Date of Award

Summer 8-2021

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical Sciences

Advisor

Melissa Maginnis

Second Committee Member

Benjamin King

Third Committee Member

Melody Neely

Additional Committee Members

Con Sullivan

Kristy Townsend

Abstract

JC polyomavirus (JCPyV) is a human-specific pathogen that infects 50-80% of the population, and can cause a deadly, demyelinating disease, known as progressive multifocal leukoencephalopathy (PML). In most of the population, JCPyV persistently infects the kidneys but during immunosuppression, it can reactivate and spread to the central nervous system (CNS), causing PML. In the CNS, JCPyV targets two cell types, astrocytes, and oligodendrocytes. Due to the hallmark pathology of oligodendrocyte lysis observed in disease, oligodendrocytes were thought to be the main cell type involved during JCPyV infection. However, recent evidence suggests that astrocytes are targeted by the virus and act as a reservoir for JCPyV. From these findings and lack of a tractable model to study JCPyV infection, the infectious cycle was characterized in primary human astrocytes (NHAs) compared to a well-studied but immortalized glial cell line, SVGA cells (SVGAs). It was discovered that the JCPyV infectious cycle was delayed in NHAs compared to SVGAs, contributed in part by the transformed properties of SVGAs.

To further define the mechanisms of JCPyV infection in NHAs, RNA sequencing (RNA-seq) analysis was performed in both cell types at time points established to be important during the infectious cycle. RNA-seq revealed a temporal difference in differential gene expression between primary and immortalized cells. Through complementary cell-based assays, the involvement of the mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) pathway was validated, and the phosphoinositide 3-kinase (PI3K)/AKT/mechanistic target of rapamycin (mTOR) pathway was discovered to be involved in JCPyV infection in NHAs. Altogether, RNA-seq revealed and confirmed pathways important for JCPyV in NHAs, yet infection and PML pathogenesis is a complex composition of variation in viral genomic sequences, contributing to a quasispecies that can impact infection and disease pathogenesis. A bioinformatic approach characterizing 181 sequences of a hypervariable region of the JCPyV genome validated and further characterized how this region is diverse from various locations in individuals and among patients with disease. Overall, this dissertation addresses how astrocytes are involved during JCPyV infection and how the virus mutates, highlighting the interplay of viral and host cell factors that contribute to pathogenesis of this deadly disease.

Available for download on Wednesday, August 16, 2023

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