Retroviral Egress via Multivesicular Bodies

描述（由申请方提供）：逆转录病毒组装和出芽由Gag蛋白驱动，Gag蛋白诱导病毒衣壳组装并募集细胞蛋白，这些蛋白对于从细胞膜上夹断至关重要。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晚期内体中的液泡蛋白分选机制。虽然Gag可以将这些因子募集到质膜，但组装和出芽也发生在称为多泡体（MVB）的细胞内晚期内体囊泡处。使用基于荧光标记病毒体的视觉方法，我们已经报道了鼠白血病病毒（MLV）和人类免疫缺陷病毒（HIV）的逆转录病毒Gag蛋白在晚期内体上积累并出芽。在哺乳动物细胞中，MVB能够与质膜融合以释放其内容物。因此，出芽成MVB提供了从质膜出芽的替代途径。现在需要解决一些重要问题。携带病毒的MVB是如何被激活与质膜融合以释放感染性病毒的？这些融合事件是否可以通过视觉捕捉来揭示活细胞中的动态？哪些细胞因子参与激活？是否有通过分泌性溶酶体在胞吐中起作用的因子参与？最后，抗原呈递细胞的MVB是否在与T细胞的相互作用中特异性动员？为了回答这些问题，我们将研究原代细胞，如巨噬细胞，其中MVB途径主要负责释放感染性病毒。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的HIV和MLV出口。为了深入了解病毒排出的细胞生物学，我们将利用分泌性溶酶体缺陷的小鼠模型的可用性，并确定在体内和体外这些模型中MLV释放是否受到影响。

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.