Nef-mediated enhancement of cellular activation and human immunodeficiency virus type 1 replication in primary T cells is dependent on association with p21-activated kinase 2.
Nef-mediated enhancement of cellular activation and human immunodeficiency virus type 1 replication in primary T cells is dependent on association with p21-activated kinase 2.
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DOI：
10.1186/1742-4690-8-64
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发表时间：
2011-08-05
期刊：
影响因子：
3.3
通讯作者：
Gabuzda D
中科院分区：
文献类型：
--
作者：
Olivieri KC;Mukerji J;Gabuzda D
HIV-1辅助蛋白Nef是慢病毒致病性的重要决定因素，通过促进病毒复制和其他鲜为人知的机制促进疾病进展。NEF介导多种功能，包括下调细胞表面CD4和MHC I类分子，增强病毒感染性，增强T细胞活化。NEF与一个多蛋白信号复合体相互作用，该复合体包括Src家族激酶、Vav1、CDC42和激活的PAK2(p21-激活的激酶2)。尽管以前的研究试图确定Nef-PAK2信号复合体的生物学作用，但该复合体及其组成蛋白在Nef功能中的重要性仍不清楚。在这里，我们表明，Nef突变体对PAK2结合有缺陷，但对CD 4和MHC I类下调和传染性增强具有功能，在感染并随后被次最大刺激(1μg/mlPHA-P)激活的原始T细胞中增强病毒复制的能力也存在缺陷。相反，这些Nef突变体对更强刺激(2μg/mlPHA-P或抗CD3/CD28包被珠)激活的T细胞中的HIV-1复制几乎没有影响。携带野生型Nef的病毒，但没有PAK2结合缺陷的Nef突变体，增强了Jurkat细胞中NFAT和IL2受体启动子的活性。此外，野生型Nef的表达，而不是PAK2结合缺陷的突变型Nef的表达，足以增强原代CD4和CD8T细胞对Nef表达和旁观者细胞对激活刺激的反应性。在Jurkat细胞中，PAK2的siRNA敲除降低了在Nef存在和不存在的情况下抗CD3/CD28刺激所诱导的NFAT激活，并且PAK2显性突变体的表达抑制了Nef介导的CD25表达的增强。Nef介导的原代T细胞激活和病毒复制的增强依赖于PAK2和激活刺激的强度，并与Nef与PAK2结合的能力相关。PAK2可能在Nef介导的体内增强病毒复制和免疫激活中发挥作用。
The HIV-1 accessory protein Nef is an important determinant of lentiviral pathogenicity that contributes to disease progression by enhancing viral replication and other poorly understood mechanisms. Nef mediates diverse functions including downmodulation of cell surface CD4 and MHC Class I, enhancement of viral infectivity, and enhancement of T cell activation. Nef interacts with a multiprotein signaling complex that includes Src family kinases, Vav1, CDC42, and activated PAK2 (p21-activated kinase 2). Although previous studies have attempted to identify a biological role for the Nef-PAK2 signaling complex, the importance of this complex and its constituent proteins in Nef function remains unclear. Here, we show that Nef mutants defective for PAK2-association, but functional for CD4 and MHC Class I downmodulation and infectivity enhancement, are also defective for the ability to enhance viral replication in primary T cells that are infected and subsequently activated by sub-maximal stimuli (1 μg/ml PHA-P). In contrast, these Nef mutants had little or no effect on HIV-1 replication in T cells activated by stronger stimuli (2 μg/ml PHA-P or anti-CD3/CD28-coated beads). Viruses bearing wild-type Nefs, but not Nef mutants defective for PAK2 association, enhanced NFAT and IL2 receptor promoter activity in Jurkat cells. Moreover, expression of wild-type Nefs, but not mutant Nefs defective for PAK2 association, was sufficient to enhance responsiveness of primary CD4 and CD8 T cells to activating stimuli in Nef-expressing and bystander cells. siRNA knockdown of PAK2 in Jurkat cells reduced NFAT activation induced by anti-CD3/CD28 stimulation both in the presence and absence of Nef, and expression of a PAK2 dominant mutant inhibited Nef-mediated enhancement of CD25 expression. Nef-mediated enhancement of cellular activation and viral replication in primary T cells is dependent on PAK2 and on the strength of the activating stimuli, and correlates with the ability of Nef to associate with PAK2. PAK2 is likely to play a role in Nef-mediated enhancement of viral replication and immune activation in vivo.
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