Kaposi's Sarcoma-Associated Herpesvirus (KSHV) is an oncogenic virus that has adapted unique mechanisms to modulate the
cellular microenvironment of its human host. The pathogenesis of KSHV is intimately linked to its manipulation of cellular signaling
pathways, including the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway. We have previously
shown that KSHV ORF45 contributes to the sustained activation of both ERK and p90 ribosomal S6 kinase (RSK, a major functional mediator of
ERK/MAPK signaling) during KSHV lytic replication. ORF45-activated RSK is required for optimal KSHV lytic gene expression and progeny
virion production, though the underlying mechanisms downstream of this activation are still unclear. We hypothesized that the activation
of RSK by ORF45 causes differential phosphorylation of cellular and viral substrates, affecting biological processes essential for
efficient KSHV lytic replication. Accordingly, we observed widespread and significant differences in protein phosphorylation upon
induction of lytic replication. Mass-spectrometry-based phosphoproteomic screening identified putative substrates of ORF45-activated RSK
in KSHV-infected cells. Bioinformatic analyses revealed that nuclear proteins, including several transcriptional regulators, were
overrepresented among these candidates. We validated the ORF45/RSK-dependent phosphorylation of several putative substrates by employing
KSHV BAC mutagenesis, kinase inhibitor treatments, and/or CRISPR-mediated knockout of RSK in KSHV-infected cells. Furthermore, we assessed
the consequences of knocking out these substrates on KSHV progeny virion production. Importantly, we investigated the regulation of gene
expression by ORF45-actvated RSK by performing RNA-seq of KSHV-infected cells. We show data to support that ORF45 regulates the
translational efficiency of a subset of viral/cellular genes with complex secondary structure in their 5' UTR. One of the few viral
substrates of ORF45-activated identified by our mass spectrometry analysis was ORF36. KSHV ORF36 encodes a serine/threonine viral protein
kinase, which is conserved throughout all herpesviruses. Although several studies have identified the viral and cellular substrates of
conserved herpesvirus protein kinases (CHPKs), the precise functions of KSHV ORF36 during lytic replication remain elusive. We report that
ORF36 interacts with another lytic protein, ORF45, in a manner dependent on ORF36 kinase activity. We mapped the regions of ORF36 and
ORF45 involved in their binding. Their association appears to be mediated by electrostatic interactions, since deletion of either the
highly basic N-terminus of ORF36 or an acidic patch of ORF45 abolished the binding. Additionally, dephosphorylation of ORF45 protein
dramatically reduced its association with ORF36. Importantly, ORF45 enhances both the stability and kinase activity of ORF36. Consistent
with previous studies of CHPK homologs, we detected ORF36 protein in extracellular virions. To investigate the roles of ORF36 in the
context of KSHV lytic replication, we employed BAC mutagenesis to engineer both ORF36-null and kinase-dead (KD) mutants. We found that
ORF36-null/mutant virions are moderately defective in viral particle production and are further deficient in primary infection. In
summary, our results uncover a functionally important interaction between ORF36 and ORF45, and indicate a significant role of ORF36 in the
production of infectious progeny virions. Altogether, these data shed light on the mechanisms by which KSHV ORF45 manipulates viral and
cellular kinase signaling to optimize lytic replication. The findings reported here have important implications for the pathobiology of
KSHV and other diseases in which RSK activity is missregulated. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the
requirements for the degree of Doctor of Philosophy. / Spring Semester 2016. / March 22, 2016. / Kaposi's sarcoma, kinase, KSHV, lytic replication, ORF45, RSK / Includes bibliographical references. / Fanxiu Zhu, Professor Directing Dissertation; Qing-Xiang Sang, University Representative; Hengli
Tang, Committee Member; David Gilbert, Committee Member; Jonathan H. Dennis, Committee Member.
| Identifer | oai:union.ndltd.org:fsu.edu/oai:fsu.digital.flvc.org:fsu_360307 |
| Contributors | Avey, Denis R. (authoraut), Zhu, Fanxiu (professor directing dissertation), Sang, Qing-Xiang Amy (university representative), Tang, Hengli (committee member), Gilbert, David M. (committee member), Dennis, Jonathan Hancock (committee member), Florida State University (degree granting institution), College of Arts and Sciences (degree granting college), Department of Biological Science (degree granting department) |
| Publisher | Florida State University |
| Source Sets | Florida State University |
| Language | English, English |
| Detected Language | English |
| Type | Text, text |
| Format | 1 online resource (132 pages), computer, application/pdf |
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