Abstract
The inherent antiviral host factors play a critical role in host defence against human pathogens, including influenza A virus (IAV). The class I host histone deacetylases (HDACs), HDAC1, HDAC2, HDAC3 and HDAC8 have been discovered to inhibit IAV infection. To this end, this thesis aims to determine (1) the role of all class I HDACs in the IAV-induced nuclear translocation of transcription factors, STAT1 and STAT3 by confocal microscopy and (2) identify the antiviral networks of HDAC1 and HDAC2 in IAV-infected cells by mass spectrometry.
To achieve the first objective, STAT1-GFP or STAT3-GFP were overexpressed from a plasmid in HeLa cells, depleted of individual HDAC expression by RNA interference (RNAi). Subsequently, cells were infected with an IAV H1N1 strain and visualized by confocal microscopy. The impact of class I HDACs on STAT signalling was determined by the change in nuclear–cytoplasmic ratio (N/C) of STAT3-GFP, in cells depleted of HDACs during IAV infection. The results suggest HDAC3 negatively regulates the nuclear localisation of STAT3 whereas HDAC1 and HDAC2 aids the nuclear retention of STAT3. Together, the differential immune regulatory mechanisms of individual class I HDACs on STAT3 expression was demonstrated.
To achieve the second objective, human lung epithelial cells, A549 were depleted of HDAC1 or HDAC2 expression by RNAi and subsequently infected with an IAV H1N1 strain. The cells were processed for Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS). Then, peptide peaks were analysed to determine the changes in protein abundances in the HDAC1 or HDAC2 depleted samples. The results have identified many antiviral proteins involved in host immune responses. In particular, STAU2 and ZC3HAV1L, proviral factors, were upregulated in response to the knockdown of HDAC1 in IAV infected cells. Notably, FAM3C, ILF3, and mTOR, members of the STAT3 immune signalling cascade, were downregulated in response to the knockdown of HDAC1.
In HDAC2 depleted cells, SERPINB1, IFIT1, and PTGES were significantly downregulated; they are immune proteins involved in inhibiting IAV replication. Furthermore, in both HDAC SWATH-MS analyses, it was revealed that AXL, an important immune response regulator, was significantly upregulated. It has been shown that HDAC1 and HDAC2 possess iii immune regulatory mechanisms through high throughput screening of proteins that are functionally aligned with the anti-viral properties of HDACs.
Overall, the results enhance the knowledge of the multilayer immune regulation of class I HDACs during IAV infection. Data obtained in this thesis indicate that class I HDACs are involved in the IAV-induced innate antiviral response. These findings will elucidate the molecular basis of the IAV pathogenesis and flu disease severity among different human populations.