Abstract
Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) is an oncogenic virus which 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 ORF45/RSK-dependent regulation of gene expression and KSHV progeny virion production. Finally, 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. Altogether, these data shed light on the mechanisms by which KSHV ORF45 manipulates components of the host cell machinery via modulation of RSK activity. Thus, this study has important implications for the pathobiology of KSHV and other diseases in which RSK activity is dysregulated.
Highlights
The dysregulation of kinase signal transduction pathways is the basis for a variety of illnesses, including infectious diseases and multiple forms of cancer
We previously discovered that the Kaposi’s sarcoma-associated herpesvirus (KSHV) protein ORF45 binds to and activates the cellular kinase RSK (p90 ribosomal S6 kinase), and that this activation is vital for optimal KSHV gene expression and virion production
We used genome editing to knock out RSK, as well as several cellular substrates identified by our screening, and characterized the consequent effect(s) on regulation of gene expression and virion production
Summary
The dysregulation of kinase signal transduction pathways is the basis for a variety of illnesses, including infectious diseases and multiple forms of cancer. KS remains the most common AIDS-associated malignancy, and there is substantial experimental and epidemiological evidence to suggest a co-regulatory relationship between KSHV and HIV [6,7,8,9,10]. KSHV and other viruses must modulate their hosts’ cellular signaling pathways in order to evade host antiviral immune responses, express viral genes and efficiently produce and disseminate progeny virions. Several KSHV genes, both latent and lytic, have been shown to manipulate various cellular signal transduction pathways (reviewed in [11,12])
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