HARC Center: HIV Accessory and Regulatory Complexes

Vpr：Vpr是一种保守的多功能蛋白，其被并入病毒体中并驱动早期（运输病毒）。 整合前复合物）和晚期（凋亡）病毒生命周期步骤。尽管经过20多年的研究， Vpr加速病毒复制的机制尚不清楚[40-42]。广泛的 Vpr的分子功能包括在G2期细胞周期阻滞和整合前的作用 复合物[43-46]，可能反映了多用途衔接蛋白。最近，SAMHDI，一种脱氧核苷 三磷酸三磷酸水解酶被发现是一种骨髓和树突细胞限制因子， 克服了HIV-2 Vpx [47，48]，一个parajog的Vpr相信执行一些功能的Vpr。就像维芙和 Vpu、Vpr和Vpx都与宿主泛素机制相互作用。Vpr结合Cullin 4 RING盒Ubiqultin 连接酶（CRL 4）复合物[49，50]，表明Vpr可以通过连接酶（CRL 4）复合物克服额外的宿主限制。 降解途径由于Vpr在许多关键的艾滋病毒感染过程中发挥着核心作用，因此Vpr代表了一种 令人兴奋的结构-功能研究的目标和立场Vpr作为一个可行的治疗目标。没有 然而，只有通过明确定义宿主蛋白和途径， 在分子水平上受到Vpr的干扰--如本提案所述--将使Vpr真正的靶向活性 被揭露。HARC蛋白质组学/基因组学核心汇编了最广泛的Vpr-宿主相互作用数据集 描述到目前为止。令人鼓舞的是，这些新的相互作用揭示了宿主复合物和伴侣（例如动力蛋白 和C 0 P9）在功能上与许多Vpr活性相关。通过评估其功能意义和能力， 形成结构化的复合物，我们将开发和测试有关Vpr主机相互作用的新假设。 PR：许多病毒编码的蛋白酶除了裂解外，还能解除宿主的防御或劫持宿主的过程 病毒底物。例如，脊髓灰质炎病毒PR通过切割翻译起始来抑制宿主蛋白质合成 因子，而鼻病毒PR切割核孔复合物和衔接蛋白参与先天性 免疫反应[51-58]。HIV-1 PR在病毒体期间加工病毒Gag和GagPol多聚蛋白 成熟，但其在影响宿主功能中的作用尚不清楚。在体外，PR切割几种哺乳动物蛋白质 [59-65]，但尚不清楚在HIV感染期间是否发生裂解或宿主中存在多少活性酶 细胞在病毒的生命周期[66，67]。HARC中心蛋白质组学/基因组学核心发现了新的宿主 与PR的非活性形式相互作用的蛋白质，包括参与免疫应答，剪接， 翻译、细胞生长和凋亡[1，29]。细胞中活跃的PR裂解了这些蛋白质中的一些，包括 真核生物翻译起始因子3复合体d亚基RNA结合域中的单个位点 （elF3d）。eIF 3d几乎与Gag一样有效地被切割。一个有趣的假设是，elF 3d招募了 翻译起始复合物进入HIV基因组，阻碍逆转录。我们的结构 这种PR-主体复合物的研究将是第一个PR与大分子底物，提供新的 底物识别知识。我们分析了潜在的宿主PR底物在HIV中的作用， 复制有可能为PR功能建立新的范例，将PR与Vit Vpu的作用联系起来， Vpr，并定义新的PR-主体复合物的结构。

VPR: Vpr is a conserved multi-functional protein that is incorporated into virions and drives early (transport of pre-integration complex) and late (apoptosis) viral life cycle steps. Despite more than 20 years of research, the mechanism by which Vpr accelerates virus replication is not well understood [40-42]. A wide range of molecular functions has been ascribed to Vpr, including roles in G2 cell cycle arrest and pre-integration complexes [43-46], perhaps reflecting a multi-purpose adaptor protein. Recently, SAMHDI, a deoxynucleoside triphosphate triphosphohydrolase, was found to be a myeloid and dendritic cell restriction factor that is overcome by HlV-2 Vpx [47, 48], a parajog of Vpr believed to carry out some functions of Vpr. Like Vif and Vpu, both Vpr and Vpx interact with the host ubiquitin machinery. Vpr binds the Cullin4 RING Box Ubiqultin Ligase (CRL4) complex [49, 50], suggesting that Vpr may overcome additional host restrictions via the degradation pathway. Due to its central roles in a number of key HIV infection processes, Vpr represents an exciting target for structure-function studies and positions Vpr as a viable therapeutic target. With no universally accepted model for its functions, however, only by clearly defining the host proteins and pathway(s) perturbed by Vpr at the molecular level-as outlined in this proposal-will the bona fide targetable activity of Vpr be revealed. The HARC Proteomics/Genomics Core compiled the most extensive Vpr-host interaction data set described to date. Encouragingly, these novel interactions reveal host complexes and partners (e.g. dynein and C0P9) functionally linked with many Vpr activities. By evaluating the functional significance and ability to form structured complexes, we will develop and test new hypotheses about Vpr-host interactions. PR: Many viruses encode proteases that disarm host defenses or hijack host processes in addition to cleaving viral substrates. For example, poliovirus PR inhibits host protein synthesis by cleaving translation initiation factors, while rhinovirus PR cleaves the nuclear pore complex and adaptor proteins involved in the innate immune response [51-58]. HIV-1 PR processes the viral Gag and GagPol polyproteins during virion maturation, but its role in affecting host functions is unclear. In vitro, PR cleaves several mammalian proteins [59-65], but it is not known if cleavage occurs during HIV infection or how much active enzyme exists in host cells during the viral life cycle [66, 67]. The HARC Center Proteomics/Genomics Core uncovered novel host proteins that interact with, an inactive version of PR, including proteins involved in immune responses, splicing, translation, cell growth, and apoptosis [1, 29]. Active PR in cells cleaved some of these proteins, including a single site in the RNA binding domain of the d subunit of the eukaryotic translation initiation factor 3 complex (elF3d). elF3d was cleaved nearly as efficiently as Gag. An intriguing hypothesis is that elF3d recruits the translation initiation complex to the entering HIV genome, obstructing reverse transcription. Our structural studies of such PR-host complexes will be the first of PR with a macromolecular substrate, providing new knowledge on substrate recognition. Our analyses of the roles of potential host PR substrates in HIV replication have the potential to establish new paradigms for PR function, link PR to the roles of Vit Vpu, and Vpr, and define the structures of novel PR-host complexes.