Department of Structural Biology, Division of Infectious Diseases, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
Retrovirology. 2010 May 7;7:40. doi: 10.1186/1742-4690-7-40.
Lemay et al recently reported that the RNA binding protein HuR directly interacts with the ribonuclease H (RNase H) domain of HIV-1 reverse transcriptase (RT) and influences the efficiency of viral reverse transcription (Lemay et al., 2008, Retrovirology 5:47). HuR is a member of the embryonic lethal abnormal vision protein family and contains 3 RNA recognition motifs (RRMs) that bind AU-rich elements (AREs). To define the structural determinants of the HuR-RT interaction and to elucidate the mechanism(s) by which HuR influences HIV-1 reverse transcription activity in vitro, we cloned and purified full-length HuR as well as three additional protein constructs that contained the N-terminal and internal RRMs, the internal and C-terminal RRMs, or the C-terminal RRM only.
All four HuR proteins were purified and characterized by biophysical methods. They are well structured and exist as monomers in solution. No direct protein-protein interaction between HuR and HIV-1 RT was detected using NMR titrations with 15N labeled HuR variants or the 15N labeled RNase H domain of HIV-1 RT. Furthermore, HuR did not significantly affect the kinetics of HIV-1 reverse transcription in vitro, even on RNA templates that contain AREs.
Our results suggest that HuR does not impact HIV-1 replication through a direct protein-protein interaction with the viral RT.
Lemay 等人最近报道，RNA 结合蛋白 HuR 可直接与 HIV-1 逆转录酶 (RT) 的核糖核酸酶 H (RNase H) 结构域相互作用，并影响病毒逆转录的效率 (Lemay 等人，2008 年，Retrovirology 5:47)。HuR 是胚胎致死异常视觉蛋白家族的成员，包含 3 个结合 AU 富含元件 (AREs) 的 RNA 识别基序 (RRMs)。为了确定 HuR-RT 相互作用的结构决定因素，并阐明 HuR 体外影响 HIV-1 逆转录活性的机制，我们克隆并纯化了全长 HuR 以及另外三个包含 N 端和内部 RRMs、内部和 C 端 RRMs 或仅 C 端 RRM 的蛋白构建体。
所有四种 HuR 蛋白均通过生物物理方法进行了纯化和表征。它们结构良好，在溶液中以单体形式存在。使用 NMR 滴定法，用 15N 标记的 HuR 变体或 HIV-1 RT 的 15N 标记 RNase H 结构域，均未检测到 HuR 与 HIV-1 RT 之间的直接蛋白-蛋白相互作用。此外，HuR 即使在含有 AREs 的 RNA 模板上，也不会显著影响 HIV-1 体外逆转录的动力学。
我们的结果表明，HuR 不会通过与病毒 RT 的直接蛋白-蛋白相互作用影响 HIV-1 复制。
