Discovery of novel host dependency factors for Middle East respiratory syndrome coronavirus infection by bioinformatics and biochemical approaches

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic coronavirus that emerged in 2012 and continues to cause severe human infections in the Middle East. Despite the clinical and public health importance of MERS-CoV, critical issues regarding virus-host interactions remain including incompletely understood. This thesis aims to identify host factors which are essential in the replication of MERS-CoV. To discover novel attachment factor involved in MERS-CoV entry, the interactions between MERS-CoV spike protein and cell surface binding molecules were investigated in the first part of the thesis. Using a combination of in vitro and ex vivo models, 78 kDa glucose-regulated protein (GRP78) was identified as an attachment factor of MERS-CoV which significantly modulated virus entry in the presence of dipeptidyl peptidase 4 (DPP4), the known receptor of MERS-CoV. Importantly, GRP78 also promoted the attachment of bat coronavirus HKU9 (bCoV-HKU9) in bat cells. The common utilization of GRP78 as an attachment factor by two betacoronaviruses implied a potential risk of interspecies transmission of bCoV-HKU9. Host non-coding RNAs such as circular RNAs (circRNAs) function in the competing endogenous RNA (ceRNA) network to exert critical regulatory roles during viral infection. The ceRNA of MERS-CoV was previously unknown. In the second part of the thesis, global transcriptomic profiling of MERS-CoV infection in human pulmonary Calu-3 cells was performed to identify a high number of virus-induced perturbations of circRNAs, miRNAs, and mRNAs involved in a wide range of biological processes. The biological relevance of two of these perturbed circRNAs (hsa_circ_0067985 and hsa_circ_0006275) identified from the computationally integrated circRNA-miRNA-mRNA co-regulatory network were investigated. In vitro validation assays demonstrated that both hsa_circ_0067985 and hsa_circ_0006275 significantly affected MERS-CoV replication through indirectly modulating the target gene expression as miRNA sponges. Based on our finding that circRNAs and their cognitively expressed mRNAs more significantly co-expressed in MERS-CoV-infected than in mock-infected human pulmonary Calu-3 cells, we postulated that MERS-CoV hijacked specific RNA binding proteins (RBPs) to regulate the expression of pathogenic circRNAs and cognitive mRNAs for efficient propagation. To this end, the interactions between RBPs and the representative circRNA-cognitive mRNA pairs upregulated upon MERS-CoV infection were systemically analysed in the third part of the thesis. Heterogeneous nuclear ribonucleoprotein C (HNRNPC) was identified as a critical host factor governing the expression of multiple MERS-CoV-perturbed circRNAs, including hsa_circ_0002846, hsa_circ_0002061, and hsa_circ_0004445. siRNA knockdown of HNRNPC potently suppressed MERS-CoV replication in vitro via the CRK-Abl2-Akt-mTOR pathway. The mTOR inhibitor OSI-027 was found to have significant antiviral activities against MERS-CoV and SARS-CoV-2 in vitro. Collectively, the findings in this thesis provided novel insights on GRP78’s role as an important attachment factor of MERS-CoV, revealed the previously unknown ceRNA network perturbations in MERS-CoV infection, and described the interactions between HNRNPC and MERS-CoV-perturbed circRNAs and their cognitive mRNAs. These new discoveries deepened understandings on MERS-CoV-host interactions and may help to identify novel host-targeting treatment strategies for MERS-CoV infection.