Determining the Entry Mechanisms of a Novel Coronavirus

Gerardi, Valeria and Munir, Muhammad and Wright, Karen (2024) Determining the Entry Mechanisms of a Novel Coronavirus. Masters thesis, Lancaster University.

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Abstract

Since the origin of the COVID-19 pandemic, numerous investigations have been triggered to identify coronaviruses in animal reservoirs that potentially hold future pathogenic potential. The study investigated the characterisation and the entry mechanism of a novel Alphacoronavirus Pak-CoV, isolated in Pakistan in 2016 from bat guano collected from Pipistrellus sp. Using a range of experimental, large-scale screening and next-generation sequencing approaches, the virus was genetically characterised. Using the genetic information, the Pak-CoV was first analysed in silico to map and characterise the spike protein which plays decisive roles in host selection and antibody-based protection. Additionally, molecular docking was performed to provide a further comprehensive view of potential residues defining receptor interaction. In order to investigate the entry mechanism, to identify patterns of viral entry across different host cell lines, and to understand the role of various receptors and co-receptors, pseudovirus particles were generated carrying spike protein on the surface and confirmed by electron microscopy. Differential entry mechanisms were observed across cell lines and the full-length Pak-S, and the truncated Pak-S exhibited varying entry efficiencies. These investigations highlight that the entry mechanisms might involve receptors other than ACE2 and TMPRSS2 and that the cytoplasmic tail plays a significant role in the virus’s fusion process. Treatment with TPCK-treated trypsin significantly enhanced viral entry highlighting the impact of protease activity on viral entry. Serum antibodies against SARS-COV-2 effectively neutralised the pseudovirus particles of Pak-S. Collectively, the study provided insights into the mechanistic pathways of Pak-CoV entry and the role of specific receptors and co-receptors. The significant role of ACE2 in facilitating viral entry aligns with entry routes of HCoV-NL63 and SARS-CoV-2, suggesting potential shared pathways between these viruses, and cross-reactivity of antibodies.