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