Greenway A, Azad A, Mills J, McPhee D
AIDS Cellular Biology Unit, Macfarlane Burnet Centre for Medical Research, Fairfield, Victoria, Australia.
J Virol. 1996 Oct;70(10):6701-8. doi: 10.1128/JVI.70.10.6701-6708.1996.
It is now well established that human immunodeficiency virus type I (HIV-1) Nef contributes substantially to disease pathogenesis by augmenting virus replication and markedly perturbing T-cell function. The effect of Nef on host cell activation could be explained in part by its interaction with specific cellular proteins involved in signal transduction, including at least a member of the src family kinase, Lck, and the serine/threonine kinase, mitogen-activated protein kinase (MAPK). Recombinant Nef directly interacted with purified Lck and MAPK in coprecipitation experiments and binding assays. A proline-rich repeat sequence [(Pxx)4] in Nef occurring between amino acid residues 69 to 78 is highly conserved and bears strong resemblance to a defined consensus sequence identified as an SH3 binding domain present in several proteins which can interact with the SH3 domain of various signalling and cytoskeletal proteins. Binding and coprecipitation assays with short synthetic peptides corresponding to the proline-rich repeat sequence [(Pxx)4] of Nef and the SH2, SH3, or SH2 and SH3 domains of Lck revealed that the interaction between these two proteins is at least in part mediated by the proline repeat sequence of Nef and the SH3 domain of Lck. In addition to direct binding to full-length Nef, MAPK was also shown to bind the same proline repeat motif. Nef protein significantly decreased the in vitro kinase activity of Lck and MAPK. Inhibition of key members of signalling cascades, including those emanating from the T-cell receptor, by the HIV-1 Nef protein undoubtedly alters the ability of the infected T cell to respond to antigens or cytokines, facilitating HIV-1 replication and contributing to HIV-1-induced disease pathogenesis.
现已充分证实，I型人类免疫缺陷病毒（HIV-1）的Nef蛋白通过增强病毒复制和显著扰乱T细胞功能，在疾病发病机制中发挥着重要作用。Nef蛋白对宿主细胞激活的影响，部分可通过其与参与信号转导的特定细胞蛋白的相互作用来解释，这些蛋白至少包括src家族激酶Lck的一个成员以及丝氨酸/苏氨酸激酶——丝裂原活化蛋白激酶（MAPK）。在共沉淀实验和结合试验中，重组Nef蛋白可直接与纯化的Lck和MAPK相互作用。Nef蛋白中位于氨基酸残基69至78之间的富含脯氨酸的重复序列[（Pxx）4]高度保守，与一个确定的共有序列非常相似，该共有序列被鉴定为存在于几种蛋白质中的SH3结合结构域，这些蛋白质可与各种信号和细胞骨架蛋白的SH3结构域相互作用。用对应于Nef蛋白富含脯氨酸重复序列[（Pxx）4]以及Lck的SH2、SH3或SH2和SH3结构域的短合成肽进行的结合和共沉淀试验表明，这两种蛋白之间的相互作用至少部分是由Nef蛋白的脯氨酸重复序列和Lck的SH3结构域介导的。除了直接与全长Nef蛋白结合外，MAPK也被证明可与相同的脯氨酸重复基序结合。Nef蛋白显著降低了Lck和MAPK的体外激酶活性。HIV-1 Nef蛋白对信号级联反应关键成员的抑制，包括那些源自T细胞受体的成员，无疑改变了被感染T细胞对抗原或细胞因子作出反应的能力，从而促进HIV-1复制，并导致HIV-1诱导的疾病发病。
