LIPID-PROTEIN INTERACTIONS IN VIRAL ASSEMBLY AND VIRUS LIKE PARTICLE FORMATION

 性状（由申请方提供）：脂质包膜病毒从宿主细胞膜复制并出芽，在宿主细胞膜处获得脂质包膜。了解几种病毒的出芽过程对阐明病毒的生命周期和确定治疗靶点具有重要影响。丝状病毒具有丝状脂质包膜，尽管在30多年前被发现，但对它们如何获得脂质外壳知之甚少。丝状病毒包括埃博拉病毒（EBOV）和马尔堡病毒（MARV），其可具有高达90%的临床致死率。丝状病毒编码七个基因，包括病毒基质蛋白VP 40，其调节宿主细胞的出芽。VP 40作为在哺乳动物细胞中表达的唯一丝状病毒蛋白足以产生与活病毒体几乎不可区分的病毒样颗粒（VLP）。因此，在本发明中， VP 40已作为研究BSL-4实验室外病毒出芽的模型。到目前为止，很少有人知道VP 40如何与生物膜相互作用，以调节出芽和从宿主细胞质膜的出口。在之前的项目期间，我们发现EBOV VP 40（eVP 40）需要质膜内叶上的PS来结合、组装和形成VLP。值得注意的是，eVP 40与质膜的相互作用诱导PS暴露于质膜外叶的出口部位;而PS通常仅暴露于内叶。PS翻转/转运到外小叶可能有助于PS呈递到新生病毒体表面，以促进TIM-1介导的进入细胞。这些发现提供了一个 脂质选择性结合和转运穿过人类细胞膜与随后病毒进入脂质的使用之间的明确联系。新的初步数据和与结构生物学家的合作表明，除了PS之外，eVP 40还需要质膜PI（4，5）P2进行组装和出芽。相比之下，MARV VP 40（mVP 40）作为一种阴离子电荷传感器，杂乱地结合阴离子脂质，与我们的合作者解决的新X射线结构一致。核心假设是eVP 40和mVP 40具有根本不同的脂质结合特性。该更新计划将研究eVP 40和mVP 40的脂质结合、组装和出芽特性。具体目标1将研究eVP 40和mVP 40与质膜脂质差异相互作用以促进出芽的机制。我们还将研究质膜胆固醇含量和膜流动性对eVP 40和mVP 40膜结合、寡聚化和出芽的作用。具体目标2将研究质膜鞘脂在eVP 40和mVP 40 VLP形成中的作用。我们的合作者将使用生物化学和细胞测定结合结构分析来阐明eVP 40和mVP 40组装事件的分子结构。总的来说，这些研究应该对丝状病毒颗粒如何从细胞质膜形成产生重要的机制见解。

 DESCRIPTION (provided by applicant): Lipid-enveloped viruses replicate and bud from host cell membranes where they acquire their lipid coat. Understanding the budding processes of several viruses has had significant impact on elucidating the viral life cycle and identifying therapeutic targets. Filoviruses have a filamentous lipid- envelope and despite being discovered more than 30 years ago, not much is known on how they acquire their lipid coat. Filoviruses include Ebola virus (EBOV) and Marburg virus (MARV), which can have up to 90% clinical fatality. Filoviruses encode seven genes including the viral matrix protein VP40, which regulates budding from the host cell. VP40 as the only filovirus protein expressed in mammalian cells is sufficient to produce virus like particles (VLPs) nearly indistinguishable from live virions. Thus, VP40 has served as a model to study viral budding outside of BSL-4 laboratories. To date, little is known about how VP40 interacts with biological membranes to regulate budding and egress from the host cell plasma membrane. During the previous project period, we found that EBOV VP40 (eVP40) required PS on the inner leaflet of the plasma membrane to bind, assemble, and form VLPs. Notably, interactions of eVP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress; whereas PS is typically only on the inner leaflet. Flipping/transport of PS to the outer leaflet may aid PS presentation on the surface of nascent virions to facilitate TIM-1-mediated entry into cells. These findings provided a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry. New preliminary data and collaboration with a structural biologist has demonstrated that in addition to PS, eVP40 requires plasma membrane PI(4,5)P2 for assembly and budding. In contrast, MARV VP40 (mVP40) acts as an anionic charge sensor, promiscuously binding anionic lipids consistent with the new X-ray structure solved by our collaborator. The central hypothesis is that eVP40 and mVP40 have fundamentally different lipid binding properties. This renewal proposal will investigate lipid binding, assembly, and budding properties of eVP40 and mVP40. Specific Aim 1 will investigate the mechanisms by which eVP40 and mVP40 differentially interact with plasma membrane lipids to facilitate budding. We will also investigate the role of plasma membrane cholesterol content and membrane fluidity on eVP40 and mVP40 membrane binding, oligomerization, and budding. Specific aim 2 will investigate the role of a plasma membrane sphingolipid in eVP40 and mVP40 VLP formation. Biochemical and cellular assays in combination with structural analysis by our collaborator will be used to elucidate the molecular architecture of the eVP40 and mVP40 assembly events. Taken together, these studies should produce important mechanistic insight into how filovirus particles form from the plasma membrane of cells.