title: Investigating host factors involved in human coronavirus infection in the search for antivirals
Creators: name: Kerr, Holly
Publisher: name: The University of Edinburgh
Year: 2025
Subjects: subject: Host factors
subject: Antivirals
subject: SARS-CoV-2
subject: Integrins
subject: Rab11a

description: In the past two decades, three highly pathogenic human coronaviruses (hCoVs) have emerged with the most recent, SARS-CoV-2, resulting in an ongoing global pandemic. Although vaccination and virus evolution have reduced the disease burden of COVID-19, effective antivirals are still sought for vulnerable or unvaccinated populations and to prepare for future coronavirus outbreaks. As an obligate intracellular parasite, host factors are recruited and repressed by SARS-CoV-2 throughout the entire viral replication cycle. Elucidating these interactions will aid in our molecular understanding of this virus and in the identification of candidates to develop host-directed antiviral therapies. This thesis aims to identify and characterise host factors of SARS-CoV-2 in vitro in relevant cell models and demonstrate the utility of targeting host factors as an antiviral strategy to treat COVID-19 and other hCoV infections. We take a hypothesis-driven approach to study a group of transmembrane proteins, integrins, as potential host factors of SARS-CoV-2 given the RGD-integrin binding motif presence in S. We show that integrin subunit ITGA5 is upregulated post-SARS-CoV-2 infection in Caki-1 cells and that downstream of integrins, the tyrosine kinases Src and FAK are phosphorylated with biphasic activity post-SARS-CoV-2 infection. Inhibiting Src with small molecule inhibitors reduces viral production of SARS-CoV-2 as well as replication of hCoV-229E in a dose-dependent manner with low IC50 values. Integrins and their downstream signalling pathways present attractive targets to further explore in the development of antiviral treatments against SARS-CoV-2 and other hCoVs. We undertake, analyse, and validate a druggable-genome RNAi screen to identify multiple host factor candidate genes involved at all stages of the viral replication cycle. Direct lysis RT-qPCR quantification of viral RNA from cell supernatant following siRNA-mediated gene knockdown allowed a comprehensive analysis of the full replication cycle, including viral release. Utilising a human druggable genome siRNA assay containing ~7000 gene targets, we performed knockdowns in human kidney Caki-1 cells infected with a clinical isolate of SARS-CoV-2 EDB-2 (B.1) at readouts 24 hpi and 48 hpi. Pathway and network analysis of our hit lists showed high confidence with expected proviral and antiviral gene clusters. Moreover, meta-analysis with other published studies of host factors in SARS-CoV-2, including CRISPR-KO screens and GWAS, demonstrated consistency and relevance of our hit lists. A cluster of proviral hits involved in vesicle-mediated transport, including multiple Rab GTPases, were validated in EDB-2 and variants Delta and Omicron. We identify Rab11a as a key host factor in SARS-CoV-2 viral release, which can be targeted using inhibitors of Rab11a cargo delivery. We demonstrate that Rab11a inhibitors have dual-inhibitory action of entry and release and is a promising antiviral for further study in targeting SARS-CoV-2. Finally, host factor interactions and antiviral candidates are assessed using a physiologically relevant cell model of human airway epithelial cells grown at air-liquid interface (ALI). ALI cultures are differentiated and a model for infection, quantification and confocal microscopy is established for SARS-CoV-2 and hCoV-229E. We begin to optimise a protocol for siRNA knockdown in ALI cultures and assess potential host factor interactions of SARS-CoV-2 through inhibitory action of candidate host-directed antivirals. Together, these results highlight the availability of host factors as candidate targets for new and effective antiviral therapies, and pave the way for further work to test interactions and antiviral candidates in vivo.