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Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication.

Avey D, Tepper S, Li W, Turpin Z, Zhu F - PLoS Pathog. (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Florida State University, Tallahassee, Florida, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

Nuclear IP further validated ORF45/RSK-dependent phosphorylation of a subset of substrates.Nuclei were isolated from the indicated cell lines at 48 hpi, then subjected to IP using anti-RxRxxS*/T* magnetic beads. The input, eluate (IP), and flow-through (FT) were analyzed by western blot with the indicated antibodies.
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ppat.1004993.g006: Nuclear IP further validated ORF45/RSK-dependent phosphorylation of a subset of substrates.Nuclei were isolated from the indicated cell lines at 48 hpi, then subjected to IP using anti-RxRxxS*/T* magnetic beads. The input, eluate (IP), and flow-through (FT) were analyzed by western blot with the indicated antibodies.

Mentions: Because there appeared to be an enrichment of nuclear substrates of ORF45-activated RSK upon KSHV lytic reactivation (Table 1, Fig 4), and phospho-specific antibodies were unavailable, we sought to validate these putative substrates by an alternative approach. We employed nuclear fractionation of iSLK.BAC16 A66F, F66A, and Stop45 cells at 2 dpi, followed by immunoprecipitation with magnetic beads conjugated to the anti-RxRxxS*/T* motif antibody. Using this approach, we found that viral ORF45 and ORF36, and cellular Lamin A, RNF20, and TAF3 are all phosphorylated in an ORF45/RSK-dependent manner (Fig 6). While H2B phosphorylation was robustly induced by lytic reactivation, it was only mildly reduced by ORF45 mutation or deletion. Significantly, all of these substrates are known or suspected to be involved in transcriptional regulation. The phosphorylation of ORF45 itself was further confirmed by immunoprecipitation with an anti-ORF45 antibody (S3 Fig).


Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication.

Avey D, Tepper S, Li W, Turpin Z, Zhu F - PLoS Pathog. (2015)

Nuclear IP further validated ORF45/RSK-dependent phosphorylation of a subset of substrates.Nuclei were isolated from the indicated cell lines at 48 hpi, then subjected to IP using anti-RxRxxS*/T* magnetic beads. The input, eluate (IP), and flow-through (FT) were analyzed by western blot with the indicated antibodies.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4489790&req=5

ppat.1004993.g006: Nuclear IP further validated ORF45/RSK-dependent phosphorylation of a subset of substrates.Nuclei were isolated from the indicated cell lines at 48 hpi, then subjected to IP using anti-RxRxxS*/T* magnetic beads. The input, eluate (IP), and flow-through (FT) were analyzed by western blot with the indicated antibodies.
Mentions: Because there appeared to be an enrichment of nuclear substrates of ORF45-activated RSK upon KSHV lytic reactivation (Table 1, Fig 4), and phospho-specific antibodies were unavailable, we sought to validate these putative substrates by an alternative approach. We employed nuclear fractionation of iSLK.BAC16 A66F, F66A, and Stop45 cells at 2 dpi, followed by immunoprecipitation with magnetic beads conjugated to the anti-RxRxxS*/T* motif antibody. Using this approach, we found that viral ORF45 and ORF36, and cellular Lamin A, RNF20, and TAF3 are all phosphorylated in an ORF45/RSK-dependent manner (Fig 6). While H2B phosphorylation was robustly induced by lytic reactivation, it was only mildly reduced by ORF45 mutation or deletion. Significantly, all of these substrates are known or suspected to be involved in transcriptional regulation. The phosphorylation of ORF45 itself was further confirmed by immunoprecipitation with an anti-ORF45 antibody (S3 Fig).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Science, Florida State University, Tallahassee, Florida, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus