A novel co-crystal structure affords the design of gain-of-function lentiviral integrase mutants in the presence of modified PSIP1/LEDGF/p75.
A novel co-crystal structure affords the design of gain-of-function lentiviral integrase mutants in the presence of modified PSIP1/LEDGF/p75.
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新型的共晶结构为在经过改进的PSIP1/LEDGF/P75的存在下提供了功能获得的慢病毒积分突变体的设计。
DOI：
10.1371/journal.ppat.1000259
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发表时间：
2009-01
期刊：
影响因子：
6.7
通讯作者：
Cherepanov P
中科院分区：
文献类型：
--
作者：
Hare S;Shun MC;Gupta SS;Valkov E;Engelman A;Cherepanov P
透镜上皮衍生生长因子（LEDGF），也称为PC 4和SFRS 1相互作用蛋白1（PSIP 1）和转录共激活因子p75，是慢病毒整合酶（IN）蛋白的细胞结合伴侣。LEDGF解释了慢病毒整合在活性转录单位内的特征倾向，并且是有效病毒复制所需的。我们现在提出了一个晶体结构，其中包含N-末端和催化核心结构域（NTD和CCD）的HIV-2 IN与IN结合结构域（IBD）的LEDGF的复合物。该结构扩展了已知的IN-LEDGF界面，主要阐明了IN的NTD和IBD之间的电荷-电荷相互作用。NTD上的一系列酸性残基是慢病毒IN的特征，IBD上带正电荷残基的突变严重影响与所有检测的慢病毒IN的相互作用。我们表明，新的NTD-IBD接触是关键的刺激协同慢病毒DNA整合LEDGF在体外和其功能在HIV-1复制的早期步骤。此外，新的结构细节使我们能够设计出HIV-1 IN的突变体，该突变体仅在与互补的LEDGF突变体一起出现时才主要起作用。这些发现为慢病毒高亲和力IN-LEDGF相互作用提供了结构基础，并为开发基于LEDGF的基因治疗靶向技术铺平了道路。逆转录病毒关键依赖于将其基因组插入宿主细胞染色体中，并且该过程由病毒酶整合酶进行。HIV和其他慢病毒也依赖于LEDGF，这是一种细胞染色质相关蛋白，它结合它们的整合酶蛋白并将它们束缚在人类染色体上。整合酶和LEDGF之间的相互作用可以潜在地用于在基因治疗应用中指导慢病毒载体的整合，以及用于开发抗逆转录病毒药物。在这里，我们提出了一个三维结构的蛋白质-蛋白质复合物含有一个片段的HIV整合酶和整合酶结合结构域的LEDGF。我们的结构阐明了迄今未知的LEDGF整合酶接口涉及的病毒酶的氨基末端部分。使用一系列互补的方法，我们进一步表明，这些新的蛋白质-蛋白质接触是必不可少的LEDGF在HIV整合的功能。新的结构细节将是非常有用的HIV抑制剂的目标整合酶-LEDGF相互作用的发展。此外，他们使我们能够设计一种依赖于LEDGF反向工程突变体的HIV整合酶突变体，为基于LEDGF的慢病毒载体靶向策略的设计提供了一种方法。
Lens epithelium derived growth factor (LEDGF), also known as PC4 and SFRS1 interacting protein 1 (PSIP1) and transcriptional co-activator p75, is the cellular binding partner of lentiviral integrase (IN) proteins. LEDGF accounts for the characteristic propensity of Lentivirus to integrate within active transcription units and is required for efficient viral replication. We now present a crystal structure containing the N-terminal and catalytic core domains (NTD and CCD) of HIV-2 IN in complex with the IN binding domain (IBD) of LEDGF. The structure extends the known IN–LEDGF interface, elucidating primarily charge–charge interactions between the NTD of IN and the IBD. A constellation of acidic residues on the NTD is characteristic of lentiviral INs, and mutations of the positively charged residues on the IBD severely affect interaction with all lentiviral INs tested. We show that the novel NTD–IBD contacts are critical for stimulation of concerted lentiviral DNA integration by LEDGF in vitro and for its function during the early steps of HIV-1 replication. Furthermore, the new structural details enabled us to engineer a mutant of HIV-1 IN that primarily functions only when presented with a complementary LEDGF mutant. These findings provide structural basis for the high affinity lentiviral IN–LEDGF interaction and pave the way for development of LEDGF-based targeting technologies for gene therapy. Retroviruses crucially rely on insertion of their genomes into a host cell chromosome, and this process is carried out by the viral enzyme integrase. HIV and other lentiviruses also depend on LEDGF, a cellular chromatin-associated protein, which binds their integrase proteins and tethers them to a human chromosome. The interaction between integrase and LEDGF can potentially be exploited for directing integration of lentiviral vectors in gene therapy applications, as well as for development of antiretroviral drugs. Herein, we present a three-dimensional structure of a protein–protein complex containing a fragment of HIV integrase and the integrase-binding domain of LEDGF. Our structure elucidates the hitherto unknown LEDGF–integrase interface involving the amino terminal portion of the viral enzyme. Using a range of complementary approaches, we further show that these novel protein–protein contacts are essential for the function of LEDGF in HIV integration. The novel structural details will be very useful for the development of HIV inhibitors that target the integrase–LEDGF interaction. Furthermore, they enabled us to design a mutant of HIV integrase that depends on a reverse-engineered mutant of LEDGF, providing an inroad to the design of LEDGF-based lentiviral vector targeting strategies.
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影响因子：
4.8
作者：
Cherepanov, P;Maertens, G;Debyser, Z
通讯作者：
Debyser, Z
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
DOI：
10.1038/nsb0995-807
发表时间：
1995-09-01
期刊：
NATURE STRUCTURAL BIOLOGY
影响因子：
--
作者：
EIJKELENBOOM, APAM;LUTZKE, RAP;HARD, K
通讯作者：
HARD, K
影响因子：
5.4
作者：
ENGELMAN, A;CRAIGIE, R
通讯作者：
CRAIGIE, R
DOI：
10.1073/pnas.0506924102
发表时间：
2005-11-29
影响因子：
11.1
作者：
Cherepanov, P;Ambrosio, ALB;Engelman, A
通讯作者：
Engelman, A