Characterization of Mortalin's interaction with HIV-1 Nef and role in Nef vesicle

描述（由申请人提供）：有证据表明，分泌的HIV-1 Nef在体内影响T细胞功能和旁观者未感染细胞的存活中起重要作用。然而，Nef的分泌机制尚未阐明。我们假设与Nef相互作用的细胞蛋白调节其分泌。在细胞内，已知Nef与内体运输途径相互作用，并已被证明上调宿主细胞的外泌体机制。此外，我们的研究小组已经在HIV和SIV Nef的n端区域绘制了高度保守的Nef基序，这些基序对分泌至关重要，并基于这些Nef分泌基序之一——分泌修饰区（SMR）设计了一种肽。该SMRwt肽仅含有SMR基序并抑制网状囊泡分泌，用于共免疫沉淀四个细胞结合伙伴。其中一种宿主细胞蛋白被鉴定为热休克蛋白70家族成员mortalin。尽管许多关于mortalin的文献都集中在它通过与肿瘤抑制蛋白p53的相互作用来阻断细胞凋亡的作用上，但有证据表明它也参与外泌体运输。由于Nef在其n端区域也含有p53结合位点，因此全长Nef蛋白可能不直接结合mortalin，而是通过与p53的相互作用与之相互作用。我们假设Nef和mortalin直接相互作用，允许Nef催化细胞内分泌过程并在外泌体样囊泡中释放自身。目前正在通过以下实验对其进行测试：(i)使用flag标记的死亡蛋白从Jurkat细胞中共免疫沉淀野生型Nef- gfp，以验证除了结合SMRwt肽外，死亡蛋白还与全长Nef蛋白相互作用；（ii）使用细菌重组野生型Nef- gfp和纯化的flag标记的mortalin结合试验来确定Nef是直接与mortalin相互作用，还是通过未识别的宿主细胞蛋白间接相互作用。由于SMRwt肽已被证明抑制Nef的分泌，可能是通过与Nef竞争细胞结合伴侣，我们假设这种抑制是破坏mortalin与Nef相互作用的结果。如果这是真的，那么mortalin对于Nef的分泌是必需的，通过其他方式破坏这种相互作用也会抑制Nef在外泌体样囊泡中的分泌。为了验证这一点，将在以下情况下评估对Nef分泌的影响：(i)使用microRNA （miRNA）敲低致死素的表达；（ii）利用抗体抑制阻断mortalin与Nef的相互作用。这些实验的结果将产生Nef分泌的细胞成分的基本知识。考虑到直接靶向Nef的困难，这一知识将开辟新的治疗途径，通过新发现的宿主细胞运输蛋白来预防艾滋病的发病机制。

DESCRIPTION (provided by applicant): There is evidence that secreted HIV-1 Nef plays an important role in affecting T cell function and in the survival of bystander uninfected cells in vivo. However, the mechanism(s) by which Nef is secreted has not been elucidated. We hypothesize that cellular proteins which interact with Nef regulate its secretion. Intracellularly, Nef is known to interact with the endosomal trafficking pathway, and has been shown to upregulate the exosomal machinery of the host cell. Furthermore, our group has mapped highly conserved Nef motifs in the N-terminal regions of both HIV and SIV Nef that are critical for secretion, and have engineered a peptide based on one of these Nef secretion motifs, the Secretion Modification Region (SMR). This SMRwt peptide, which contains only the SMR motif and which inhibits Nef vesicle secretion, was used to co-immunoprecipitate four cellular binding partners. One of these host cell proteins was identified as heat shock protein 70 family member, mortalin. Though much of the literature on mortalin centers on its role in blocking apoptosis through its interaction with the tumor suppressor protein p53, there is evidence that it is also involved in exosomal trafficking. As Nef also contains a p53-binding site in its N-terminal region, it is possible that the full-length Nef protein does not bind mortalin directly, but rather interacts with it via their mutual interactions with p53. We hypothesize that Nef and mortalin directly interact allowing Nef to catalyze the intracellular secretary process and release itself in exosomes-like vesicles. This is currently being tested through the following experiments, (i) FLAG-tagged mortalin is being used to co-immunoprecipitate wild type Nef-GFP from Jurkat cells to verify that, in addition to binding the SMRwt peptide, mortalin interacts with the full-length Nef protein; and, (ii) a binding assay with bacterial recombinant wild type Nef-GFP and purified FLAG-tagged mortalin is being used to determine if Nef interacts directly with mortalin, or indirectly via an unidentified host cell protein. Since the SMRwt peptide has been shown to inhibit Nef secretion, presumably by out-competing it for a cellular binding partner, we hypothesize that this inhibition is a result of disrupting mortalin's interaction with Nef. If true, mortalin is necessary for Nef secretion, and disrupting this interaction by other means will also inhibit Nef's secretion in exosome-like vesicles. To test this, the effect on Nef secretion will be assessed following (i) knockdown of the expression of mortalin using microRNA (miRNA); and, (ii) blocking of mortalin's interaction with Nef using antibody inhibition. The results of these experiments will generate fundamental knowledge of the cellular component(s) of Nef secretion. Given the proven difficulty in targeting Nef directly, this knowledge will open new therapeutic avenues to prevent AIDS pathogenesis through the newly identified host cell trafficking proteins. PUBLIC HEALTH RELEVANCE: The development of an HIV/AIDS vaccine has proven to be more difficult than anticipated, and the production of new antiviral drugs, many of them targeting viral proteins, has provided the greatest success in reducing AIDS deaths. This research will confirm the identification of a new set of cellular trafficking proteins involved in progression to AIDS, which could be potential therapeutic targets.