Recruitment of BST-2/tetherin to HIV-1 assembly sites

描述(申请人提供)：最近的研究表明，一种称为HM1.24、CD317、BST-2或tetherin(以下称为BST-2/tetherin)的细胞蛋白通过将新组装的病毒颗粒拴在病毒产生细胞的质膜(PM)上来抑制HIV-1颗粒的释放。然而，BST-2/tetherin被整合到组装病毒颗粒中的机制仍有待确定。值得注意的是，BST-2/tetherin可以抑制多种包膜病毒的释放，包括逆转录病毒、丝状病毒、疱疹病毒、横纹病毒、副粘病毒和阿雷纳病毒。因此，确定将BST-2/tetherin招募到HIV-1组装部位的机制不仅将促进我们对导致艾滋病的HIV生物学的理解，还将有助于我们开发针对广泛包膜病毒的抗病毒策略。值得注意的是，一种与BST-2/tetherin有相同关键结构特征但没有序列同源性的人造蛋白质能够阻止HIV-1的释放。因此，BST-2/tetherin与新生病毒颗粒的关联可能依赖于共同的细胞结构，而不是特定的病毒或细胞蛋白。现在认为PM由多个微域组成，而不是以随机方式嵌入蛋白质的统一脂质双层。这些微域包含特定的脂类和蛋白质组，并且可能具有不同的寿命、大小和动态。其中，脂筏与HIV-1组装有关是众所周知的。值得注意的是，BST-2/tetherin在其C端被GPI锚点修饰，众所周知，GPI锚点将蛋白质靶向脂筏。再加上许多被包裹的病毒与脂筏或其他微域相关的概念，可以想象，BST-2/tetherin与脂筏的结合可能会促进这种蛋白质的结合，以组装颗粒。然而，BST-2/tetherin敏感病毒常见的其他膜相关结构也可能在BST-2/tetherin募集中发挥作用，如膜曲、膜性颈/柄或ESCRT(运输所需的内体分类复合体)蛋白。为了更好地了解BST-2/tetherin的抗病毒活性，在这项提议中，我们计划检验以下假设：BST-2/tetherin与特定的PM结构相关，这些结构被招募到病毒组装部位。为了验证这一假设，在目标1中，将确定在病毒组装过程中与RAFT、ESCRT和BST-2/tetherin结合有关的事件的顺序。利用遗传方法和先进的显微技术相结合，我们计划确定BST-2/tetherin被招募到组装位置的组装步骤。AIM 2中的实验旨在确定脂筏和ESCRT是否对BST-2/Tetherin的功能及其在病毒组装部位的招募至关重要。总之，在本申请中提出的实验中获得的信息可能有助于我们阐明BST-2/tetherin介导的限制HIV-1释放的机制。

DESCRIPTION (provided by applicant): Recent studies showed that a cellular protein known as HM1.24, CD317, BST-2, or tetherin (hereafter referred to as BST-2/tetherin) inhibits HIV-1 particle release by tethering newly assembled virus particles to the plasma membrane (PM) of virus-producing cells. However, the mechanism by which BST-2/tetherin is incorporated into assembling virus particles remains to be determined. Notably, BST-2/tetherin can inhibit release of a wide variety of enveloped viruses including retroviruses, filoviruses, herpesviruses, rhabdoviruses, paramyxoviruses, and arenaviruses. Therefore, determining the mechanism that recruits BST-2/tetherin to HIV-1 assembly sites will not only advance our understanding of the biology of HIV that causes AIDS but also will help us develop antiviral strategies targeting a broad range of enveloped viruses. Notably, an artificial protein that shares key structural features with BST-2/tetherin with no sequence homology was capable of blocking HIV-1 release. Therefore, association of BST-2/tetherin to nascent virus particles is likely dependent on a common cellular structure rather than specific viral or cellular proteins. The PM is now thought to consist of multiple microdomains rather than a uniform lipid bilayer with proteins embedded in a random manner. These microdomains contain specific sets of lipids and proteins and may have various lifetimes, sizes, and dynamics. Among them, lipid rafts are well known to associate with HIV-1 assembly. Notably, BST-2/tetherin is modified at its C terminus with a GPI anchor, which is known to target proteins to lipid rafts. Together with the notion that many enveloped viruses associate with lipid rafts or other microdomains, it is conceivable that association of BST-2/tetherin with lipid rafts may promote incorporation of this protein to assembling particles. However, it is also possible that other membrane-associated structures common to BST-2/tetherin-susceptible viruses, such as membrane curvature, membranous neck/stalk, or ESCRT (endosomal sorting complex required for transport) proteins, play a role in BST-2/tetherin recruitment. Toward better understanding of the antiviral activity of BST-2/tetherin, in this proposal, we plan to test the following hypothesis: BST-2/tetherin associates with specific PM structures that are recruited to virus assembly sites. To test this hypothesis, in Aim 1, the order of events with regard to incorporation of rafts, ESCRTs, and BST-2/tetherin during virus assembly will be determined. Using combination of genetic approaches with advanced microscopy techniques, we plan to identify the assembly step at which BST-2/tetherin are recruited to assembly sites. Experiments in Aim 2 are designed to determine whether lipid rafts and ESCRTs are essential for BST- 2/tetherin function and its recruitment to virus assembly sites. Altogether, information gained in experiments proposed in this application will likely help us elucidate the mechanism that initiates BST-2/tetherin-mediated restriction of HIV-1 release.