In vitro activities of purified visna virus integrase.
In vitro activities of purified visna virus integrase.
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纯化的维斯纳病毒整合酶的体外活性。
DOI：
10.1128/jvi.68.6.3558-3569.1994
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发表时间：
1994
影响因子：
5.4
通讯作者：
Sudol,M
中科院分区：
文献类型：
--
作者：
Katzman,M;Sudol,M
虽然整合通常被认为是逆转录病毒生命周期中的关键步骤，但据报道，导致绵羊退行性神经系统疾病的visna病毒可以有效地感染绵羊脉络丛细胞，而不会检测到整合。为了确定visna病毒整合酶（IN）是否是一种固有缺陷酶，并为进一步研究系统发育相关的人类免疫缺陷病毒1型（HIV-1）的整合提供工具，我们利用细菌表达系统纯化了visna病毒整合酶，并应用各种体外寡核苷酸检测方法对该蛋白进行了研究。我们发现visna病毒具有逆转录病毒整合酶的全部体外功能。特别是，visna病毒In表现出位点特异性内切酶活性，在不变的CA从病毒DNA的3'端发现两个核苷酸（加工活性）之后，将加工过的寡核苷酸连接到其他寡核苷酸上的不同位点（链转移或整合活性），并通过分解一个模仿病毒DNA一端整合到宿主DNA的复合体来逆转整合反应（分解活性）。此外，虽然有报道称纯化的HIV-1 In不能特异性地破坏visna病毒的DNA末端，但纯化的visna病毒In确实特异性地加工和整合HIV-1 DNA末端。
Although integration generally is considered a critical step in the retrovirus life cycle, it has been reported that visna virus, which causes degenerative neurologic disease in sheep, can productively infect sheep choroid plexus cells without detectable integration. To ascertain whether the integrase (IN) of visna virus is an inherently defective enzyme and to create tools for further study of integration of the phylogenetically related human immunodeficiency virus type 1 (HIV-1), we purified visna virus IN by using a bacterial expression system and applied various in vitro oligonucleotide-based assays to studying this protein. We found that visna virus IN demonstrates the full repertoire of in vitro functions characteristic of retroviral integrases. In particular, visna virus IN exhibits site-specific endonuclease activity following the invariant CA found two nucleotides from the 3' ends of viral DNA (processing activity), joins processed oligonucleotides to various sites on other oligonucleotides (strand transfer or integration activity), and reverses the integration reaction by resolving a complex that mimics one end of viral DNA integrated into host DNA (disintegration activity). In addition, although it has been reported that purified HIV-1 IN cannot specifically nick visna virus DNA ends, purified visna virus IN does specifically process and integrate HIV-1 DNA ends.