Two amino acid substitutions in the SIV Nef protein mediate associations with distinct cellular kinases.
Two amino acid substitutions in the SIV Nef protein mediate associations with distinct cellular kinases.
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SIV Nef 蛋白中的两个氨基酸取代介导与不同细胞激酶的关联。
DOI：
10.1006/viro.2000.0558
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发表时间：
2000
期刊：
影响因子：
3.7
通讯作者：
Clements,JE
中科院分区：
文献类型：
--
作者：
Barber,SA;Maughan,MF;Roos,JW;Clements,JE
功能性 Nef 蛋白对于体内病毒复制至关重要，从而导致 SIV 感染的猕猴发病。此外，全长 Nef 蛋白是原代细胞中病毒最佳复制所必需的，HIV 和 SIV Nef 蛋白都增强病毒粒子的感染性。感染性增强的部分原因可能是 Nef 将细胞激酶整合到病毒粒子中的能力。在之前的两份报告中，我们比较了 SIV 重组克隆的体外激酶谱，这些重组克隆表达源自原型淋巴细胞 SIVmac239、克隆 SIV/Fr-2 或我们的神经毒力克隆 SIV/17E-Fr 的 nef 基因。虽然 SIV/Fr-2 Nef 蛋白与先前描述的 PAK 相关激酶和 Nef 相关激酶复合物 (NAKC) 中存在的未鉴定的丝氨酸激酶相关，但发现 SIV/17E-Fr Nef 与一种在生化上不同于 PAK 和 NAKC 的新型丝氨酸激酶活性相关。有趣的是，虽然两种 Nef 蛋白都被整合到病毒颗粒中，但仅在含有 SIV/17E-Fr Nef 蛋白的病毒体中检测到 Nef 相关激酶活性。由于序列分析仅鉴定出 SIV/Fr-2 和 SIV/17E-Fr 的 Nef 蛋白之间存在差异的 5 个氨基酸，因此我们能够通过构建一组 SIV 克隆来评估每个氨基酸对 Nef 相关激酶活性以及病毒感染性的贡献，这些克隆包含 SIV/17E-Fr Nef 中每个不同氨基酸与 SIV/Fr-2 中相应氨基酸的单独回复。 内夫。在本报告中，我们鉴定了 N 末端先前未表征的氨基酸和 Nef 的保守核心结构域，这些氨基酸对于检测 Nef/激酶相互作用以及 SIV 感染期间的 Nef 磷酸化至关重要。此外，通过我们实验室最近开发的一种新型感染性测定，该测定利用稳定表达 SIV/LTR-荧光素酶构建体的 CEMX174 报告细胞，我们发现特定 Nef 激酶关联与增强的病毒体感染性之间没有直接相关性。
A functional Nef protein is crucial in vivo for viral replication leading to pathogenesis in SIV-infected macaques. Moreover, a full-length Nef protein is required for optimal virus replication in primary cells, and both HIV and SIV Nef proteins enhance virion infectivity. Enhanced infectivity may result in part from the ability of Nef to incorporate cellular kinases into virions. In two previous reports, we compared in vitro kinase profiles of SIV recombinant clones that express nef genes derived either from the prototypic lymphocyte-tropic SIVmac239, clone SIV/Fr-2, or from our neurovirulent clone SIV/17E-Fr. While the SIV/Fr-2 Nef protein associated with the previously described PAK-related kinase and an unidentified serine kinase present in a Nef-associated kinase complex (NAKC), SIV/17E-Fr Nef was found to associate with a novel serine kinase activity that was biochemically distinct from both PAK and NAKC. Interestingly, while both Nef proteins were incorporated into virus particles, Nef-associated kinase activity was detected only in virions containing the SIV/17E-Fr Nef protein. Because sequence analysis identified only five amino acids that differed between the Nef proteins of SIV/Fr-2 and SIV/17E-Fr, we were able to evaluate the contribution of each amino acid to Nef-associated kinase activity as well as virus infectivity by constructing a panel of SIV clones containing individual reversions of each differing amino acid in SIV/17E-Fr Nef to the corresponding amino acid in SIV/Fr-2 Nef. In this report, we identify previously uncharacterized amino acids in the N terminus and the conserved core domain of Nef that are essential for the detection of Nef/kinase interactions as well as Nef phosphorylation during SIV infection. Further, via a novel infectivity assay recently developed in our laboratory that utilizes CEMX174 reporter cells stably expressing an SIV/LTR-luciferase construct, we find no direct correlation between specific Nef kinase associations and enhanced virion infectivity.