Villanueva Rodrigo A, Jonsson Colleen B, Jones Jennifer, Georgiadis Millie M, Roth Monica J
Department of Biochemistry, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, 675 Hoes Lane, Piscataway, NJ 08854, USA.
Virology. 2003 Nov 10;316(1):146-60. doi: 10.1016/s0042-6822(03)00559-2.
Retroviral integrases (IN) catalyze the integration of the reverse-transcribed viral DNA into the host genome, an essential process leading to virus replication. For Moloney murine leukemia virus (M-MuLV) IN, the limited solubility of the recombinant protein has restricted the development of biophysical and structural analyses. Herein, recombinant M-MuLV IN proteins, either full length or two nonoverlapping domain constructs, were purified under non-denaturing conditions from solubilized bacterial extracts by Ni(2+)-NTA resins. Additionally, WT IN was further purified by heparin chromatography. All of the purified proteins were shown to be active and stable. WT M-MuLV IN chromatographed with a peak corresponding with a dimer by gel filtration chromatography. In contrast, the single point mutant C209A IN migrated predominantly as a tetramer. For both proteins, fractions in equilibrium between dimers and tetramers were competent to assemble concerted two-end integrations and yielded a unique strand-transfer profile in the presence of a 28-mer U5 oligonucleotide substrate, indicative of a distinct conformation within the synaptic complex. This specific target-site selection was not observed with a shorter 20-mer U5 substrate. These studies provide the foundation for biophysical and structural analysis on M-MuLV IN and the mechanism of retroviral integration.
逆转录病毒整合酶（IN）催化逆转录病毒DNA整合到宿主基因组中，这是病毒复制的一个关键过程。对于莫洛尼鼠白血病病毒（M-MuLV）整合酶而言，重组蛋白的低溶解度限制了生物物理和结构分析的进展。在此，全长或两个不重叠结构域构建体的重组M-MuLV整合酶蛋白在非变性条件下，通过Ni(2+)-NTA树脂从可溶的细菌提取物中纯化得到。此外，野生型（WT）整合酶通过肝素柱层析进一步纯化。所有纯化的蛋白均表现出活性且稳定。野生型M-MuLV整合酶在凝胶过滤层析中呈现出与二聚体相对应的峰。相比之下，单点突变体C209A整合酶主要以四聚体形式迁移。对于这两种蛋白，处于二聚体和四聚体平衡状态的组分能够进行协同的两端整合，并且在存在28聚体U5寡核苷酸底物时产生独特的链转移图谱，这表明突触复合物内存在独特的构象。使用较短的20聚体U5底物时未观察到这种特定的靶位点选择。这些研究为M-MuLV整合酶的生物物理和结构分析以及逆转录病毒整合机制奠定了基础。
