Efficiency and fidelity of full-site integration reactions using recombinant simian immunodeficiency virus integrase
Efficiency and fidelity of full-site integration reactions using recombinant simian immunodeficiency virus integrase
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DOI：
10.1128/jvi.73.10.8104-8111.1999
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发表时间：
1999-10-01
影响因子：
5.4
通讯作者：
Grandgenett, D
中科院分区：
文献类型：
--
作者：
Goodarzi, G;Pursley, M;Grandgenett, D
以线形逆转录病毒样DNA(469bp)为供体底物，以环状DNA(2867bp)为靶底物，研究了野生型和突变型重组猴免疫缺陷病毒(SN)整合酶(IN)的全位整合。在优化的反应条件下，重组SIV IN以相同的频率产生与全位(两个供体端)和半位(一个供体端)反应一致的供体-靶产物。对3.8-kBP全部位反应产物的限制性内切酶分析证实，来自不同供体的两个末端协同插入到单个靶分子中。携带病毒U5末端的供体末端比U3末端更适合生产半部位和全部位产物。用细菌遗传选择法分离单个供体-靶向重组子，并对克隆产物的供体-靶向连接进行测序。对149个重组子的分析表明，适当的猴逆转录病毒5-bp宿主复制的保真度约为84%。如先前从裂解的病毒粒子与人类免疫缺陷病毒1型(HIV-1)IN的类似反应中看到的那样，用重组SN IN产生的供体靶向重组体中约有8%发生了特定的17-18或27-29个核苷酸的缺失。由纯化的重组SIV LN介导的全部位整合反应的效率和保真度与来自病毒粒子的HIV-1 IN相当。这些观察表明，纯化的重组慢病毒IN本身就足以概括全部位整合过程。
Full-site integration by recombinant wild-type and mutant simian immunodeficiency virus (SN) integrase (IN) was investigated with linear retrovirus-like DNA (469 bp) as a donor substrate and circular DNA (2,867 bp) as a target substrate. Under optimized conditions, recombinant SIV IN produced donor-target products consistent with full-site (two donor ends) and half-site (one donor end) reactions with equivalent frequency. Restriction enzyme analysis of the 3.8-kbp full-site reaction products confirmed the concerted insertion of two termini from separate donors into a single target molecule. Donor ends carrying the viral U5 termini were preferred over U3 termini for producing both half-site and full-site products. Bacterial genetic selection was used to isolate individual donor-target recombinants, and the donor-target junctions of the cloned products were characterized by sequencing. Analysis of 149 recombinants demonstrated approximately 84% fidelity for the appropriate simian retrovirus 5-bp host duplication. As seen previously in similar reactions with human immunodeficiency virus type 1 (HIV-1) IN from lysed virions, approximately 8% of the donor-target recombinants generated with recombinant SN IN incurred specific 17- to 18- or 27- to 29-bp deletions. The efficiency and fidelity of the full-site integration reaction mediated by the purified, recombinant SIV LN is comparable to that of HIV-1 IN from virions. These observations suggest that a purified recombinant lentivirus IN is itself sufficient to recapitulate the full-site integration process.