Underpinning the Molecular Barriers to Emergence of Novel Viruses

Nasr, Asmaa and Munir, Muhammad and Copeland, Nikki (2026) Underpinning the Molecular Barriers to Emergence of Novel Viruses. PhD thesis, Lancaster University.

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Abstract

Over the last two decades, bats have been linked to the emergence of several human viral pandemics. Molecular barriers to virus replication govern the viral host range, interspecies transmission, and potential spillover. N6-methyladenosine (m6A) is a post-transcriptional modification regulated by a wide range of cellular proteins, collectively referred to as the m6A machinery, which add (writers), remove (erasers), and recognise (readers) the modification on the target RNA. Angiotensin-converting enzyme 2 (ACE2) is an essential component of the renin-angiotensin system (RAS), and it also serves as a gateway for the replication of several coronaviruses. This study hypothesised the potential role of ACE2 and m6A as key barriers to the emergence of viruses in bats. Thus, the study examined the roles of these two barriers in the replication of recombinant Cedar virus green fluorescent protein (rCedPV-GFP) and bat coronavirus HKU5 carrying a SARS-CoV-1 spike ectodomain (BtCoV HKU5-SE) using Rousettus aegyptiacus (R. aegyptiacus)-derived cell lines, accompanied by comparisons to human m6A (hm6A) machinery and human ACE2 (hACE2). The study also incorporated comparative bioinformatic analyses of m6A components of R. aegyptiacus, Pteropus alecto (P. alecto), and Homo sapiens (H. sapiens). Initial bioinformatics and in silico analyses revealed that the identity matrices of m6A machinery of R. aegyptiacus, P. alecto, and H. sapiens have a high degree of similarity in the amino acid sequences of the machinery across the three species. Additionally, several syntenic genes were similarly detected in the m6A loci of these mammals. At the structural level, the m6A writer group in R. aegyptiacus and P. alecto was identical to that of H. sapiens. In contrast, erasers and readers exhibited polymorphism between the two bat species and between bats and humans. Adaptation of rCedPV-GFP in R. aegyptiacus cell lines (R06E and R05T) was performed using VeroE6 cells, while the virus failed to adapt using BHK-21. Screening of the human 6A machinery against rCedPV-GFP in human A549 cells revealed antiviral activity of methyltransferase-like 3 (METTL3) and Wilms' tumour 1-associating protein (WTAP) writers, the eraser AlkB homolog 5 (ALKBH5), and all reader proteins, with YTH domain-containing 2 (YTHDC2) being the most potent inhibitor of the virus. Establishment of R06E cell lines that overexpressed R. aegyptiacus METTL3 (RE 4 METTL3) and its associated domains was performed using the piggyBac (pB) transposon system. Infection of these cell lines with rCedPV-GFP revealed that the full-length protein and its domains exerted comparable antiviral activity. Moreover, the full-length protein was translocated from the nucleus to the cytoplasm upon rCedPV-GFP infection. Infection of R06E wild-type (R06E WT) with the BtCoV HKU5-SE virus resulted in abortive infection due to the low expression level of ACE2. Using the pB system, the roles of both R. aegyptiacus ACE2 (RE-ACE2) and hACE2 in viral replication were investigated. While overexpression of RE-ACE2 significantly increased infection in R06E, it did not reach the level achieved with hACE2. Nevertheless, both receptors induced virus syncytia under the fluorescence microscope; moreover, both receptors underwent degradation upon virus infection, although hACE2 showed more pronounced degradation than RE-ACE2. Using protease inhibitors, BtCoV HKU5-SE was detected to depend on the transmembrane protease serine 2 (TMPRSS2) for its entry in R06E rather than cathepsin L (CTSL). Whereas qPCR results indicated significant upregulation of RE-ACE2 and TMPRSS2 at the peak of viral infection in the cells, the heat map analysis showed co-expression of RE-ACE2 with the interferon-stimulated genes (ISGs). These results collectively provide fundamental approaches for understanding the role of m6A and the ACE2 receptor as key barriers to the emergence of novel viruses in bats.