Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication.
Phosphoproteomic Analysis of KSHV-Infected Cells Reveals Roles of ORF45-Activated RSK during Lytic Replication.
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DOI：
10.1371/journal.ppat.1004993
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发表时间：
2015-07
期刊：
影响因子：
6.7
通讯作者：
Zhu F
中科院分区：
文献类型：
--
作者：
Avey D;Tepper S;Li W;Turpin Z;Zhu F
卡波西肉瘤相关疱疹病毒（KSHV）是一种致癌病毒，它具有独特的机制来调节其人类宿主的细胞微环境。KSHV的发病机制与其对细胞信号通路的调控密切相关，包括细胞外信号调节激酶（ERK）、丝裂原活化蛋白激酶（MAPK）通路。我们之前已经证明，在KSHV裂解复制过程中，KSHV ORF45有助于ERK和p90核糖体S6激酶（RSK， ERK/MAPK信号的主要功能介质）的持续激活。orf45激活的RSK是最佳的KSHV裂解基因表达和子代病毒粒子产生所必需的，尽管这种激活的下游潜在机制尚不清楚。我们假设ORF45激活RSK会导致细胞和病毒底物的不同磷酸化，从而影响有效的KSHV裂解复制所必需的生物过程。因此，我们观察到在诱导裂解复制时蛋白质磷酸化的广泛而显著的差异。基于质谱的磷酸化蛋白组学筛选鉴定了kshv感染细胞中orf45激活的RSK的推定底物。生物信息学分析显示，核蛋白，包括几个转录调节因子，在这些候选中被过度代表。我们通过KSHV BAC诱变、激酶抑制剂治疗和/或crispr介导的KSHV感染细胞中RSK的敲除，验证了几种假定底物的ORF45/RSK依赖性磷酸化。此外，我们评估了敲除这些底物对ORF45/ rsk依赖性基因表达调控和KSHV子代病毒粒子产生的影响。最后，我们展示的数据支持ORF45调节在其5 ' UTR中具有复杂二级结构的病毒/细胞基因子集的翻译效率。总之，这些数据揭示了KSHV ORF45通过调节RSK活性来操纵宿主细胞机制成分的机制。因此，这项研究对KSHV和其他RSK活性失调的疾病的病理生物学具有重要意义。卡波西肉瘤相关疱疹病毒（KSHV）是一种人类肿瘤病毒，它劫持宿主信号通路以维持持续感染。我们之前发现KSHV蛋白ORF45结合并激活细胞激酶RSK （p90核糖体S6激酶），并且这种激活对于优化KSHV基因表达和病毒粒子产生至关重要。在这里，我们对kshv感染的细胞进行了磷酸化蛋白质组学分析，以进一步表征orf45激活的RSK的特定底物。生物信息学分析提供了对这些底物的功能作用的见解。我们通过各种手段验证了这些底物的一个子集的ORF45/ rsk依赖性磷酸化。最后，我们使用基因组编辑技术敲除RSK，以及通过筛选鉴定的几种细胞底物，并表征了对基因表达和病毒粒子产生调控的后续影响。因此，这项工作进一步阐明了KSHV调节的关键信号节点之一，并暗示orf45介导的RSK激活在KSHV裂解复制过程中调控病毒和宿主基因表达。
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. Kaposi’s sarcoma-associated herpesvirus (KSHV) is a human tumor virus which hijacks the host signaling pathways in order to maintain persistent infection. We previously discovered that the 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. Here, we performed a phosphoproteomic analysis of KSHV-infected cells to further characterize the specific substrates of ORF45-activated RSK. Bioinformatic analyses provided insights into the functional roles of these substrates. We verified the ORF45/RSK-dependent phosphorylation of a subset of these substrates by various means. Finally, 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. Thus, this work further elucidates one of the key signaling nodes modulated by KSHV, and implicates ORF45-mediated activation of RSK in the regulation of viral and host gene expression during KSHV lytic replication.
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