PROTEINS pV AND pVIII ENCODED BY BOVINE ADENOVIRUS-3 ARE POTENTIAL VIRAL SUPPRESSORS OF THE INTERFERON RESPONSE
Date
2017-09-19
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ORCID
Type
Thesis
Degree Level
Masters
Abstract
Bovine adenovirus 3 (BAdV-3), which belongs to the Mastadenovirus genus, is a non-enveloped virus with an icosahedral capsid of 75 nm diameter, which wraps a double-stranded linear DNA genome with size of 34,446 base pairs (Reddy et al., 1999). It has been studied for potential application as gene delivery vector (Zakhartchouk et al., 1999). But one of the major limitations of adenovirus vector application is the induction of innate immune response. It has been reported that multiple adenoviral components could be detected by several host sensors leading to multi-layered innate immune response, which limit the viral infection (Hendrickx et al., 2014a, Thaci et al., 2011). Like many other viruses, it has also been shown that adenoviruses have developed strategies to modulate the innate antiviral by expression of adenoviral early proteins E1A, E1B, E3, E4 as well as structural proteins pVI and pVII (Avgousti et al., 2016, Burgert et al., 2002, Schreiner et al., 2012).
Here, the objective of this study is to identify BAdV-3 viral proteins that are involved in regulating the primary signaling cascade of innate immune response. This could provide insight into methods of engineering BAdVs in future with attenuated innate response and enhanced vaccine delivery features. The initial luciferase reporter assay results of monitoring type I interferon (IFN) response suggest that several BAdV-3 viral proteins may act as potential suppressors of the interferon response. Since both pV (a core protein connecting the viral capsid and DNA genome) and pVIII (a structural protein connecting the core with the viral capsid) significantly reduced the expression of luciferase due to reduced activation of IFN- promoter in both cell lines tested, we choose pV and pVIII for further analysis (Ayalew et al., 2016, Gaba, 2016).
Overexpression of pattern recognition receptors (RIG-1, MDA-5, TLR-3) did not increase the IFN- activity in cells expressing pV, suggesting that pV acts at a common step(s) of IFN- signaling pathways used by different PRRs. Overexpression of TBK1 but not IKK significantly down-regulated the IFN- promoter activity in pV expressing cells suggesting that pV may suppress the activation of IFN-β promoter by targeting the activity down-stream of TBK1 activation including activation of interferon regulatory factor(s) (IRF) or at the level of IKKε kinase activation. Using co-immunoprecipitation assays, we demonstrated that pV interacts with IRF3. Further analysis using Western blotting and immunofluorescence microscopy suggested that expression of pV leads to significant reduction in the phosphorylation and translocation of transcription factor IRF3 to the nucleus. These results suggest that BAdV-3 pV modulate the activation of IFN- promoter by affecting the required activation of IRF3 for its transcription activation function.
Although pV significantly reduced the transcription factor NF-kB or IRF7 responsive promoter activity, there was no effect on the nuclear translocation of NF-kB and IRF7 in the presence of pV, suggesting that pV may not act to modulate IFN- promoter activity by targeting NF-kB and IRF7 transcriptional factors.
Similar experiments using overexpression of different PRRs (RIG-1, MDA-5, TLR-3) or kinases (TBK1 and IKKε) in pVIII expressing cells suggested that pVIII may act down-steam of TBK1 and IKKε in the signaling pathway of IFN- promoter activation. Moreover, although pVIII significantly reduced the transcription factor NF-kB responsive promoter activity, there was no effect on the nuclear translocation of NF-kB in the presence of pVIII, suggesting that pVIII may not act to modulate IFN- promoter activity by targeting NF-kB and IRF7 transcriptional factors. Further experiments are needed to explore the possibility that pVIII interferes with the DNA binding activity of IRF3 by assessing the function of CBP/p300 transcriptional co-activating proteins.
Description
Keywords
Adenovirus, interferon response
Citation
Degree
Master of Science (M.Sc.)
Department
School of Public Health
Program
Vaccinology and Immunotherapeutics