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。这些限制因子(FR)可以结构性表达，但经常被宿主细胞上调以应对病毒感染，在病毒跨物种传播的背景下形成特别有效的障碍。该项目的重点是宿主RF CD317(也称为BST-2/HM1.24/tetherin)，它通过有效地阻止成熟的HIV-1颗粒从生产性感染细胞中释放，在艾滋病毒复制周期后期发挥作用。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介导的限制及其拮抗HIV-1 VPU的分子机制。我们的第一个资助期的结果与其他实验室的结果一起证实，VPU通过两种机制对抗CD317施加的颗粒释放限制：(I)损害向宿主细胞质膜的整体输送和(Ii)改变质膜内的亚膜横向分布，以排除病毒出芽部位。虽然VPU螺旋2是这两种VPU效应的决定因素，但VPU高度保守的磷酸二丝氨酸基序只参与抑制CD317的顺行运输和再循环。在另外的前期工作中，我们确定了宿主细胞蛋白Arl6IP1是一种新型的VPU相互作用因子，可以增强CD317介导的限制作用，并建立了基于全身-组织芯片的表达谱方法，以确定相关宿主因子在HIV传播的生理部位的原位表达模式。基于这些发现，第二个资助期的主要目标是(I)剖析VPU影响CD317细胞内转运和膜分离的分子机制和宿主细胞配体，(Ii)表征新发现的细胞VPU相互作用元件Arl6IP1在HIV-1限制中的作用，以及(Iii)确定CD317和Arl6IP1介导的体内外限制的生理相关位置。为了解决这些关键问题，我们将在我们两个实验室之间良好合作的背景下，整合生物化学、细胞内转运、超分辨率显微镜、病毒学和免疫组织学方法。这些研究的结果有望极大地促进我们理解HIV-1VPU和CD317之间的相互作用通过哪些分子机制和哪些生理位点影响HIV-1的致病机制。

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.