Hsp70 Isoforms Are Essential for the Formation of Kaposi's Sarcoma-Associated Herpesvirus Replication and Transcription Compartments.
Hsp70 Isoforms Are Essential for the Formation of Kaposi's Sarcoma-Associated Herpesvirus Replication and Transcription Compartments.
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DOI：
10.1371/journal.ppat.1005274
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发表时间：
2015-11
期刊：
影响因子：
6.7
通讯作者：
Whitehouse A
中科院分区：
文献类型：
--
作者：
Baquero-Pérez B;Whitehouse A
卡波西肉瘤相关疱疹病毒(KSHV)是一种与多种艾滋病相关的恶性肿瘤相关的致瘤性疱疹病毒。与其他疱疹病毒一样，KSHV裂解复制所需的多个过程，包括病毒转录、病毒DNA合成和衣壳组装，都发生在病毒诱导的核内结构中，称为复制和转录间隔室(RTC)。在这里，我们使用了一种新的方法，结合亚细胞分离和定量蛋白质组学，来鉴定被招募到KSHV诱导的RTCs中的细胞蛋白，从而在KSHV裂解复制中发挥关键作用。我们发现，HSP70伴侣家族的几种亚型Hsc70和iHsp70从细胞质重新分布到细胞核中，这与KSHV诱导的RTCs的最初形成一致。我们证明了核伴侣焦点是动态的，最初形成于新形成的KSHV RTC附近，但在后来的时间点上，伴侣在KSHV RTC内移动，并与活跃复制的病毒DNA完全共存。用特异性Hsp70亚型小分子抑制剂VER-155008检测了Hsp70亚型重新整合到KSHV RTCs中的功能意义。有趣的是，结果强调了Hsp70亚型在KSHV复制周期中的重要作用，与蛋白质的稳定性和成熟度无关。值得注意的是，抑制Hsp70亚型阻止了KSHV RTC的形成和RNA聚合酶II(RNAPII)到病毒基因组的重新定位，导致KSHV的整体转录和随后的病毒蛋白质合成和DNA复制的取消。这些新的发现揭示了调节KSHV裂解复制的新机制，并突出了HSP70抑制剂作为新型抗病毒药物的潜力。HSP70和HSP90家族的分子伴侣在细胞存活中起着重要作用。最近的证据还表明，它们的功能与包括癌症在内的各种疾病有关。因此，它们已被确定为新兴的药物靶点。卡波西肉瘤相关疱疹病毒(KSHV)是一种致癌性疱疹病毒，与其他疱疹病毒一样，在核内由病毒诱导的结构中裂解复制，称为复制和转录间隔室(RTCs)。在这里，我们发展了一种新的蛋白质组学方法，通过亚细胞分级增强来研究KSHV诱导的RTCs的细胞蛋白组成。结果显示，在KSHV诱导的RTCs中，结构性表达的Hsc70和应激诱导的iHsp70伴侣蛋白显著增加。重要的是，抑制这些伴侣的ATPase功能导致KSHV RTCs的形成和KSHV裂解复制显著减少。值得注意的是，这些结果突出了HSP70抑制剂用于治疗KSHV相关疾病的治疗潜力，如卡波西肉瘤。
Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus associated with various AIDS-related malignancies. Like other herpesviruses, multiple processes required for KSHV lytic replication, including viral transcription, viral DNA synthesis and capsid assembly occur in virus-induced intranuclear structures, termed replication and transcription compartments (RTCs). Here we utilised a novel methodology, combining subcellular fractionation and quantitative proteomics, to identify cellular proteins which are recruited to KSHV-induced RTCs and thus play a key role in KSHV lytic replication. We show that several isoforms of the HSP70 chaperone family, Hsc70 and iHsp70, are redistributed from the cytoplasm into the nucleus coinciding with the initial formation of KSHV-induced RTCs. We demonstrate that nuclear chaperone foci are dynamic, initially forming adjacent to newly formed KSHV RTCs, however during later time points the chaperones move within KSHV RTCs and completely co-localise with actively replicating viral DNA. The functional significance of Hsp70 isoforms recruitment into KSHV RTCs was also examined using the specific Hsp70 isoform small molecule inhibitor, VER-155008. Intriguingly, results highlight an essential role of Hsp70 isoforms in the KSHV replication cycle independent of protein stability and maturation. Notably, inhibition of Hsp70 isoforms precluded KSHV RTC formation and RNA polymerase II (RNAPII) relocalisation to the viral genome leading to the abolishment of global KSHV transcription and subsequent viral protein synthesis and DNA replication. These new findings have revealed novel mechanisms that regulate KSHV lytic replication and highlight the potential of HSP70 inhibitors as novel antiviral agents. Molecular chaperones from the HSP70 and HSP90 families have important roles in cell survival. Recent evidence has also implicated their functioning in a variety of diseases, including cancer. As such they have been identified as emerging drug targets. Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic herpesvirus which, like other herpesviruses, lytically replicates in virus-induced structures within the nucleus, termed replication and transcription compartments (RTCs). Here we developed a novel proteomic approach enhanced by subcellular fractionation to study the cellular protein composition of KSHV-induced RTCs. Results revealed that the constitutively expressed Hsc70 and the stress-inducible iHsp70 chaperones were significantly increased in the KSHV-induced RTCs. Importantly, inhibition of the ATPase function of these chaperones led to a marked reduction in KSHV RTCs formation and KSHV lytic replication. Notably, these results highlight the therapeutic potential of HSP70 inhibitors for the treatment of KSHV-related diseases, such as Kaposi’s sarcoma.