Retroviral Egress via Multivesicular Bodies

描述（由申请人提供）：逆转录病毒的组装和出芽是由Gag蛋白驱动的，Gag蛋白诱导病毒衣壳的组装，并招募细胞蛋白，这些细胞蛋白是从细胞膜上剪下来所必需的。这些宿主因子包括介导衣壳泛素化的泛素连接酶和负责将蛋白质分选到后期核内体的空泡蛋白分选机制。虽然Gag可以将这些因子招募到质膜上，但组装和出芽也发生在称为多泡体（MVB）的细胞内内体晚期囊泡中。利用基于荧光标记病毒粒子的视觉方法，我们报道了小鼠白血病病毒（MLV）和人类免疫缺陷病毒（HIV）的逆转录病毒Gag蛋白在晚期核内体上积累并萌发。在哺乳动物细胞中，MVBs能够与质膜融合释放其内容物。因此，出芽进入MVBs提供了从质膜出芽的另一种途径。现在需要解决若干重要问题。携带MVBs的病毒是如何被激活与质膜融合从而释放感染性病毒的？这些融合事件能否通过视觉捕捉来揭示活细胞中的动态？哪些细胞因子参与了激活？通过分泌溶酶体参与胞吐作用的因素是否参与其中？最后，抗原呈递细胞的MVBs是否在与t细胞相互作用时被特异性动员？为了回答这些问题，我们将研究巨噬细胞等原代细胞，其中MVB途径主要负责感染性病毒的释放。为此，我们已经建立了技术，使我们能够使用全内反射荧光显微镜观察原代细胞中HIV和MLV的输出。为了深入了解病毒输出的细胞生物学，我们将利用分泌溶酶体缺陷的小鼠模型的可用性，并确定这些模型在体内和体外是否影响MLV的释放。

DESCRIPTION (provided by applicant): Retroviral assembly and budding are driven by the Gag protein that induces assembly of the viral capsid and recruits cellular proteins that are essential for pinching off from the cellular membrane. These host factors include ubiquitin ligases that mediate ubiquitination of the capsid and the vacuolar protein sorting machinery responsible for the sorting of proteins into the late endosome. While Gag can recruit these factors to the plasma membrane, assembly and budding also occur at intracellular late endosomal vesicles called multivesicular bodies (MVB). Using a visual approach based on fluorescently labeled virions, we have reported that retroviral Gag proteins of the murine leukemia virus (MLV) and the human immunodeficiency virus (HIV) accumulate on and bud into late endosomes. In mammalian cells, MVBs are capable of fusing with the plasma membrane to release their contents. As such, budding into MVBs provides an alternative pathway to budding from the plasma membrane. A number of important questions now need to be addressed. How are MVBs carrying virus activated to fuse with the plasma membrane in order to release infectious virus? Can these fusion events be captured visually to reveal the dynamics in living cells? What cellular factors are involved in the activation? Are factors that play a role in exocytosis via secretory lysosomes involved? Finally, are the MVBs of antigen-presenting cells specifically mobilized in response to an interaction with T-cells? To answer these questions, we will study primary cells such as macrophages in which the MVB pathway is primarily responsible for the release of infectious viruses. To this end, we have established technologies that allow us to visualize HIV and MLV egress in primary cells using total internal reflection fluorescent microscopy. To gain insights into the cell biology of viral egress we will take advantage of the availability of mouse models for defects in secretory lyososomes and determine if MLV release is affected in these models in vivo and in vitro.