The role of host factor Transportins in Influenza virus entry and infection

Abstract

Influenza viruses are RNA viruses of the Orthomyxoviridae family of viruses and remain a
global threat, contributing a significant disease and healthcare burden globally. Influenza
viruses rely heavily on host proteins and cellular machinery to successfully infect its host and
complete its replication cycle. A previous study identified host protein Transportin-1 (TNPO1)
as an influenza A virus (IAV) uncoating factor, which interacts with viral protein Matrix protein
1 (M1) during virus entry. TNPO1, along with TNPO2 and TNPO3 are members of the
karyopherin-β family of nuclear import receptors, however the dependency and interaction
between TNPO2, TNPO3 and IAV remains unclear. In the first part of this study, an siRNA
targeting approach was used to determine the breadth of dependency on TNPO1, 2 and 3
on IAV entry and infection.
An opposing effect on IAV entry and infection was observed between TNPO1 and TNPO3
depleted cells. As expected, TNPO1-depletion resulted in a decrease in IAV entry and
infection, which was also seen during influenza B virus (IBV) infection. Conversely, TNPO3
depletion resulted in a significant increase in IAV entry and infection. Through further
investigation, it was demonstrated that this effect on IAV entry and infection was
endocytosis-dependent, revealing potential novel insights into IAV entry.
Secondly, the M1-TNPO1 interaction was investigated in more detail by utilising reverse
genetics to generate IAV M1 variants with substitutions in residues we hypothesised to be
important for the TNPO1 interaction. A small number of M1 variants were attenuated in
mammalian cell culture passage upon virus rescue. However, second-site substitutions were
quickly acquired in mammalian cell culture passage which coincided with an increase in viral
titre. It was observed that the M1 variants had reduced virus entry, which may involve a
defect in endosomal escape, but further work is needed to validate this conclusion.
Lastly, the use of a proximity-labelling technique was explored to map the M1 interactome,
identifying potential interactors that can be investigated further. Furthering our understanding
of viral-host protein interactions and the dependence of influenza virus on its host through
identification of novel host factors may in turn lead to novel host-targeted therapeutic
strategies.

Date of Award	10 Dec 2024
Original language	English
Awarding Institution	University of Bristol
Supervisor	Yohei Yamauchi (Supervisor), Jason Long (Supervisor) & Othmar Engelhardt (Supervisor)