Development of an ORF45-Derived Peptide To Inhibit the Sustained RSK Activation and Lytic Replication of Kaposi's Sarcoma-Associated Herpesvirus
Development of an ORF45-Derived Peptide To Inhibit the Sustained RSK Activation and Lytic Replication of Kaposi's Sarcoma-Associated Herpesvirus
复制标题
开发 ORF45 衍生肽来抑制卡波西肉瘤相关疱疹病毒的持续 RSK 激活和裂解复制
DOI：
10.1128/jvi.02154-18
复制
发表时间：
2019-05-01
影响因子：
5.4
通讯作者：
Kuang, Ersheng
中科院分区：
文献类型：
--
作者：
Li, Xiaojuan;Huang, Lu;Kuang, Ersheng
ORF45 诱导的 RSK 激活在 KSHV 裂解性复制中发挥重要作用，ORF45 缺失或 ORF45 F66A 突变会消除持续的 RSK 激活，而 RSK 抑制剂会显着降低裂解性复制，表明 ORF45-RSK 关联是 KSHV 相关疾病的独特靶点。然而，RSK调节剂的副作用、亲和力低、疗效差限制了其临床应用。在这项研究中，我们从 RSK 结合区开发了一种无毒的细胞渗透性 ORF45 衍生肽，以破坏 ORF45-RSK 关联并阻断 ORF45 诱导的 RSK 激活，而不干扰 S6K1 激活。该肽可有效抑制自发、缺氧诱导或化学诱导的 KSHV 裂解性复制，并增强雷帕霉素对裂解性 KSHV 感染细胞中裂解性复制的抑制作用和对雷帕霉素的敏感性。我们的结果表明，短肽可以有效靶向 ORF45-RSK 信号轴和 KSHV 裂解性复制，并为治疗 KSHV 裂解性和持续性感染提供特定方法。摘要 卡波西肉瘤相关疱疹病毒 (KSHV) 的裂解复制需要持续的细胞外信号调节激酶 (ERK)-p90 核糖体 S6 激酶 (RSK) 激活，这是由立即早期 (IE) 基因编码的被膜蛋白 ORF45 诱导的，以促进病毒裂解基因的晚期转录和翻译。 ORF45 中的 ORF45 缺失或单点 F66A 突变消除了 ORF45-RSK 相互作用，并在裂解重新激活过程中维持了 ERK-RSK 激活，随后导致晚期裂解基因表达和病毒颗粒产生显着减少，表明 ORF45 介导的 RSK 激活在 KSHV 裂解复制中发挥着关键作用。在这里，我们证明 RSK 结合区域中的短 ORF45 衍生肽足以破坏 ORF45-RSK 相互作用，从而抑制裂解基因表达和病毒颗粒产生。我们设计了一种源自ORF45的无毒细胞渗透性肽TAT-10F10，该肽由ORF45 56至76个氨基酸（aa）区域和HIV Tat蛋白转导结构域组成，该肽显着抑制iSLK.219和BCBL1细胞中的KSHV裂解性复制。重要的是，该肽增强雷帕霉素对 KSHV 感染细胞的抑制作用，并减少 KSHV 阳性淋巴瘤细胞中自发和缺氧诱导的裂解复制。这些发现表明，破坏 ORF45-RSK 相互作用的小肽可能是控制 KSHV 裂解性感染和发病机制的有前途的药物。重要性 ORF45 诱导的 RSK 激活在 KSHV 裂解性复制中发挥重要作用，ORF45 缺失或 ORF45 F66A 突变会消除持续的 RSK 激活，而 RSK 抑制剂会显着降低裂解性复制，表明 ORF45-RSK 关联是 KSHV 相关疾病的独特靶点。然而，RSK调节剂的副作用、亲和力低、疗效差限制了其临床应用。在这项研究中，我们从 RSK 结合区开发了一种无毒的细胞渗透性 ORF45 衍生肽，以破坏 ORF45-RSK 关联并阻断 ORF45 诱导的 RSK 激活，而不干扰 S6K1 激活。该肽可有效抑制自发、缺氧诱导或化学诱导的 KSHV 裂解性复制，并增强雷帕霉素对裂解性 KSHV 感染细胞中裂解性复制的抑制作用和对雷帕霉素的敏感性。我们的结果表明，短肽可以有效靶向 ORF45-RSK 信号轴和 KSHV 裂解性复制，并为治疗 KSHV 裂解性和持续性感染提供特定方法。
ORF45-induced RSK activation plays an essential role in KSHV lytic replication, and ORF45-null or ORF45 F66A mutagenesis that abolishes sustained RSK activation and RSK inhibitors significantly decreases lytic replication, indicating that the ORF45-RSK association is a unique target for KSHV-related diseases. However, the side effects, low affinity, and poor efficacy of RSK modulators limit their clinical application. In this study, we developed a nontoxic cell-permeable ORF45-derived peptide from the RSK-binding region to disrupt ORF45-RSK associations and block ORF45-induced RSK activation without interfering with S6K1 activation. This peptide effectively suppresses spontaneous, hypoxia-induced, or chemically induced KSHV lytic replication and enhances the inhibitory effect of rapamycin on lytic replication and sensitivity to rapamycin in lytic KSHV-infected cells. Our results reveal that the ORF45-RSK signaling axis and KSHV lytic replication can be effectively targeted by a short peptide and provide a specific approach for treating KSHV lytic and persistent infection. ABSTRACT The lytic replication of Kaposi’s sarcoma-associated herpesvirus (KSHV) requires sustained extracellular signal-regulated kinase (ERK)-p90 ribosomal S6 kinase (RSK) activation, which is induced by an immediate early (IE) gene-encoded tegument protein called ORF45, to promote the late transcription and translation of viral lytic genes. An ORF45-null or single-point F66A mutation in ORF45 abolishes ORF45-RSK interaction and sustained ERK-RSK activation during lytic reactivation and subsequently results in a significant decrease in late lytic gene expression and virion production, indicating that ORF45-mediated RSK activation plays a critical role in KSHV lytic replication. Here, we demonstrate that a short ORF45-derived peptide in the RSK-binding region is sufficient for disrupting ORF45-RSK interaction, consequently suppressing lytic gene expression and virion production. We designed a nontoxic cell-permeable peptide derived from ORF45, TAT-10F10, which is composed of the ORF45 56 to 76 amino acid (aa) region and the HIV Tat protein transduction domain, and this peptide markedly inhibits KSHV lytic replication in iSLK.219 and BCBL1 cells. Importantly, this peptide enhances the inhibitory effect of rapamycin on KSHV-infected cells and decreases spontaneous and hypoxia-induced lytic replication in KSHV-positive lymphoma cells. These findings suggest that a small peptide that disrupts ORF45-RSK interaction might be a promising agent for controlling KSHV lytic infection and pathogenesis. IMPORTANCE ORF45-induced RSK activation plays an essential role in KSHV lytic replication, and ORF45-null or ORF45 F66A mutagenesis that abolishes sustained RSK activation and RSK inhibitors significantly decreases lytic replication, indicating that the ORF45-RSK association is a unique target for KSHV-related diseases. However, the side effects, low affinity, and poor efficacy of RSK modulators limit their clinical application. In this study, we developed a nontoxic cell-permeable ORF45-derived peptide from the RSK-binding region to disrupt ORF45-RSK associations and block ORF45-induced RSK activation without interfering with S6K1 activation. This peptide effectively suppresses spontaneous, hypoxia-induced, or chemically induced KSHV lytic replication and enhances the inhibitory effect of rapamycin on lytic replication and sensitivity to rapamycin in lytic KSHV-infected cells. Our results reveal that the ORF45-RSK signaling axis and KSHV lytic replication can be effectively targeted by a short peptide and provide a specific approach for treating KSHV lytic and persistent infection.