Tait-Burkard, Christine

Digard, Paul

Keane, Susana

Biotechnology and Biological Sciences Research Council (BBSRC)

2023-02-14T13:07:22Z

2023-02-14T13:07:22Z

2023-02-14

Influenza A virus (IAV) causes a contagious respiratory disease which can be fatal in at risk populations. Seasonal epidemics of IAV infection are responsible for as many as 650,000 human deaths annually. Antiviral drugs currently available for the treatment of IAV infection target viral polypeptides. However, as an RNA virus, IAV is able to mutate and rapidly develop resistance to these drugs. For this reason, new approaches which focus on targeting the host are being explored. IAV interacts with many host proteins which are necessary for efficient completion of the viral lifecycle; these host factors potentially make attractive drug targets for the treatment of IAV, as development of resistance to host-targeted treatments is predicted to be slower. Host factors required for IAV replication have been identified through a number of routes including targeted research based on previous knowledge of the virus and the host, and large scale screens. The work presented in this thesis aimed to investigate three cellular proteins that had been previously proposed as pro-viral host factors for IAV. Using RNA interference (RNAi) and chemical inhibitors, I sought to dissect the role of these proteins in the viral lifecycle and to assess their suitability as drug targets in the treatment of IAV infection. The arginine demethylase JMJD6 had been suggested to have a proviral role in IAV replication. In the process of refining previously developed RNAi procedures for depleting JMJD6, I found that a published siRNA used to study JMJD6 induced an interferon (IFN) response in cells. Here, I showed that IAV was able to replicate normally in cells in which JMJD6 was depleted by alternative RNAi methods that did not induce an IFN response and in JMJD6⁻/⁻ cells, strongly suggesting that the previous finding was likely an artefact of the approach used to deplete JMJD6. Thus JMJD6 was determined not to be a host factor of IAV or a suitable target for the treatment of IAV infection. Next, a host factor that had been identified in a genome-wide CRISPR screen was investigated. The cap 2’-O-methyltransferase, CMTR1, had been proposed to be required for preventing detection of viral mRNA via the IFN signalling pathway. In my hands, knockdown of CMTR1 using RNAi did not lead to enhanced IFN response to infection, nor did it inhibit the replication of IAV in cell culture. This did not support CMTR1 alone as an effective target for IAV treatment. However, preliminary results investigating CMTR1 knockdown in combination with the viral cap-dependent endonuclease inhibitor Baloxavir, suggested that future work should examine CMTR1 as a target to enhance other antiviral treatment. Finally, the N6-methyladenosine (m⁶A) reader protein IGF2BP1 was examined. IAV RNAs, both genomic and messenger, are known to contain the m⁶A modification, which is required for optimal replication efficiency. IGF2BP1, which binds to m⁶A modified mRNA to enhance stability, has been linked to IAV replication in host interaction screens. However, using RNAi and chemical inhibition, I did not find that IGF2BP1 was required for IAV replication or spread. Furthermore, overexpression of IGF2BP1 showed no effect on IAV replication. Together these findings rule out IGF2BP1 as an IAV host factor and drug target. Overall, the work presented in this study did not support the three host proteins investigated as being suitable drug targets for the treatment of IAV, although CMTR1 could still be investigated as a target for combination therapy. The ruling out of JMJD6 and IGF2BP1 can help to focus future work investigating host targets for IAV treatment.

https://hdl.handle.net/1842/39844

http://dx.doi.org/10.7488/era/3092

en

The University of Edinburgh

Influenza A virus

IAV

contagious respiratory disease

Seasonal epidemics

Antiviral drugs

viral lifecycle

host-targeted treatment

RNA interference

RNAi

chemical inhibitors

arginine demethylase JMJD6

siRNA

JMJD6

cap 2’-O-methyltransferase, CMTR1

CMTR1

interferon signalling pathway

N⁶-methyladenosine

m⁶A

IGF2BP1

Investigation of host factors in influenza a virus infection

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy