CD63 Regulates Epstein-Barr Virus LMP1 Exosomal Packaging, Enhancement of Vesicle Production, and Noncanonical NF-κB Signaling
CD63 Regulates Epstein-Barr Virus LMP1 Exosomal Packaging, Enhancement of Vesicle Production, and Noncanonical NF-κB Signaling
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DOI：
10.1128/jvi.02251-16
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发表时间：
2017-03-01
影响因子：
5.4
通讯作者：
Meckes, David G., Jr.
中科院分区：
文献类型：
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作者：
Hurwitz, Stephanie N.;Nkosi, Dingani;Meckes, David G., Jr.
潜伏膜蛋白1 （LMP1）是一种eb病毒（EBV）编码的癌蛋白，被包装在称为外泌体的小细胞外囊泡（ev）中。LMP1转运到多泡体（MVBs）会改变外泌体的内容和功能。lmp1修饰的外泌体增强恶性细胞的生长、迁移和侵袭，显示出操纵肿瘤微环境和促进ebv相关癌症进展的能力。尽管越来越多的证据表明LMP1修饰的外泌体在癌症中的重要性，但对LMP1整合到这些囊泡中的机制知之甚少。最近，LMP1被证明与CD63（一种富集于内体晚期和溶酶体区室的保守的四跨蛋白蛋白）共同增殖。在这里，我们证明了CD63的存在对于LMP1的外泌体包装的重要性。纳米颗粒跟踪分析和梯度纯化显示，在LMP1表达后，细胞外囊泡分泌和外泌体蛋白增加。cd63阳性外泌体的免疫分离显示LMP1在该囊泡群中积累。功能上，CRISPR/Cas9敲除CD63导致lmp1诱导的颗粒分泌减少。此外，LMP1的包装在CD63敲除细胞中严重受损，同时LMP1的核周定位被破坏。重要的是，在敲除CD63后，LMP1转运到脂筏，NF-kappa B和PI3K/Akt通路的激活保持完整，而丝裂原活化蛋白激酶/细胞外信号调节激酶（MAPK/ERK）和非典型NF-kappa B激活被观察到增加。这些结果表明，CD63在LMP1外泌体运输和LMP1介导的外泌体生成增强中起关键作用，并可能在限制下游LMP1信号传导中发挥进一步的作用。EBV是一种普遍存在的伽玛疱疹病毒，与鼻咽癌、伯基特淋巴瘤和霍奇金淋巴瘤等恶性肿瘤有关。在癌症的背景下，EBV通过将病毒成分（如癌蛋白LMP1）分泌到宿主细胞膜结合的ev中，劫持外泌体途径来调节细胞间信号传导。LMP1转运到外泌体与这些分泌囊泡的致癌性增加有关。然而，我们对外泌体货物包装（包括病毒蛋白）的机制了解有限。在这里，我们描述了LMP1在需要CD63的EV生产中的作用，并提供了CD63介导的外泌体LMP1释放不同于脂质筏运输的广泛证明。最后，我们提出了CD63在限制lmp1诱导的非典型nf - κ B和ERK激活中的作用的进一步证据。我们的发现对未来研究外泌体生物发生、蛋白质运输和信号转导的生理和病理机制，特别是在病毒相关的肿瘤发生中具有重要意义。
Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded oncoprotein that is packaged into small extracellular vesicles (EVs) called exosomes. Trafficking of LMP1 into multivesicular bodies (MVBs) alters the content and function of exosomes. LMP1-modified exosomes enhance the growth, migration, and invasion of malignant cells, demonstrating the capacity to manipulate the tumor microenvironment and enhance the progression of EBV-associated cancers. Despite the growing evidence surrounding the significance of LMP1-modified exosomes in cancer, very little is understood about the mechanisms that orchestrate LMP1 incorporation into these vesicles. Recently, LMP1 was shown to be copurified with CD63, a conserved tetraspanin protein enriched in late endosomal and lysosomal compartments. Here, we demonstrate the importance of CD63 presence for exosomalpackaging of LMP1. Nanoparticle tracking analysis and gradient purification revealed an increase in extracellular vesicle secretion and exosomal proteins following LMP1 expression. Immunoisolation of CD63-positive exosomes exhibited accumulation of LMP1 in this vesicle population. Functionally, CRISPR/Cas9 knockout of CD63 resulted in a reduction of LMP1-induced particle secretion. Furthermore, LMP1 packaging was severely impaired in CD63 knockout cells, concomitant with a disruption in the perinuclear localization of LMP1. Importantly, LMP1 trafficking to lipid rafts and activation of NF-kappa B and PI3K/Akt pathways remained intact following CD63 knockout, while mitogen- activated protein kinase/ extracellular signal- regulated kinase (MAPK/ERK) and noncanonical NF-kappa B activation were observed to be increased. These results suggest that CD63 is a critical player in LMP1 exosomal trafficking and LMP1mediated enhancement of exosome production and may play further roles in limiting downstream LMP1 signaling.IMPORTANCE EBV is a ubiquitous gamma herpesvirus linked to malignancies such as nasopharyngeal carcinoma, Burkitt's lymphoma, and Hodgkin's lymphoma. In the context of cancer, EBV hijacks the exosomal pathway to modulate cell-to-cell signaling by secreting viral components such as an oncoprotein, LMP1, into host cell membrane-bound EVs. Trafficking of LMP1 into exosomes is associated with increased oncogenicity of these secreted vesicles. However, we have only a limited understanding of the mechanisms surrounding exosomal cargo packaging, including viral proteins. Here, we describe a role of LMP1 in EV production that requires CD63 and provide an extensive demonstration of CD63-mediated exosomal LMP1 release that is distinct from lipid raft trafficking. Finally, we present further evidence of the role of CD63 in limiting LMP1-induced noncanonical NF-kappa B and ERK activation. Our findings have implications for future investigations of physiological and pathological mechanisms of exosome biogenesis, protein trafficking, and signal transduction, especially in viral-associated tumorigenesis.