Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, Florida, USA.
Department of Biomedical Sciences, Florida State University College of Medicine, Tallahassee, Florida, USA
J Virol. 2018 Aug 16;92(17). doi: 10.1128/JVI.00280-18. Print 2018 Sep 1.
EBV latent membrane protein 1 (LMP1) is released from latently infected tumor cells in small membrane-enclosed extracellular vesicles (EVs). Accumulating evidence suggests that LMP1 is a major driver of EV content and functions. LMP1-modified EVs have been shown to influence recipient cell growth, migration, differentiation, and regulation of immune cell function. Despite the significance of LMP1-modified exosomes, very little is known about how this viral protein enters or manipulates the host EV pathway. In this study, LMP1 deletion mutants were generated to assess protein regions required for EV trafficking. Following transfection of LMP1 or mutant plasmids, EVs were collected by differential centrifugation, and the levels of specific cargo were evaluated by immunoblot analysis. The results demonstrate that, together, the N terminus and transmembrane region 1 of LMP1 are sufficient for efficient sorting into EVs. Consistent with these findings, a mutant lacking the N terminus and transmembrane domains 1 through 4 (TM5-6) failed to be packaged into EVs, and exhibited higher colocalization with endoplasmic reticulum and early endosome markers than the wild-type protein. Surprisingly, TM5-6 maintained the ability to colocalize and form a complex with CD63, an abundant exosome protein that is important for the incorporation of LMP1 into EVs. Other mutations within LMP1 resulted in enhanced levels of secretion, pointing to potential positive and negative regulatory mechanisms for extracellular vesicle sorting of LMP1. These data suggest new functions of the N terminus and transmembrane domains in LMP1 intra- and extracellular trafficking that are likely downstream of an interaction with CD63. EBV infection contributes to the development of cancers, such as nasopharyngeal carcinoma, Burkitt lymphoma, Hodgkin's disease, and posttransplant lymphomas, in immunocompromised or genetically susceptible individuals. LMP1 is an important viral protein expressed by EBV in these cancers. LMP1 is secreted in extracellular vesicles (EVs), and the transfer of LMP1-modified EVs to uninfected cells can alter their physiology. Understanding the cellular machinery responsible for sorting LMP1 into EVs is limited, despite the importance of LMP1-modified EVs. Here, we illustrate the roles of different regions of LMP1 in EV packaging. Our results show that the N terminus and TM1 are sufficient to drive LMP1 EV trafficking. We further show the existence of potential positive and negative regulatory mechanisms for LMP1 vesicle sorting. These findings provide a better basis for future investigations to identify the mechanisms of LMP1 targeting to EVs, which could have broad implications in understanding EV cargo sorting.
EBV 潜伏膜蛋白 1（LMP1）从潜伏感染的肿瘤细胞中以小膜包裹的细胞外囊泡（EV）的形式释放。越来越多的证据表明，LMP1 是 EV 内容物和功能的主要驱动因素。已经表明，LMP1 修饰的 EV 会影响受体细胞的生长、迁移、分化和免疫细胞功能的调节。尽管 LMP1 修饰的外泌体意义重大，但对于这种病毒蛋白如何进入或操纵宿主 EV 途径知之甚少。在这项研究中，生成了 LMP1 缺失突变体，以评估 EV 运输所需的蛋白区域。转染 LMP1 或突变质粒后，通过差速离心收集 EV，并通过免疫印迹分析评估特定货物的水平。结果表明，LMP1 的 N 端和跨膜区 1 共同足以有效地分拣到 EV 中。与这些发现一致，缺乏 N 端和跨膜结构域 1 至 4（TM5-6）的突变体未能被包装到 EV 中，并且与内质网和早期内体标记物的共定位高于野生型蛋白。令人惊讶的是，TM5-6 保持与 CD63 共定位和形成复合物的能力，CD63 是一种丰富的外泌体蛋白，对于将 LMP1 掺入 EV 中很重要。LMP1 中的其他突变导致分泌水平升高，这表明 LMP1 细胞外囊泡分拣的潜在正调节和负调节机制。这些数据表明，LMP1 的 N 端和跨膜结构域在 LMP1 的细胞内和细胞外运输中具有新的功能，这可能是与 CD63 相互作用的下游事件。EBV 感染会导致癌症的发展，例如鼻咽癌、伯基特淋巴瘤、霍奇金病和移植后淋巴瘤，在免疫功能低下或遗传易感个体中。LMP1 是 EBV 在这些癌症中表达的一种重要病毒蛋白。LMP1 在细胞外囊泡（EV）中分泌，将 LMP1 修饰的 EV 转移到未感染的细胞中会改变它们的生理学。尽管 LMP1 修饰的 EV 非常重要，但负责将 LMP1 分拣到 EV 中的细胞机制仍知之甚少。在这里，我们说明了 LMP1 不同区域在 EV 包装中的作用。我们的结果表明，N 端和 TM1 足以驱动 LMP1 EV 运输。我们进一步显示了 LMP1 囊泡分拣的潜在正调节和负调节机制的存在。这些发现为进一步研究确定 LMP1 靶向 EV 的机制提供了更好的基础，这可能对理解 EV 货物分拣具有广泛意义。
