Influenza A virus (IAV) is a global health threat, causing seasonal epidemics and potential pandemics leading to morbidity, death and economic losses. Currently, there are two main classes of licensed antivirals against IAV available in the US and Europe; adamantanes and neuraminidase inhibitors, both of which are hindered by the generation of resistant virus variants. The viral polymerase has a high error rate leading to mutations that allow the virus to overcome selection pressures directed at its own genome from conventional antivirals. The prospect of inhibiting host proteins that the virus exploits to facilitate its replication is of increasing interest as an antiviral strategy as the emergence of resistance has been predicted to be slower when targeting a host cellular factor. IAV utilizes the host nuclear export protein CRM1 to transport viral ribonucleoproteins (vRNPs) from the nucleus to the cytoplasm of an infected cell, a critical late stage of the influenza lifecycle. Leptomycin B (LMB), a Streptomyces metabolite, has been previously shown to target this pathway, resulting in reduced viral propagation; however, LMB's potent cytotoxicity has limited its use as a therapeutic agent. This thesis examined two novel selective inhibitors of nuclear export (SINE), KPT-335 and KPT-185, with less cytotoxicity. In vitro, KPT-335 inhibited replication of human and animal IAV strains in a dose-dependent manner with minimal cytotoxicity. To assess the resistance potential of KPT-335, IAV viruses were serially passaged in the presence of a sub-optimal concentration of the compound and assayed for the development of resistance. Resistance to KPT-335 became evident at 8-10 rounds of passage. Sequencing analysis of independently derived resistant virus clones identified 4 single amino acid changes on a surface exposed patch of the viral nucleoprotein (NP). Introduction of these amino acid changes, into otherwise wild type viruses by reverse genetics, confirmed that changes Q311R and N309T conferred a drug-resistant phenotype. However, these substitutions came at a fitness cost to virus replication. The molecular basis for resistance was unclear but Q311R and N309T NP-mutant viruses produced increased levels of M1 during infection as well as producing virus particles with increased M1:NP ratios. Furthermore, the KPT-335-resistance mutations were surprisingly similar to NP sequence polymorphisms previously associated with susceptibility to the innate defence protein MxA. Consistent with this, viruses harbouring the Q311R mutation displayed increased susceptibility to MxA inhibition compared to wild-type virus. Altogether this study confirms that SINEs have the potential to be successful therapeutic agents against IAV replication and that although resistance could be generated, it may be difficult for the virus to overcome both drug selection pressures and the human innate immune response restrictions by escape mutations.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:756630 |
| Date | January 2018 |
| Creators | Dewar, Rebecca Amy |
| Contributors | Digard, Paul ; Dutia, Bernadette |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/31349 |
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