Influence of the host cell factors CK2, hTERT, and PML, on the antiviral response to herpes simplex virus type I infection
Smith, Miles Christian
University of Kansas
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Herpes simplex virus type I (HSV-1) is a significant human pathogen that infects a large portion of the human population. As an obligate intracellular parasite, HSV-1 requires certain cellular factors for its replication; on the other hand, the cell deploys a variety of defenses to limit the extent to which the virus can replicate. This thesis is the summation of projects that examined the impact of three host cell factors - those being CK2, hTERT, and PML - on HSV-1 replication. CK2 is a cellular kinase that, through its phosphorylation of a large number of targets, broadly functions to promote cellular growth and survival. HSV-1 encodes nine proteins that are either potential or bona fide substrates of CK2, the virus packages the kinase into progeny virions, and CK2 activity is stimulated during infection, suggesting that this kinase is important for viral replication. Two viral substrates (i.e., ICP0 and ICP27) act to counter the interferon response while a number of cellular targets function against HSV-1. I show as part of this thesis that, while the activity of CK2 is largely dispensable for viral replication, the use of CK2 pharmacological inhibitors sensitized HSV-1 to the interferon (IFN) response, an innate cellular antiviral pathway. This effect appears to function through the viral E3 ubiquitin ligase, ICP0, though it did not affect ICP0's ability to induce the loss of two of its targets. Additionally, the effect these inhibitors had on IFN sensitivity was specific to HSV-1, as there was no such result on adenovirus or vesicular stomatitis virus. hTERT is a cellular reverse transcriptase that functions in a complex termed telomerase to regenerate the ends of human chromosomes in pluripotent cells but whose lack of expression in the major factor limiting the lifespan of terminally differentiated cells. While the reintroduction of hTERT into primary cells has been reported to extend the proliferative capacity of differentiated fibroblasts, other reports have demonstrated non-telomerase activities for hTERT and changes in the transcription profile in hTERT-restored cells. To date, however, cellular antiviral pathways have not been examined in these studies. In this thesis, I demonstrate that the IFN response is largely unchanged in fibroblasts immortalized by hTERT and that, unlike transformation by oncogenes, the requirement for HSV-1 proteins involved in overcoming cellular antiviral effectors remained high. PML is the nucleating and central organizing factor of a nuclear suborganelle, ND10s, and coordinates the cellular efforts in response to a number of stress stimuli. Available evidence suggests that PML serves in intrinsic and innate cellular antiviral defenses against HSV-1. While the role of PML in a number of pathways is regulated through post-translational modifications, the effects of phosphorylation on its antiviral activity toward HSV-1 has been largely unexplored. As part of this thesis, a mapping of phosphorylation sites on PML was undertaken and sites of phosphorylation then examined for their effects on a number of PML's activities. I have identified a number of novel sites of phosphorylation and found that sites near a SUMO interaction motif influence the ability of PML to respond to HSV-1 infection.
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