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Discovery of Cellular Kinase Inhibitors that Impair the Early Phase of HSV-1 Lytic Infection
Ly, Cindy Yang
Ly, Cindy Yang
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Abstract
Herpes simplex virus 1 (HSV-1) is a prevalent human pathogen infecting approximately 80-90% of the global population. HSV-1 establishes life-long infections due to its ability to cycle between a lytic and latent phase. During infection, symptoms can include painful recurrent cold sores, corneal scarification, viral encephalitis, or high infant mortality. In recent years antiviral resistance has occurred against nucleoside analogs, the main class of HSV-1 therapeutics. This has resulted in the need for identifying new strategies, targets, and antivirals against HSV-1. Herein, we developed a high-throughput assay to rapidly screen multiple small molecule libraries to identify potential antivirals against HSV-1 gene expression. Infected cell protein 0 (ICP0) is a critical immediate-early (IE) gene involved in transactivation of the HSV-1 gene cascade. ICP0 is a potent and specific inducer of ICP6. We utilized an HSV-1 reporter virus containing an ICP6 promoter::lacZ expression cassette, allowing us to analyze β-galactosidase as a measure of viral gene expression. After screening an array of structurally diverse compounds, 76 compounds were identified as potent hits against HSV-1 gene expression. From this screen I discovered that three aurora kinase inhibitors substantially reduce HSV-1 replication. It was determined that these aurora kinase inhibitors impaired the transcription of immediate-early (IE) genes, which are critical for HSV-1 replication. The loss of IE viral transcription led to significant impairment of viral protein expression in HSV-1. This study demonstrates, for the first time, a pivotal role for aurora kinases during HSV-1 lytic cycle. HSV-1 strategically modulates and redirects cellular factors to aid in its viral replication. One of aurora B kinase activities include alleviating cellular transcriptional repression by phosphorylating Histone H3 on the serine 10 position neighboring a tri-methylated lysine 9, this is known as a methyl/phospho switch. The methyl/phospho switch occurring at serine 10 has been shown to result in the disassociation of heterochromatin protein 1, a transcriptional repressor, bound to the H3K9me3 modification. Aurora B kinase may be exploited by HSV-1 to relieve transcriptional repression on IE viral promoters during the early phase of HSV-1 replication. Also, we observed significant reductions with aurora kinase inhibitor treatment in the replication of diverse DNA and RNA viruses, suggesting the aurora kinase inhibitors have a broader antiviral effect. As another approach to identify novel inhibitors and cellular kinases associated with HSV-1, we screened a broad-spectrum kinase inhibitor library. We utilized HSV-tk12 reporter virus, which expresses lacZ under the control of the HSV-1 ICP4 promoter. Four potent kinase inhibitors reduced β-galactosidase activity of the HSV-1 reporter virus. Interestingly, these inhibitors had irreversible effects on HSV-1 gene expression up to 48 hours with only six hours of treatment. This result demonstrates these inhibitors have a lasting effect on HSV-1 with limited treatment. These kinase inhibitors also potently reduced HSV-1 replication. The restriction in HSV-1 replication appears to occur between viral transcription and translation. I did observe a significant impairment of IE gene ICP27 transcript levels and viral protein levels from all 3 classes of HSV-1 genes. Interestingly, all four kinase inhibitors, either directly or indirectly, affect protein kinase C activity, suggesting that protein kinase C may have a role early in the HSV-1 lytic cycle. Overall, this dissertation used high-throughput cell-based assays to efficiently screen small molecule libraries and identify potential compounds that impair HSV-1 replication. I have demonstrated the antiviral effects with aurora kinase inhibitors and determined their effects abrogate the transcription of immediate-early genes. In collaboration with other research groups, aurora kinase inhibitors impair the replication of other DNA and RNA viruses, illustrating their broader antiviral effects. We have also identified other host kinase inhibitors that diminish HSV-1 progeny production and determined their restriction is at a step between viral transcription and translation. Altogether, these studies have provided innovative assays for discovering new antivirals and molecular probes in elucidating novel and potentially wide-ranging virus-host interactions.
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Date
2022-05-31
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University of Kansas
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Keywords
Microbiology, Virology, Biology, Antiviral, Aurora Kinase, Drug discovery, Herpes simplex virus 1, Kinase, Virus-host interactions