Functional analysis of HIV-1 integrase and attachment site

整合是建立包括人类免疫缺陷病毒1型（HIV-1）在内的逆转录病毒原病毒状态的必要步骤。在这项研究中，整合依赖病毒基因表达的程度和整合酶（In）在体内的可能功能得到了解决。为此，我们通过在HIV-1 IN高度保守的HHCC（锌指结构域）或D，D35E（催化位点）基序中引入单或双氨基酸取代，制造了几个IN突变体。此外，还制备了附着（att）位点突变体，其中In保持完整，但U3或U5端区序列被改变或删除。采用包络假型病毒单步感染系统检测各突变的效果。通过定量PCR方法检测每个新合成病毒DNA的稳定性，估计每个突变体的相对整合效率。催化位点和att位点缺失突变体的效率估计低至WT水平的0.5%。利用高灵敏度荧光素酶测定法测量的每个突变体的基因表达水平与每个整合效率密切相关，表明整合是HIV-1基因表达的必要步骤。同时，我们还发现，所有三个锌指突变体在感染后病毒DNA的重新合成中都存在严重缺陷，这表明这些突变体在逆转录（RT）过程之前或过程中都存在缺陷。最后，at位点的突变分析表明，末端11bp是特异性IN相互作用所需的最小顺式元件，并且IN独立识别每个att位点，这表明IN多聚化对于体内保守整合的重要性。

Integration is an essential step to establish the proviral state of retroviruses including human immunodeficiency virus type 1 (HIV-1). In this study, the extent of integration dependency for viral gene expression and possible function (s) of the integrase (IN) of HIV-1 in vivo were addressed. Towards these ends, we made several IN mutants by introducing single or double amino acid substitution into the highly conserved HHCC (zinc-finger domain) or D,D35E (catalytic site) motif of HIV-1 IN.In addition, the attachment (att) site mutants were also made, in which the IN was kept intact but the sequences of the U3 or U5 teminal region was altered or deleted. The effect of each mutation was examined by using single-step infection system with emvelope psuedotype virus. The relative integration efficiency of each mutant was estimated by examining the stability of each de novo synthesized viral DNA with quantitative PCR method. The efficiency of the catalytic site and the att site deletion mutants were estimated to be as low as 0.5 % of WT level. The gene expression level of each mutant measured by using highly sensitive luciferase assay was shown to well correlate with each integration efficiency, demonstrating that integration is obligate step for HIV-1 gene expression. Meanwhile, we also found that all the three zinc-finger mutants examined here, were sevearly defective in the de novo synthesis of viral DNA after the infection, suggesting impairment of these mutants before or in the reverse transcription (RT) process. Finally from mutational analysis of att site indicated that terminal 11 bp is minimum cis element required for spesific IN interaction, and that IN recognize each att site indipendently, suggesting importance of IN multimerization for conserted integration in vivo.