Characterization of the antiviral immunity factor CD317/tetherin

哺乳动物已经进化出一套内在的细胞防御机制，能够抑制病毒病原体的复制。这些限制性因子（RFs）可以组成性表达，但在病毒感染的反应中经常被宿主细胞上调，在病毒跨物种传播的背景下，它们形成了特别有效的屏障。这个项目的重点是宿主RF CD317（也被称为BST-2/HM1.24/tetherin），它通过有效地阻止成熟的HIV-1颗粒从有效感染的细胞中释放出来，在HIV复制周期的后期起作用。CD317的活性可以被HIV-1蛋白Vpu拮抗，本项目的总体目标是明确CD317介导的限制及其被HIV-1 Vpu拮抗的分子机制。我们在第一个资助期的研究结果以及其他实验室的研究结果表明，Vpu通过两种机制拮抗CD317施加的颗粒释放限制：(i)损害宿主细胞质膜的整体递送；（ii）改变质膜内的膜下侧向分布，以排除病毒出芽位点。虽然Vpu螺旋2是Vpu效应的决定因素，但Vpu高度保守的磷酸二丝氨酸基序仅参与抑制CD317的顺行运输和再循环。在进一步的初步工作中，我们发现宿主细胞蛋白Arl6IP1是一种新的Vpu相互作用物，可以增强cd317介导的限制，并建立了一种基于全身组织微阵列的表达谱方法，以确定HIV传播生理部位相关宿主因子的原位表达模式。在这些发现的基础上，第二个资助期的主要目标是：(1)Vpu影响细胞内转运和CD317膜分离的分子机制和宿主细胞配体的解剖，(2)新发现的细胞Vpu相互作用物Arl6IP1在HIV-1限制中的作用的表征，以及(3)鉴定CD317和Arl6IP1介导的体内和体外限制的生理相关位点。为了解决这些基本问题，我们将整合生物化学、细胞内运输、超分辨率显微镜、病毒学和免疫组织学方法，在我们两个实验室之间建立了良好的合作关系。这些研究结果有望显著推进我们对HIV-1 Vpu和CD317在哪些分子机制和哪些生理部位相互作用形成HIV-1发病机制的理解。

Mammals have evolved a set of intrinsic cellular defense mechanisms capable of inhibiting the replication of viral pathogens. These restriction factors (RFs), which can be constitutively expressed but are frequently up-regulated by host cells in response to virus infection, impose particularly effective barriers in the context of cross-species transmission of viruses. The focus of this project is on the host RF CD317 (also referred to as BST-2/HM1.24/tetherin) that acts late in the HIV replication cycle by potently blocking the release of mature HIV-1 particles from productively infected cells. The activity of CD317 can be antagonized by the HIV-1 protein Vpu and the overall goal of this project is to define the molecular mechanisms of CD317-mediated restriction and its antagonism by HIV-1 Vpu. Our results of the first funding period together with those by other laboratories established that Vpu antagonizes the particle release restriction imposed by CD317 via two mechanisms: (i) impairing overall delivery to the host cell plasma membrane and (ii) altering the submembrane lateral distribution within the plasma membrane for exclusion from viral budding sites. While Vpu helix 2 emerges as a determinant for both Vpu effects, the highly conserved phospho-di-serine motif of Vpu is only involved in the inhibition of CD317 anterograde transport and recycling. In additional preliminary work we identified the host cell protein Arl6IP1 as novel Vpu interactor that enhances the CD317-mediated restriction and established a whole body-tissue microarray-based expression profiling approach to define in situ expression patterns of relevant host factors at physiological sites of HIV transmission. Building on these findings the main goals of the second funding period are (i) the dissection of the molecular mechanisms and host cell ligands employed by Vpu to affect intracellular transport and membrane segregation of CD317, (ii) characterization of the role of the newly identified cellular Vpu interactor Arl6IP1 in HIV-1 restriction, and (iii) the identification of physiologically relevant sites of CD317-and Arl6IP1-mediated restriction ex vivo and in vivo. To address these cardinal questions we will integrate biochemistry, intracellular transport, super-resolution microscopy, virology and immunohistology approaches in the context of the well-established collaboration between our two laboratories. Results of these studies are expected to significantly advance our understanding by which molecular mechanisms and at which physiological sites the interplay between HIV-1 Vpu and CD317 shape HIV-1 pathogenesis.