosis by utilizing a combination of shRNA knockdown cell lines and specific inhibitors against RIPK1 and RIPK3 and MLKL. Importantly, the inflammatory response in mice lung and human lung tissues was significantly reduced when necroptosis were inhibited, revealing necroptosis as a key pathogenic mechanism of MERS.

In December 2019, the third highly pathogenic coronavirus capable of infecting human emerged, which was later named SARS-CoV-2. This thesis investigated the replication, cell tropism, and immune activation profile of SARS-CoV-2 infection in ex vivo human lung tissues and ex vivo human intestine tissues using SARS-CoV-1 as a control. In ex vivo human lung tissues, our results showed that SARS-CoV-2 infected and replicated three times more efficiently than that of SARS-CoV-1. Both viruses target type I and II pneumocytes and alveolar macrophages but SARS-CoV-2 did not significantly trigger the expression of types I, II, or III interferons in infected human lung tissues. SARS-CoV-2 also induced lower pro-inflammatory response in ex vivo lung tissues. Replication kinetics and gene expression of interferons and representative pro-inflammatory cytokines and chemokines were similarly detected in infected intestinal tissues to measure immune activation. In ex vivo human intestinal tissues, the replication of SARS-CoV-2 was less effective than SARS-CoV-1, causing less cytopathology in the epithelium, and elicited a stronger innate immune response, including activation of both type I and type III interferons. These findings revealed key pathogenic features of SARS-CoV-2 that in part explained the respiratory and intestinal manifestations of COVID-19.

Overall, this thesis revealed important host and viral determinants for the infection and pathogenesis of MERS-CoV and SARS-CoV-2. Our studies substantially advanced our knowledge on the biology of these highly pathogenic coronaviruses and provided potential targets of interventions for the treatment of these important viral pathogens.