Shun Ming-Chieh, Raghavendra Nidhanapati K, Vandegraaff Nick, Daigle Janet E, Hughes Siobhan, Kellam Paul, Cherepanov Peter, Engelman Alan
Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Division of AIDS, Harvard Medical School, Boston, Massachusetts 02115, USA.
Genes Dev. 2007 Jul 15;21(14):1767-78. doi: 10.1101/gad.1565107.
LEDGF/p75 directly interacts with lentiviral integrase proteins and can modulate their enzymatic activities and chromosomal association. A novel genetic knockout model was established that allowed us for the first time to analyze HIV-1 integration in the absence of LEDGF/p75 protein. Supporting a crucial role for the cofactor in viral replication, HIV-1 vector integration and reporter gene expression were significantly reduced in LEDGF-null cells. Yet, integrase processed the viral cDNA termini normally and maintained its local target DNA sequence preference during integration. Preintegration complexes extracted from knockout cells moreover supported normal levels of DNA strand transfer activity in vitro. In contrast, HIV-1 lost its strong bias toward integrating into transcription units, displaying instead increased affinity for promoter regions and CpG islands. Our results reveal LEDGF/p75 as a critical targeting factor, commandeering lentiviruses from promoter- and/or CpG island-proximal pathways that are favored by other members of Retroviridae. Akin to yeast retrotransposons, disrupting the lentiviral targeting mechanism significantly perturbs overall integration.
LEDGF/p75 直接与慢病毒整合酶蛋白相互作用，并可调节其酶活性和染色体关联。建立了一种新型基因敲除模型，首次使我们能够在不存在 LEDGF/p75 蛋白的情况下分析 HIV-1 整合。支持该辅助因子在病毒复制中起关键作用的是，在 LEDGF 缺失的细胞中，HIV-1 载体整合和报告基因表达显著降低。然而，整合酶正常处理病毒 cDNA 末端，并在整合过程中保持其对局部靶 DNA 序列的偏好。此外，从敲除细胞中提取的整合前复合物在体外支持正常水平的 DNA 链转移活性。相比之下，HIV-1 失去了对整合到转录单元的强烈偏好，转而对启动子区域和 CpG 岛表现出更高的亲和力。我们的结果表明，LEDGF/p75 是一种关键的靶向因子，可将慢病毒从逆转录病毒科其他成员所青睐的启动子和/或 CpG 岛近端途径中引导出来。与酵母逆转座子类似，破坏慢病毒靶向机制会显著扰乱整体整合。
