Investigations into the Entry Mechanisms of Emergent and Pre-emergent Betacoronaviruses
Year: 2023
Type: dissertation
Abstract: SARS-CoV-2 is the third highly pathogenic coronavirus (CoV) to emerge in the past 20 years, highlighting the continued risk of cross-species CoV transmission. Bats harbour many novel CoVs that pose a threat for spillover to humans, such as RaTG-13, which is highly related to SARS-CoV-2, and WIV1-CoV and WIV16-CoV, which are highly related to SARS-CoV-1. In the past, ACE2 receptor usage has been extensively explored as a determinant for cross-species transmission; however, most bat-CoVs may not utilize ACE2 as an entry receptor, and it has been recently shown that some bat-CoVs infect human cells in the absence of ACE2. As such, the detailed cell entry mechanisms of bat CoVs remain to be defined. In this study, I worked to investigate potential broadly conserved beta-CoV entry factors, such as glycans and entry proteases. Through heparin competition assays and genetic depletion of a key heparan sulfate biosynthesis enzyme (EXT1), we demonstrated that heparan sulfate is a conserved entry factor for beta-CoVs. Furthermore, we provided preliminary characterization of the role that specific heparan sulfate proteoglycans (syndecans and glypicans) play in beta-CoV entry through stable knockdown. Conversely, we showed that removal of terminal sialic acid from complex glycans does not broadly affect beta-CoV entry. We additionally explored the varying requirement of different entry proteases in beta-CoV entry. We demonstrated that similar to SARS-CoV-1, the bat CoVs WIV1-CoV and WIV16-CoV prefer the endosomal entry pathway mediated by cathepsin L, contrasting SARS-CoV-2 and MERS-CoV, which favor the TMPRSS2-mediated cell surface entry route. We also confirmed a synergistic relationship between the existence of the furin cleavage site on SARS-CoV-2 spike and its ability to use the TMPRSS2 cell-surface pathway, as the disruption of its conserved furin cleavage site led to reliance of SARS-CoV-2 on the endosomal pathway for entry. By characterizing the conserved entry mechanisms employed by beta-CoVs, we can provide a framework for the rational design and development of pan-CoV antivirals, thus equipping us to effectively combat future beta-CoV outbreaks.
Author: Siwak, Kimberley
Language: English
Subfield: Infectious Diseases
Field: Medicine
Domain: Health Sciences
Open Access status: green
OpenAlex ID: W7029360864