THE ROLE OF HIV-1 VPU IN THE REGULATION OF CD40

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 本研究的目的是了解HIV-1蛋白VPU在HIV发病机制中的作用。我们以前的研究表明，VPU通过诱导细胞因子受体CD40来调节内皮细胞(EC)上黏附分子VCAM-1的表达。以这种方式调节内皮细胞可能有助于艾滋病相关B细胞淋巴瘤的特征结外表现。正在进行的研究表明，VPU对CD40的影响可能是VPU调节另一种蛋白质或途径的下游结果。因此，我们采用了一种基于MS的蛋白质组学方法，即细胞培养中氨基酸稳定同位素标记(SILAC)，以鉴定受VPU影响的新的细胞蛋白。我们比较了表达野生型VPU的Hela-CD4细胞和表达VPU突变体的同一细胞系的蛋白质谱，该突变体不与β-TrCP相互作用，因此不能与泛素-蛋白质体途径相互作用以稳定或降解VPU靶标。我们鉴定了7种野生型VPU上调或下调至少1.5倍的蛋白质。其中一种蛋白质BST-2最近被鉴定为宿主抗病毒因子，其功能是限制HIV病毒粒子从感染细胞中释放。因此，这种蛋白质被重新命名为tetherin。我们新发现的HIV VPU可以下调BST-2/tetherin的表面表达，揭示了HIV抵消宿主限制的机制。基于这些发现的潜在意义，我们目前的工作重点是确定VPU介导的BST-2下调的机制和意义。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The goal of this study is to understand the role of the HIV-1 protein Vpu in HIV pathogenesis. Our previous studies demonstrated that Vpu modulates expression of the adhesion molecule VCAM-1 on endothelial cells (EC) via induction of the cytokine receptor CD40. Modulation of the endothelium in this manner may contribute to the characteristic extranodal presentation of AIDS-associated B cell lymphomas. Ongoing studies have shown that Vpu's effect on CD40 is likely the downstream consequence of Vpu modulation of another protein or pathway. We thus undertook an MS-based proteomics approach, stable isotope labeling with amino acids in cell culture (SILAC), to identify novel cellular proteins affected by Vpu. We compared the protein profiles of Hela-CD4 cells expressing wildtype Vpu to the same cell line expressing a mutant of Vpu that does not interact with beta-TrCP and thus cannot interact with the Ubiquitin-proteosome pathway for stabilization or degradation of Vpu targets. We identified 7 proteins that were up- or downregulated at least 1.5-fold by wildtype Vpu. One of these proteins, BST-2, was recently identified as a host antiviral factor whose function is to restrict the release of HIV virions from infected cells. The protein was thus renamed tetherin. Our novel finding that HIV Vpu could downregulate surface expression of BST-2/tetherin reveals a mechanism for HIV to counteract the host restriction. Based on the potential significance of these findings, we are focusing our current efforts on determining the mechanism and significance of Vpu-mediated BST-2 downregulation.