Programmed cell death during norovirus infection and the development of a reverse genetic tool to study viral replication
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2022
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Melbourne Authors
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Microbiology & Immunology
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PhD thesis
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Open Access
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© 2022 Joshua Mark Deerain
Abstract
Human norovirus (HuNoV) is the leading cause of acute gastroenteritis worldwide with over 680 million cases each year resulting in an estimated $65 billion in associated costs and over 200,000 deaths. Despite the significance of this pathogen to global health, challenges in culturing noroviruses have significantly impeded our understanding of how these viruses infect, cause disease, and modulate the innate immune response. Programmed cell death (PCD) is an important component of the innate response to invading pathogens, but little is known about how specific PCD pathways contribute to norovirus replication and facilitate clearance and inflammation.
In these studies, we characterised PCD during murine norovirus (MNV) infection of bone marrow-derived macrophages and determined that a dramatic reduction in cell viability correlated with the exponential release of infectious virus. We showed, genetically in both immortalized and primary macrophages, and chemically, that MNV-induced cell death and replication occurs independent of the essential effectors of pyroptosis: caspase-1, caspase-11 and gasdermin D; necroptosis: RIPK1, RIPK3 and MLKL; and extrinsic apoptosis: caspase-8. Intriguingly we observed that during infection with MNV the initiators of necroptosis, namely RIPK1 and RIPK3, were reduced in abundance as the infection progressed. Further analysis revealed that MNV infection promoted the cleavage of apoptotic caspase-3 and PARP. Correspondingly, pan-caspase inhibition, or BAX and BAK deficiency, perturbed viral replication rates and delayed virus release and cell death. Overall, these observations confirmed that MNV infection promoted the induction of intrinsic apoptosis facilitating viral replication and release.
Additional investigations revealed a previously unrecognised mechanism of apoptosis induction by MNV. We showed that either infection or over-expression of the viral protein NS3 was sufficient to induce apoptosis through repression of host cell translation and reduction of pro-survival BCL-2 family proteins including MCL-1. Treatment with the proteosome inhibitor MG-132 or the caspase inhibitor QVD protected cells against apoptosis but only MG-132 prevented MCL-1 loss and apoptosis induction. Furthermore, through generation of truncation mutants and tripartite alanine mutants we mapped the region of NS3 responsible for translational shut-off and apoptosis and identified key residues between amino acids 67-100 of the protein. Additionally, we found that HuNoV homologue of NS3 also induced translation repression and apoptosis, signifying a conserved function and possible antiviral target for potential drug design.
Lastly, we describe the development of a novel reverse genetics tool to study noroviruses. Through application of circular polymerase extension reaction (CPER), we optimised the system for propagation of MNV and demonstrate that it could be used to isolate HuNoV directly from clinical material. Further advancing the approach, we designed and constructed multiple recombinant, chimeric and reporter-tagged viruses. While many of these failed to yield infectious virus due to replication constraints, NS3-mutant viruses could be recovered offering a powerful reagent to further our investigations into MNV-induced apoptosis. Together we describe the benefits and immense potential of CPER for NoVs.
Overall, this research has contributed to the understanding of virus-host interactions and has provided a molecular and biochemical mechanism that results in MNV-induced translational control and virus release. Incorporating our new CPER approach we can interrogate not only the functional capacity of the MNV NS3, but provide an application to pave the way in the recovery of HuNoV directly from patient samples.
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Keywords
Norovirus, Virology, Programmed cell death, Apoptosis