HIV-1 Nef regulates activity of the ER chaperone calnexin

描述(申请人提供)：HIV-1蛋白Nef是一种多功能蛋白，参与调节病毒的传染性，并对HIV感染的许多致病作用负责，包括下调CD4、MHC I和ABCA1。Nef对宿主细胞蛋白质的影响归因于Nef能够作为宿主细胞运输蛋白和靶蛋白之间的桥梁，从而促进后者通过细胞溶酶体或蛋白酶体机制的运输和降解。在我们的初步实验中，我们确定了Nef的一个新的相互作用伙伴，内质网(ER)伴侣Calnexin。Calnexin是一种完整的内质网跨膜蛋白，其主要功能是在N-连接的糖蛋白成熟过程中协助蛋白质折叠和进行质量控制。我们发现Nef干扰了Calnexin和胆固醇转运体ABCA1之间的相互作用，导致非功能性ABCA1从内质网释放，并抑制胆固醇外流。然而，Nef的这种作用是选择性的，因为Calnexin与另一种糖蛋白HIV-1 gp160的相互作用没有中断。造成这种选择性的机制尚不清楚，但已知Nef会影响其结合的蛋白质的定位。在这些初步研究的基础上，我们假设Nef与calnexin的相互作用改变了calnexin的定位，促进了其与gp16的相互作用，但代价是某些宿主细胞蛋白的成熟和功能受到损害。这一假设将在提案的目标1中得到检验。在目标2中，我们将使用生物信息学、分子模拟和Nef和calnexin的突变，以确定负责这些蛋白质之间相互作用的基序和结构域，并将测试现有的Nef靶向药物和通过虚拟筛选确定的新化合物的干扰能力。 这种互动。生物信息学研究将由我们的俄罗斯合作者阿德朱贝博士进行。在目标3中，我们将表征Nef对Calnexin的影响对病毒、细胞和宿主生物体的功能后果。使用质谱仪，我们将评估在Nef存在和不存在的情况下gp160和ABCA1的糖基化。我们还将在Nef转基因小鼠身上测试针对Nef-calnexin相互作用的药物，Nef转基因小鼠可以在另一位俄罗斯合作者Nedospasov博士的实验室获得。这些研究将表征Nef对宿主细胞和病毒蛋白作用的新机制，可能为Nef刺激HIV感染性的作用提供解释，将发现新的抗HIV药物，并可能为抗HIV疫苗的设计提供参考。

DESCRIPTION (provided by applicant): HIV-1 protein Nef is a multifunctional protein involved in regulation of viral infectivity and responsible for many pathogenic effects of HIV infection, including downregulation of CD4, MHC I and ABCA1. The effect of Nef on host cell proteins has been ascribed to the ability of Nef to function as a bridge between the host cell transport proteins and the target protein, thus promoting transport and degradation of the latter by the cellular lysosomal or proteasomal machinery. In our preliminary experiments, we identified a novel interaction partner of Nef, the endoplasmic reticulum (ER) chaperone calnexin. Calnexin is an integral ER transmembrane protein the main function of which is to assist protein folding and perform quality control during maturation of N-linked glycoproteins. We found that Nef disrupts interaction between calnexin and cholesterol transporter ABCA1, resulting in release from ER of non-functional ABCA1 and inhibition of cholesterol efflux. However, this effect of Nef was selective, as calnexin interaction with another glycoprotein, HIV-1 gp160, was not disrupted. The mechanism responsible for this selectivity is unknown, but Nef is known to affect localization of proteins to which it binds. Based on these preliminary studies, we hypothesize that Nef interaction with calnexin alters calnexin localization promoting its interaction with gp16 at the expense of a certain repertoire of host cell proteins whose maturation and function are impaired. This hypothesis will be tested in Aim 1 of the proposal. In Aim 2, we will use bioinformatics, molecular modeling and mutagenesis of Nef and calnexin, to identify the motifs and domains responsible for the interaction between these proteins and will test available Nef-targeting drugs and new compounds identified by virtual screening for the ability to interfere with this interaction. The bioinformatics studies will be performed by our Russian collaborator, Dr. Adzhubei. In Aim 3, we will characterize functional consequences of Nef effects on calnexin for the virus, cell and host organism. Using mass-spectrometry, we will assess glycosylation of gp160 and ABCA1 in the presence and absence of Nef. We will also test drugs targeting Nef-calnexin interaction in Nef-transgenic mice available in the laboratory of another Russian collaborator, Dr. Nedospasov. These studies will characterize novel mechanism behind the effects of Nef on host cell and viral proteins, may provide an explanation for the stimulatory effect of Nef on HIV infectivity, will identify new anti-HIV agents, and may inform anti-HIV vaccine design efforts.