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

描述(申请人提供)：有证据表明，分泌的HIV-1 Nef在影响T细胞功能和体内旁观者未感染细胞的生存中发挥重要作用。然而，Nef的分泌机制(S)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与内体转运途径相互作用，并已被证明上调宿主细胞的外体机制。此外，我们的团队已经在HIV和SIV Nef的N端定位了对分泌至关重要的高度保守的Nef基序，并根据这些Nef分泌基序之一-分泌修饰区域(SMR)设计了一个肽。这种只含有SMR基序并抑制Nef囊泡分泌的SMRwt多肽被用来免疫共沉淀四个细胞结合伙伴。其中一个宿主细胞蛋白被鉴定为热休克蛋白70家族成员mortalin。虽然关于mortalin的大部分文献都集中在它通过与肿瘤抑制蛋白P53的相互作用来阻止细胞凋亡的作用上，但有证据表明它也参与了胞外转运。由于Nef在其N末端还含有一个P53结合位点，因此全长Nef蛋白可能不直接与mortalin结合，而是通过与P53的相互作用而与之相互作用。我们假设Nef和mortalin直接相互作用，使Nef催化细胞内的分泌过程，并在外体样囊泡中释放自己。目前正在通过以下实验来测试这一点：(I)标记了标志的mortalin用于免疫共沉淀来自Jurkat细胞的野生型Nef-GFP，以证实除了结合SMRwt肽外，mortalin还与全长Nef蛋白相互作用；以及(Ii)用细菌重组野生型Nef-GFP和纯化的标记了标志的mortalin进行结合试验来确定Nef是直接与Mortalin相互作用，还是通过未知宿主细胞蛋白间接作用。由于SMRwt多肽已被证明抑制Nef的分泌，推测是通过竞争细胞结合伙伴来抑制Nef的分泌，我们假设这种抑制是由于干扰mortalin与Nef的相互作用造成的。如果是真的，mortalin对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在外切体样小泡中的分泌。为了测试这一点，将在以下情况下评估对Nef分泌的影响：(I)使用microRNA(MiRNA)抑制mortalin的表达；以及(Ii)使用抗体抑制来阻断mortalin与Nef的相互作用。这些实验的结果将产生关于Nef分泌的细胞成分(S)的基础知识。鉴于直接靶向Nef已被证明是困难的，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。 与公共卫生有关：艾滋病毒/艾滋病疫苗的开发已被证明比预期的更困难，新的抗病毒药物的生产在减少艾滋病死亡方面取得了最大成功，其中许多药物针对病毒蛋白。这项研究将确认一组与艾滋病进展有关的新的细胞运输蛋白，这些蛋白可能成为潜在的治疗靶点。

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.