DNA requirements for assembly and stability of HIV-1 intasomes.
DNA requirements for assembly and stability of HIV-1 intasomes.
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HIV-1 嵌体的组装和稳定性的 DNA 要求。
DOI：
10.1002/pro.2010
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
Craigie,Robert
中科院分区：
文献类型：
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作者：
Li,Min;Ivanov,Vassili;Mizuuchi,Michiyo;Mizuuchi,Kiyoshi;Craigie,Robert
病毒DNA整合到宿主基因组中是逆转录病毒复制的重要步骤，由稳定的核蛋白复合物介导，该复合物包含整合酶四聚体，在稳定的突触复合物（SSC）或整合体中桥接病毒DNA的两端。除了在足迹实验中受到保护的末端病毒 DNA 序列之外，HIV-1 嵌体的组装还需要数百个碱基对的非特异性内部 DNA。我们发现，嵌体中只有一个病毒 DNA 末端需要长非特异性内部 DNA 来进行嵌体组装。尽管当非特异性内部DNA在组装后被切断时，整合体在溶液中不稳定，但它们在琼脂糖凝胶中是稳定的。在 Förster 共振能量转移 (FRET) 实验中，这些复合物与具有非特异性内部 DNA 的 SSC 无法区分，这表明无论是否存在非特异性内部 DNA，与病毒 DNA 和整合酶四聚体的相互作用都是相同的。我们讨论了内部 DNA 如何促进整合体组装和稳定性的模型。 FRET 对荧光团之间的距离非常敏感，并且给定某些假设可以转化为距离测量。我们预计一组这样的距离约束将提供嵌体内 DNA 路径的图谱。事实上，我们可以从 FRET 数据中施加的约束非常弱，从而为可能的路径提供了较宽的范围。我们讨论了将 FRET 信号转换为核蛋白复合物内绝对距离的困难。
Integration of viral DNA into the host genome is an essential step in retroviral replication that is mediated by a stable nucleoprotein complex comprising a tetramer of integrase bridging the two ends of the viral DNA in a stable synaptic complex (SSC) or intasome. Assembly of HIV‐1 intasomes requires several hundred base pairs of nonspecific internal DNA in addition to the terminal viral DNA sequence that is protected in footprinting experiments. We find that only one of the viral DNA ends in the intasome requires long‐nonspecific internal DNA for intasome assembly. Although intasomes are unstable in solution when the nonspecific internal DNA is cut off after assembly, they are stable in agarose gels. These complexes are indistinguishable from SSCs with nonspecific internal DNA in Förster resonance energy transfer (FRET) experiments suggesting the interactions with the viral DNA and integrase tetramer are the same regardless of the presence of nonspecific internal DNA. We discuss models of how the internal DNA contributes to intasome assembly and stability. FRET is exquisitely sensitive to the distance between the fluorophores and given certain assumptions can be translated to distance measurements. We anticipated that a set of such distance constraints would provide a map of the DNA path within the intasome. In reality, the constraints we could impose from the FRET data were quite weak allowing a wide envelope for the possible path. We discuss the difficulties of converting the FRET signal to absolute distance within nucleoprotein complexes.