Degree
Master of Science
Program
Microbiology and Immunology
Supervisor
Dr. JS Mymryk
Abstract
Viruses rely on host cell machinery, often mimicking cellular components, in order to circumvent host cell defenses and hijack cellular processes. DNA viruses, such as human Adenovirus (hAdV), rely on RNA Polymerase II (RNAPII) to transcribe viral genes. RNAPII has a C-terminal domain (CTD), made up of highly conserved heptad repeats of tyrosine-serine-proline-threonine-serine-proline-serine (YSPTSPS). Post-translational modifications of residues within the CTD, including phosphorylation, coordinates the transcription cycle. Several viruses, including Human Immunodeficiency Virus (HIV), Human Cytomegalovirus (hCMV), Epstein-Bar Virus (EBV) and Herpes Simplex Virus (HSV), modify the phosphorylation state of the RNAPII CTD by hijacking cellular cyclin dependent kinases (CDKs) to enhance viral transcription. The hAdV E1A protein, specifically the conserved region 3 (CR3), is a potent transcriptional activator. This region contains a sequence (YSPVSEP), highly conserved across hAdV subtypes, that has considerate sequence similarity to the heptad repeat of the RNAPII CTD. Two serine residues in E1A (S185 and S188) are phosphorylated, mimicking the phosphorylation of serine at positions 2 and 5 of the RNAPII CTD. We believe that given the sequence similarity, and the phosphorylation at conserved serine residues, that E1A CR3 acts as a mimic of the CTD to promote transcriptional elongation of viral genes. This study has demonstrated the role of the putative CTD mimic, and the specific phosphorylation of serine residues in this region, for maximal transcription activation. This study examined how this region may structurally and functionally mimic the RNAPII CTD. To assess functional mimicry, the E1A putative CTD mimic was investigated for it’s ability to interact with CDK9, a protein known to interact with the RNAPII CTD. Although this interaction is not specific to the phosphorylation of S185 and S188, it may still rely on the putative CTD mimic region within E1A CR3. Finally, I constructed a novel hAdV point mutant (JM17-E1A S185/188A) and characterized the kinetics of viral gene expression so that it can be used as a tool to examine the role of the putative CTD mimic in transcription of viral genes. Cells infected with JM17-E1A S185/188A showed decreased amounts of E1A production, but not other viral genes, when compared to wild type (WT) virus. This study investigated a novel example of viral mimicry, and the mechanism by which it enhances viral transcription.
Recommended Citation
Galpin, Kristianne JC, "Investigating Adenovirus E1A as an RNA Polymerase II C-Terminal Domain Mimic and its Role in Transcription Activation" (2017). Electronic Thesis and Dissertation Repository. 4680.
https://ir.lib.uwo.ca/etd/4680