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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: 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.

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

ORF45/eIF4B-dependent translational control of mRNAs with complex 5’ UTR structure.(A and B) 293T WT cells or cells stably transduced with the indicated lentiCRISPR were transfected with the indicated plasmids, as well as HCV IRES-FL (firefly luciferase), and subjected to luciferase assays (A) or western blot analysis (B) at 24 hpt. Values for (A) are represented as the renilla/firefly luciferase ratio normalized to the WT sample. (C) iSLK.BAC16 A66F, F66A, or Stop45 cells were uninduced or induced with dox/butyrate, and cells were harvested at 48 hpi. Lysates were fractionated by sucrose gradient centrifugation and RNAs were extracted. RNAs associated with either monosome or polysome fractions were pooled, reverse transcribed, and subjected to qRT-PCR using the indicated primers. Values shown are for mRNA level relative to the uninduced control, as a ratio of polysome:monosome (normalized to β-Actin). Genes are arranged in order of decreasing minimum free energy (left to right), as predicted by RNAFold software [109].
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ppat.1004993.g009: ORF45/eIF4B-dependent translational control of mRNAs with complex 5’ UTR structure.(A and B) 293T WT cells or cells stably transduced with the indicated lentiCRISPR were transfected with the indicated plasmids, as well as HCV IRES-FL (firefly luciferase), and subjected to luciferase assays (A) or western blot analysis (B) at 24 hpt. Values for (A) are represented as the renilla/firefly luciferase ratio normalized to the WT sample. (C) iSLK.BAC16 A66F, F66A, or Stop45 cells were uninduced or induced with dox/butyrate, and cells were harvested at 48 hpi. Lysates were fractionated by sucrose gradient centrifugation and RNAs were extracted. RNAs associated with either monosome or polysome fractions were pooled, reverse transcribed, and subjected to qRT-PCR using the indicated primers. Values shown are for mRNA level relative to the uninduced control, as a ratio of polysome:monosome (normalized to β-Actin). Genes are arranged in order of decreasing minimum free energy (left to right), as predicted by RNAFold software [109].

Mentions: All of our data indicated that eIF4B is robustly phosphorylated during viral lytic reactivation, and that it represents one of the most specific and functionally significant substrates of ORF45-activated RSK. Thus, we aimed to further characterize the role(s) of the ORF45/RSK/eIF4B signaling axis in translational regulation. Recent reports that the RNA helicase eIF4A regulates the translation of mRNAs with G-quadruplex (G4) structures in their 5’ UTR [46], in addition to the known role of eIF4B phosphorylation in stimulating the helicase activity of eIF4A [42,47,48], led us to hypothesize that eIF4B may also contribute to this phenomenon. To test this, we transfected 293T (WT or eIF4B-knockout) cells with a reporter construct containing G4 structures in the 5’ UTR (pRL-G4) or random sequence with equal G/C content (pRL-con) in the presence or absence of ORF45 overexpression (Fig 9A and 9B). Interestingly, we found that ORF45 significantly increased luciferase activity of pRL-G4, and that loss of eIF4B almost completely abrogated this effect (Fig 9A and 9B). The ORF45-dependent induction of luciferase activity of the negative control (pRL-con) is mild by comparison and insensitive to eIF4B knockout. Thus, this difference probably represents transcriptional regulation by ORF45, whereas the more significant effect on the G4-containing reporter can be attributed primarily to translational regulation.


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)

ORF45/eIF4B-dependent translational control of mRNAs with complex 5’ UTR structure.(A and B) 293T WT cells or cells stably transduced with the indicated lentiCRISPR were transfected with the indicated plasmids, as well as HCV IRES-FL (firefly luciferase), and subjected to luciferase assays (A) or western blot analysis (B) at 24 hpt. Values for (A) are represented as the renilla/firefly luciferase ratio normalized to the WT sample. (C) iSLK.BAC16 A66F, F66A, or Stop45 cells were uninduced or induced with dox/butyrate, and cells were harvested at 48 hpi. Lysates were fractionated by sucrose gradient centrifugation and RNAs were extracted. RNAs associated with either monosome or polysome fractions were pooled, reverse transcribed, and subjected to qRT-PCR using the indicated primers. Values shown are for mRNA level relative to the uninduced control, as a ratio of polysome:monosome (normalized to β-Actin). Genes are arranged in order of decreasing minimum free energy (left to right), as predicted by RNAFold software [109].
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004993.g009: ORF45/eIF4B-dependent translational control of mRNAs with complex 5’ UTR structure.(A and B) 293T WT cells or cells stably transduced with the indicated lentiCRISPR were transfected with the indicated plasmids, as well as HCV IRES-FL (firefly luciferase), and subjected to luciferase assays (A) or western blot analysis (B) at 24 hpt. Values for (A) are represented as the renilla/firefly luciferase ratio normalized to the WT sample. (C) iSLK.BAC16 A66F, F66A, or Stop45 cells were uninduced or induced with dox/butyrate, and cells were harvested at 48 hpi. Lysates were fractionated by sucrose gradient centrifugation and RNAs were extracted. RNAs associated with either monosome or polysome fractions were pooled, reverse transcribed, and subjected to qRT-PCR using the indicated primers. Values shown are for mRNA level relative to the uninduced control, as a ratio of polysome:monosome (normalized to β-Actin). Genes are arranged in order of decreasing minimum free energy (left to right), as predicted by RNAFold software [109].
Mentions: All of our data indicated that eIF4B is robustly phosphorylated during viral lytic reactivation, and that it represents one of the most specific and functionally significant substrates of ORF45-activated RSK. Thus, we aimed to further characterize the role(s) of the ORF45/RSK/eIF4B signaling axis in translational regulation. Recent reports that the RNA helicase eIF4A regulates the translation of mRNAs with G-quadruplex (G4) structures in their 5’ UTR [46], in addition to the known role of eIF4B phosphorylation in stimulating the helicase activity of eIF4A [42,47,48], led us to hypothesize that eIF4B may also contribute to this phenomenon. To test this, we transfected 293T (WT or eIF4B-knockout) cells with a reporter construct containing G4 structures in the 5’ UTR (pRL-G4) or random sequence with equal G/C content (pRL-con) in the presence or absence of ORF45 overexpression (Fig 9A and 9B). Interestingly, we found that ORF45 significantly increased luciferase activity of pRL-G4, and that loss of eIF4B almost completely abrogated this effect (Fig 9A and 9B). The ORF45-dependent induction of luciferase activity of the negative control (pRL-con) is mild by comparison and insensitive to eIF4B knockout. Thus, this difference probably represents transcriptional regulation by ORF45, whereas the more significant effect on the G4-containing reporter can be attributed primarily to translational regulation.

Bottom Line: 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.

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