Structure-based mutagenesis of the human immunodeficiency virus type 1 DNA attachment site: Effects on integration and cDNA synthesis
Structure-based mutagenesis of the human immunodeficiency virus type 1 DNA attachment site: Effects on integration and cDNA synthesis
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DOI：
10.1128/jvi.73.11.9011-9020.1999
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发表时间：
1999-11-01
影响因子：
5.4
通讯作者：
Engelman, A
中科院分区：
文献类型：
--
作者：
Brown, HEV;Chen, HM;Engelman, A
线性逆转录病毒cDNA末端的序列对整合很重要，它们定义了病毒DNA附着（aft）位点。而人类免疫缺陷病毒1型（HIV-1）的整合酶的复制在T淋巴细胞中的重要的决定因素已被广泛的特点，ATT网站的病毒传播的重要区域还没有得到彻底的检查。以前的转座子介导的足迹分离从感染细胞的整合前复合物显示增强的区域噬菌体Mu插入HIV-1的cDNA的末端附近，在该地区的att网站，在这里，我们确定了亚末端的cDNA序列切割在体外足迹和使用这种基于结构的信息与以前的工作结果一起构建和表征24个att网站突变病毒。我们发现，虽然亚末端的cDNA序列有助于HIV-1的复制，这些基地的身份并不重要的整合。相比之下，位于HIV-1末端的系统发育保守的CA二核苷酸对病毒复制和整合做出了显着贡献。与野生型相比，含有一个完整CA末端的突变体显示病毒生长峰值延迟。相比之下，缺乏两个CA的双突变病毒是复制缺陷型的。CA的A似乎是整合的最关键决定因素，因为含有TG取代CA的两种不同的U 5突变病毒通过将G变回A而部分回复。我们还鉴定了一个U 5缺失突变体，其中CA在逆转录中起着至关重要的作用。
Sequences at the ends of linear retroviral cDNA important for integration define the viral DNA attachment (aft) site. Whereas determinants of human immunodeficiency virus type 1 (HIV-1) integrase important for replication in T lymphocytes have been extensively characterized, regions of the att site important for viral spread have not been thoroughly examined. Previous transposon-mediated footprinting of preintegration complexes isolated from infected cells revealed enhanced regions of bacteriophage Mu insertion near the ends of HIV-1 cDNA, in the regions of the att sites, Here, we identified the subterminal cDNA sequences cleaved during in vitro footprinting and used this structure-based information together with results of previous work to construct and characterize 24 att site mutant viruses. We found that although subterminal cDNA sequences contributed to HIV-1 replication, the identities of these bases were not critical for integration. In contrast, the phylogenetically conserved CA dinucleotides located at the ends of HIV-1 contributed significantly to virus replication and integration. Mutants containing one intact CA end displayed delays in peak virus growth compared to the wild type. In contrast, double mutant viruses lacking both CAs were replication defective, The A of the CA appeared to be the most critical determinant of integration, because two different U5 mutant viruses containing the substitution of TG for CA partially reverted by changing the G back to A. We also identified a U5 deletion mutant in which the CA played a crucial role in reverse transcription.