Human Rhinovirus 3C protease cleaves receptor-interacting protein kinase-1 to modulate extrinsic apoptosis

  • Sarah Croft

Student thesis: Doctoral Thesis


Human rhinovirus (HRV) causes human respiratory illnesses, such as the common cold, asthma exacerbations and is often followed by secondary bacterial infections. Induction of apoptosis is an innate immune response to HRV infection. The apoptotic signal is generated either via receptors (extrinsic) or disruption of homeostasis within the cell (intrinsic) and is propagated via caspase cascades. Cell death reduces viral replication as HRV relies on cellular machinery for replication. As such, the overall aim of this study was to elucidate the effect of HRV16 on apoptosis.
To first elucidate the method of cell death induced in response to HRV16, caspase activation, cellular membrane changes and apoptosis-associated caspase 8 complexes were analysed. This data shows that early apoptotic events are induced in response to HRV16, however late apoptotic changes are not, indicating that apoptosis may be induced in response to HRV16 infection but a viral countermeasure suppresses this response.
The HRV 3C protease is key in not only viral protein maturation but also in altering the cellular environment to promote viral replication through proteolysis of key cellular substrates. I used inhibitors of the 3C protease in infected cells, cell-free protease assays and expression of 3C protease in transfected cells to show that HRV16 and HRV14 3C protease cleaves RIPK1. RIPK1 is a key adaptor protein that promotes different cell death pathways, and thus this cleavage event may provide a mechanism by which HRV suppresses apoptosis pathways.
Next, the effect of Actinomycin D (ActoD) induced apoptosis on viral replication was investigated by infecting cells with HRV16, followed by treatment with ActoD. The effect of apoptosis on viral replication parallels previous reports that ActoD treatment reduces viral replication. The treatment of infected cells with ActoD and a caspase 8 or RIPK1 inhibitor did not reduce virus titre, indicating that the apoptosis pathway that is detrimental to HRV16 occurs through RIPK1. Further work to elucidate the apoptotic consequence that restricts viral replication showed that viral protein synthesis and viral release were not reduced with ActoD treatment, hence apoptosis may reduce viral RNA synthesis.
The 3C mediated cleavage of RIPK1 was also examined in representative HRV strains from major and minor groups of HRV A and B species. The cleavage of RIPK1 was conserved across all tested HRVs and may occur at two cleavage sites in HRV B species viruses. Additionally, HRV A species viruses were shown to induce necrosis-like cell death, even in the presence of an apoptosis inducer. While HRV B species viruses were not sensitive to the induction of apoptosis.
Ex vivo and in vivo models of HRV16 infection were also used to examine RIPK1 cleavage in a biologically relevant model. While data is inconclusive, we hypothesise that the modulation of apoptosis may result in a skewing of cell death pathways towards an inflammatory response and subsequently perpetuate viral-induced apoptosis.
These results suggest RIPK1 cleavage may be a mechanism by which HRV regulates the apoptotic process. Taken together, the results presented in this study highlight the importance of viral regulation of apoptosis for optimal viral replication.
Date of Award2018
Original languageEnglish
SupervisorReena Ghildyal (Supervisor) & Erin Walker (Supervisor)

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