The anti-influenza viral mechanism and viral antagonism of host histone deacetylase 6
Influenza virus is a causative agent of respiratory illness called "flu" and is a prototypic member of Orthomyxoviridae family. Influenza viruses are enveloped, negative-sense single stranded RNA viruses with a segmented genome. It causes regular seasonal epidemics, intermittent but unpredictable pandemics and outbreaks leading to significant mortality and morbidity worldwide. Influenza virus has been categorized in four types: A, B, C and D, among which, influenza A virus (IAV) has caused pandemics in 1918, 1957, 1968 and recently in 2009. Besides vaccines, neuraminidase inhibitors (Osaltamivir, Zanamivir, Peramivir and Laninamivir) are approved for the clinical use against influenza virus and cases with drug-resistant influenza have also been reported. Therefore, it is critical to understand the influenza-host interactions to discover the novel pro and anti-viral host factors that can be exploited as drug targets. Histone deacetylases (HDACs) are a broad array of enzymes, which catalyze the hydrolysis of an N(6)-acetyl-lysine residue of histone or non-histone proteins and consist of four classes. Previously, our laboratory and others have discovered that HDAC1 and HDAC2 (Class I HDACs), HDAC6 (Class II HDAC) and HDAC11 (Class IV HDAC) have anti-IAV potential. HDAC6 restricts the release of IAV by deacetylating alpha-tubulin, decreases IAV replication by inhibiting and degrading viral polymerase (PA), and regulates the RIG-I dependent innate immune response. Here, we investigate the HDAC6 dependent regulation of the innate immune response in A549 cells during IAV infection. We also investigate IAV mediated dysregulation of HDAC6 at the mRNA and polypeptide level. We observed a decreased phosphorylated STAT1 level in HDAC6 depleted A549 cells during IAV infection, which led to decreased expression of subsequent interferon effector genes (IFITM3 and ISG15). The inhibition of HDAC6 by Tubacin during IAV infection indicated that the deacetylase function of HDAC6 is required for the regulation of STAT1 phosphorylation. The decreased phosphorylation of STAT1 in HDAC6 depleted and interferon-alpha treated cells showed that HDAC6 controls the phosphorylation of STAT1 in the type I interferon signaling pathway. We discovered that IAV downregulates HDAC6 at the mRNA and polypeptide levels in a dose-and-time dependent manner, and strain independent manner. The replication potential of IAV was required to dysregulate HDAC6 at the polypeptide level. Time course kinetics analysis showed that IAV induced the cleavage of HDAC6 into polypeptides of ~120 kDa and ~98 kDa. Both cleavages appeared as temporally separate events. The potential role of lysosomal and proteasomal protein degradation pathways, and caspase-3 enzyme in degradation of HDAC6 during IAV infection was determined by employing NH4Cl, MG132 and z-DEVD-fmk, respectively. This inhibitor based analysis suggested that lysosomal protein degradation pathway and apoptotic pathways were involved in the cleavage of HDAC6 during IAV infection. The dose-dependent inhibition of the lysosomal pathway showed that this pathway induces the apoptotic pathway in a dose dependent manner during IAV infection, which also led to complete recovery of full-length HDAC6. The siRNA based knockdown of caspase-3, caspase-6 and caspase-7 showed that caspase-3 and caspase-6 are involved in the cleavage of HDAC6 during IAV infection. In summary, this PhD research shows that HDAC6 regulates STAT1 phosphorylation dependent type I interferon signaling during IAV infection. We also show that IAV restricts the anti-viral host factor HDAC6 at the mRNA and polypeptide levels using a multi-pronged molecular approach.
Advisor: Husain, Matloob; Fleming, Stephen
Degree Name: Doctor of Philosophy
Degree Discipline: Microbiology and Immunology
Publisher: University of Otago
Keywords: influenza; HDAC6; ISG15; Viperin; IFITM3; antiviral; apoptosis; caspases; STAT1
Research Type: Thesis