Herbein Georges, Varin Audrey, Larbi Anis, Fortin Carl, Mahlknecht Ulrich, Fulop Tamas, Aggarwal Bharat B
Department of Virology, University of Franche-Comte, Besançon, France.
Curr HIV Res. 2008 Mar;6(2):117-29. doi: 10.2174/157016208783884985.
The human immunodeficiency virus (HIV) Nef protein is myristoylated and plays a critical role in AIDS pathogenesis by enhancing viral replication, survival of the virus within infected cells and by facilitating its spread in vivo. We observed that, in the promonocytic cell line U937, myristoylated exogenous Nef protein activates NF-kappaB and AP-1, whereas unmyristoylated exogenous Nef protein does not. Using methyl-beta-cyclodextrin (MbetaC) treatment, we observed that the activation of NF-kappaB and AP-1 by exogenous Nef protein is mediated primarily via lipid rafts both in U937 cells and in primary human macrophages. In agreement with this observation, exogenous Nef protein colocalized with GM1 ganglioside, a major component of lipid rafts, in U937 cells as detected by confocal microscopy. Since tumor necrosis factor alpha (TNFalpha) activates NF-kappaB and AP-1, we investigated the role of exogenous Nef protein in TNFalpha-stimulated U937 cells and primary macrophages. We observed that exogenous Nef and TNFalpha synergistically activate NF-kappaB and AP-1 in U937 cells and primary macrophages resulting in enhanced stimulation of the HIV-1 long terminal repeat (LTR), and subsequently in enhanced viral replication in both chronically infected promonocytic U1 cells and acutely HIV-1-infected primary macrophages. Both enhanced LTR stimulation and viral replication following treatment with exogenous Nef and TNFalpha were mediated via lipid rafts. Therefore, our results indicate that exogenous Nef protein and enhanced TNFalpha production detected in HIV-infected subjects could synergize to fuel the progression of the disease via lipid raft-dependent stimulation of the HIV-1 provirus present in such cellular reservoirs as mononuclear phagocytes.
人类免疫缺陷病毒（HIV）Nef蛋白经肉豆蔻酰化修饰，通过增强病毒复制、在受感染细胞内的存活能力以及促进其在体内传播，在艾滋病发病机制中发挥关键作用。我们观察到，在单核细胞系U937中，经肉豆蔻酰化修饰的外源性Nef蛋白可激活核因子κB（NF-κB）和活化蛋白-1（AP-1），而未经肉豆蔻酰化修饰的外源性Nef蛋白则无此作用。通过使用甲基-β-环糊精（MβC）处理，我们观察到外源性Nef蛋白对NF-κB和AP-1的激活主要通过脂筏介导，这一现象在U937细胞和原代人巨噬细胞中均有发现。与该观察结果一致，通过共聚焦显微镜检测发现，外源性Nef蛋白在U937细胞中与脂筏的主要成分GM1神经节苷脂共定位。由于肿瘤坏死因子α（TNFα）可激活NF-κB和AP-1，我们研究了外源性Nef蛋白在TNFα刺激的U937细胞和原代巨噬细胞中的作用。我们观察到，外源性Nef蛋白和TNFα在U937细胞和原代巨噬细胞中协同激活NF-κB和AP-1，导致对HIV-1长末端重复序列（LTR）的刺激增强，随后在慢性感染的单核细胞系U1细胞和急性HIV-1感染的原代巨噬细胞中病毒复制均增强。用外源性Nef蛋白和TNFα处理后，LTR刺激增强和病毒复制增强均通过脂筏介导。因此，我们的结果表明，在HIV感染受试者中检测到的外源性Nef蛋白和增强的TNFα产生可能通过脂筏依赖性刺激存在于单核吞噬细胞等细胞储存库中的HIV-1前病毒，协同推动疾病进展。
