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Al-Beltagi, Sarah El-Sayed Abd El-Wahab (2025) Investigation of host innate antiviral mechanisms that block replication of influenza A virus and coronaviruses. PhD thesis, University of Nottingham.

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Abstract

Influenza A virus (IAV) and coronaviruses, including OC43 and SARS-CoV-2 variants, are

RNA respiratory viruses that pose a substantial global health challenge due to their high

transmissibility, potential for severe illness, and risk of co-infections. Despite the

availability of vaccines and antiviral treatments, the continuous emergence of viral

variants and resistance mechanisms underscores the urgent need for innovative host

targeted antiviral therapies, which are less likely to induce viral revertants. Increasing

evidence supports the antiviral activity of thapsigargin (TG), an inhibitor of the

sarcoplasmic/endoplasmic reticulum (ER) Ca2+ ATPase pump and an inducer of ER stress,

against a variety of viruses, including tick-borne encephalitis virus (TBEV), foot-and-mouth

disease virus (FMDV), and IAV. However, the precise antiviral mechanisms of TG remain

to be fully elucidated.

The aim of this thesis was to investigate the potential antiviral mechanisms of TG against

IAV, OC43, and SARS-CoV-2 variants. The first objective was to assess how TG influences

the replication of these viruses. TG was found to inhibit these viruses at various stages of

their life cycles; with post-translational inhibition observed for IAV, and transcriptional

inhibition for OC43 and SARS-CoV-2 variants. The second objective focused on evaluating

the induction of ER stress-related genes, both under basal conditions and during IAV,

OC43, and SARS-CoV-2 infections. qPCR analysis revealed that TG increased the

expression of ER stress-related genes both basally and during IAV, OC43, and SARS-CoV

2 infections. Furthermore, TG demonstrated extended antiviral activity accompanied with

activation of ER stress response during OC43 and SARS-Cov-2 infections. The third

objective was to examine the activation of the host innate immune response both basally

and during IAV, OC43, SARS-CoV-2 infection using qPCR. TG enhanced early but also

regulated activation of innate immune response. Moreover, in a preliminary study to

evaluate if TG induces paracrine antiviral effect between macrophages and epithelial

cells, supernatants from TG-treated macrophages, when used to prime epithelial cells,

effectively inhibited SARS-CoV-2 infection.

The final objective was to investigate the impact of TG on ER-synthesized IAV proteins—

HA, NA, and M2—in both pig and human cells, and to elucidate TG's antiviral mechanisms

on cellular processes exploited by these IAV proteins, including ubiquitination,

glycosylation, autophagy, and their influence on virus morphology and budding. This

objective was achieved using various techniques such as western blotting,

immunofluorescence, a ubiquitin enrichment kit, a glycoprotein isolation kit, and

transmission electron microscopy (TEM). It was found that TG induces cell-type specific

effects on ER-synthesized IAV HA, NA, and M2 proteins. In human cells (A549), TG reduced

the production of HA, NA, and M2 viral proteins and caused their accumulation in the

perinuclear region compared to controls. In contrast, in pig cells (NPTr), TG did not affect

the production or subcellular localization of HA, NA, and M2 viral proteins.

In A549 cells, TG-induced M2 reduction was accompanied by a reduction in LC3 II levels,

i.e. a decrease in autophagy, and absence of M2 ubiquitination. While, in NPTr cells, TG

did not influence M2 protein production, which exhibited a notable double-band pattern.

This TG-induced double banding of M2 was associated with (1) reduced co-localisation

between M2 and LC3, (2) interference with M2 ubiquitination, (3) reduced M2 levels in

the supernatant, and (4) a reduction in the length of the filamentous virus produced.

Similarly, TG induced a double banded pattern for NP protein, a cytoplasmic-synthetized

IAV protein, in both A549 and NPTr cells. Moreover, HA2 of HA and NP glycosylation was

reduced by TG in NPTr cells. Consequently, in both A549 and NPTr cells, TG disrupted the

assembly of viral proteins into new virions, as evidenced by a reduction in the quantity of

IAV proteins (NA, HA, NP, M1, M2) present in the supernatant. This was accompanied by

a decrease in viral RNA and infectious virus particles released, without affecting virus

budding or causing significant changes to virus morphology.

In conclusion, the findings demonstrate TG’s potential as a broad-spectrum antiviral

against these viruses, acting through multiple mechanisms that target critical host

pathways. TG induces several antiviral mechanisms, thus it offers a promising strategy for

targeting other RNA viruses that similarly exploit these pathways during replication. However, further research is required to explore TG's efficacy as a host-targeted antiviral

across a wider range of RNA viral pathogens and to assess its therapeutic potential in vivo.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Foster, Toshana Mellits, Kenneth Dunham, Stephen
Keywords:	Influenza A virus, Coronaviruses, Antiviral mechanisms, Antivirals
Subjects:	Q Science > QR Microbiology > QR355 Virology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Veterinary Medicine and Science
Item ID:	81203
Depositing User:	Al-Beltagi, Sarah
Date Deposited:	24 Jul 2025 04:40
Last Modified:	24 Jul 2025 04:40
URI:	https://eprints.nottingham.ac.uk/id/eprint/81203

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