Chen Daming, Nicholas John
Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
J Virol. 2015 Aug;89(15):7979-90. doi: 10.1128/JVI.00375-15. Epub 2015 May 27.
The interleukin-6 homologue (viral interleukin-6 [vIL-6]) of human herpesvirus 8 is implicated in viral pathogenesis due to its proproliferative, inflammatory, and angiogenic properties, effected through gp130 receptor signaling. In primary effusion lymphoma (PEL) cells, vIL-6 is expressed latently and is essential for normal cell growth and viability. This is mediated partly via suppression of proapoptotic cathepsin D (CatD) via cocomplexing of the endoplasmic reticulum (ER)-localized CatD precursor, pro-CatD (pCatD), and vIL-6 with the previously uncharacterized ER membrane protein vitamin K epoxide reductase complex subunit 1 variant 2 (VKORC1v2). vIL-6 suppression of CatD occurs also during reactivated productive replication in PEL cells and is likely to contribute to proreplication functions of vIL-6. Here, we report that vIL-6 suppresses CatD through vIL-6, VKORC1v2, and pCatD association with components of the ER-associated degradation (ERAD) machinery. In transfected cells, expression of vIL-6 along with CatD led to proteasome-dependent (inhibitor-sensitive) decreases in CatD levels and the promotion of pCatD polyubiquitination. Depletion of particular ERAD-associated isomerases, lectins, and translocon components, including ERAD E3 ubiquitin ligase HRD1, diminished suppression of CatD by vIL-6. Coprecipitation assays identified direct or indirect interactions of VKORC1v2, vIL-6, and pCatD with translocon proteins (SEL1L and/or HRD1) and ERAD-associated lectins OS9 and XTP3-B. Endogenous CatD expression in PEL cells was increased by depletion of ERAD components, and suppression of CatD by vIL-6 overexpression in PEL cells was dependent on HRD1. Our data reveal a new mechanism of ER-localized vIL-6 activity and further characterize VKORC1v2 function.
Human herpesvirus 8 (HHV-8) viral interleukin-6 (vIL-6), unlike cellular IL-6 proteins, is secreted inefficiently and sequestered mainly in the endoplasmic reticulum (ER), from where it can signal through the gp130 receptor. We have recently reported that vIL-6 also associates with a novel membrane protein termed vitamin K epoxide reductase complex subunit 1 variant 2 (VKORC1v2) and mediates suppression of VKORC1v2-cointeracting cathepsin D, a stress-released proapoptotic protein negatively impacting HHV-8 latently infected primary effusion lymphoma (PEL) cell viability and reactivated virus productive replication. Here, we have examined the mechanistic basis of the VKORC1v2-vIL-6 interaction-dependent suppression of cathepsin D and have found that this novel activity of vIL-6 is mediated through coassociation of VKORC1v2, procathepsin D, and vIL-6 with components of the ER-associated degradation (ERAD) machinery. Our findings provide information of significance for potential antiviral and therapeutic targeting of VKORC1v2-mediated vIL-6 activities and also indicate the nature of VKORC1v2 function in normal cell biology.
人类疱疹病毒8的白细胞介素-6同源物（病毒白细胞介素-6 [vIL-6]）因其具有通过gp130受体信号传导发挥的促增殖、炎症和血管生成特性而与病毒发病机制有关。在原发性渗出性淋巴瘤（PEL）细胞中，vIL-6以潜伏状态表达，对正常细胞生长和活力至关重要。这部分是通过内质网（ER）定位的组织蛋白酶D（CatD）前体、组织蛋白酶D原（pCatD）与vIL-6和此前未明确的ER膜蛋白维生素K环氧化物还原酶复合物亚基1变体2（VKORC1v2）共复合来抑制促凋亡组织蛋白酶D实现的。在PEL细胞重新激活的生产性复制过程中也会发生vIL-6对CatD的抑制，这可能有助于vIL-6的促复制功能。在此，我们报告vIL-6通过vIL-6、VKORC1v2和pCatD与内质网相关降解（ERAD）机制的组分结合来抑制CatD。在转染细胞中，vIL-6与CatD一起表达导致CatD水平依赖蛋白酶体（对抑制剂敏感）下降，并促进pCatD多聚泛素化。特定ERAD相关异构酶、凝集素和转运体组分（包括ERAD E3泛素连接酶HRD1）的缺失减少了vIL-6对CatD的抑制。共沉淀试验确定了VKORC1v2、vIL-6和pCatD与转运体蛋白（SEL1L和/或HRD1）以及ERAD相关凝集素OS9和XTP3-B的直接或间接相互作用。通过缺失ERAD组分可增加PEL细胞中内源性CatD的表达，并且在PEL细胞中通过过表达vIL-6抑制CatD依赖于HRD1。我们的数据揭示了内质网定位的vIL-6活性的新机制，并进一步明确了VKORC1v2的功能。
人类疱疹病毒8（HHV-8）的病毒白细胞介素-6（vIL-6）与细胞白细胞介素-6蛋白不同，其分泌效率低下，主要被隔离在内质网（ER）中，它可从内质网通过gp130受体发出信号。我们最近报告vIL-6还与一种名为维生素K环氧化物还原酶复合物亚基1变体2（VKORC1v2）的新型膜蛋白结合，并介导对与VKORC1v2共同相互作用的组织蛋白酶D的抑制，组织蛋白酶D是一种应激释放的促凋亡蛋白，对HHV-8潜伏感染的原发性渗出性淋巴瘤（PEL）细胞活力和重新激活的病毒生产性复制产生负面影响。在此，我们研究了VKORC1v2 - vIL-6相互作用依赖的组织蛋白酶D抑制的机制基础，发现vIL-6的这种新活性是通过VKORC1v2、组织蛋白酶D原和vIL-6与内质网相关降解（ERAD）机制的组分共同结合介导的。我们的发现为针对VKORC1v2介导的vIL-6活性进行潜在抗病毒和治疗靶向提供了重要信息，也表明了VKORC1v2在正常细胞生物学中的功能性质。
