Moreau Karen, Faure Claudine, Violot Sébastien, Verdier Gérard, Ronfort Corinne
Université Claude Bernard, Centre National de la Recherche Scientifique, Institut National de la Recherche Agronomique, Lyon, France.
Eur J Biochem. 2003 Nov;270(22):4426-38. doi: 10.1046/j.1432-1033.2003.03833.x.
Integrase (IN) is the retroviral enzyme responsible for the integration of the DNA copy of the retroviral genome into the host cell DNA. The C-terminal domain of IN is involved in DNA binding and enzyme multimerization. We previously performed single amino acid substitutions in the C-terminal domain of the avian leukemia and sarcoma viruses (ALSV) IN. Here, we modelled these IN mutants and analysed their ability to mediate concerted DNA integration (in an in vitro assay) as well as to form dimers (by size exclusion chromatography and protein-protein cross-linking). Mutations of residues located at the dimer interface (V239, L240, Y246, V257 and K266) have the greatest effects on the activity of the IN. Among them: (a) the L240A mutation resulted in a decrease of integration efficiency that was concomitant with a decrease of IN dimerization; (b) the V239A, V249A and K266A mutants preferentially mediated non-concerted DNA integration rather than concerted DNA integration although they were found as dimers. Other mutations (V260E and Y246W/DeltaC25) highlight the role of the C-terminal domain in the general folding of the enzyme and, hence, on its activity. This study points to the important role of residues at the IN C-terminal domain in the folding and dimerization of the enzyme as well as in the concerted DNA integration of viral DNA ends.
整合酶（IN）是一种逆转录病毒酶，负责将逆转录病毒基因组的DNA拷贝整合到宿主细胞DNA中。IN的C末端结构域参与DNA结合和酶的多聚化。我们之前在禽白血病和肉瘤病毒（ALSV）IN的C末端结构域中进行了单氨基酸替换。在此，我们对这些IN突变体进行建模，并分析它们介导协同DNA整合的能力（通过体外测定）以及形成二聚体的能力（通过尺寸排阻色谱法和蛋白质-蛋白质交联）。位于二聚体界面的残基（V239、L240、Y246、V257和K266）的突变对IN的活性影响最大。其中：（a）L240A突变导致整合效率降低，同时IN二聚化减少；（b）V239A、V249A和K266A突变体优先介导非协同DNA整合而非协同DNA整合，尽管它们以二聚体形式存在。其他突变（V260E和Y246W/ΔC25）突出了C末端结构域在酶的整体折叠中的作用，进而影响其活性。这项研究指出了IN C末端结构域中的残基在酶的折叠、二聚化以及病毒DNA末端的协同DNA整合中的重要作用。
