Abstract
Influenza A virus (IAV) remains an ongoing threat and challenge to the global public health. The ever-rapid evolving nature of IAV restricts the development of effective long-term vaccines and antiviral drugs. This also provides IAV with the ability to evade host antiviral mechanisms, allowing continual replication of different IAV strains.
A growing interest on enhancing host antiviral mechanisms as a new antiviral strategy against IAV has developed over the years. Host histone deacetylase 1 and 2 (HDAC1 and HDAC2) have shown strong antiviral abilities. The expression of these HDACs is known to be downregulated and antagonised by IAV, however the exact mechanism of these manipulations remains unknown. The main objective of this study is to identify the IAV proteins and host factors, involved with the manipulation of HDAC1 and HDAC2 expression.
The first section of this thesis involves a series of individual IAV internal gene expression knockdowns, in an attempt to recover the antagonism of HDAC1 and HDAC2 expression in IAV infected cells. Suppressed gene expression of the IAV nucleoprotein (NP) recovered HDAC1 on the polypeptide level, to > 100% in IAV infected cells but this recovery was not detected on the mRNA level. Additionally, the recovery of HDAC2 antagonism on both the polypeptide and mRNA levels was not observed within this series. Furthermore, the manipulation of HDAC1 and HDAC2 expression in IAV infected cells was inconsistent over multiple experiments. Hence, the limitations of studying endogenous HDAC1 and HDAC2 proteins were discovered through protocol optimisations.
The second section of this thesis utilises high-throughput screening by mass spectrometry (SWATH-MS), to identify unique protein interactors of HDAC1 and HDAC2 in IAV infected cells. The coimmunoprecipitation of HDAC1 lead to discoveries of two unique interacting proteins, TRIM21 and IMPDH2. The interferon induced E3 ligase, TRIM21 was identified as a novel interactor of HDAC1 in IAV infected cells. This was further confirmed through western blot analysis in HDAC1 coimmunoprecipitates. However, preliminary analysis on this interaction, has suggested that TRIM21 does not regulate the antagonism of HDAC1 on the polypeptide level in IAV infected cells. Instead, the findings suggest that TRIM21 could facilitate IAV replication in a strain dependent manner, and its expression is instead regulated by HDAC1. Further, no novel interactors were found in HDAC2 coimmunoprecipitates.
Overall, these finding suggest the need for alternate experimental approaches, to further validate the manipulation of HDAC1 and HDAC2 expression in IAV infected cells. The findings in the first section of this thesis have led to uncertainties about the manipulations of HDAC1 and HDAC2 during IAV infection. However, based upon literature reviews, coupled with the findings in this thesis, HDAC1 and HDAC2 remain promising candidates for future studies relating to IAV infection.