Leavitt A D, Robles G, Alesandro N, Varmus H E
Department of Laboratory Medicine, University of California, San Francisco 94143-0100, USA.
J Virol. 1996 Feb;70(2):721-8. doi: 10.1128/JVI.70.2.721-728.1996.
The viral integrase (IN) protein is the only viral protein known to be required for integration of the human immunodeficiency virus type 1 (HIV-1) genome into the host cell DNA, a step in the viral life cycle that is essential for viral replication. To better understand the relationship between in vitro IN activity and IN-mediated integration of viral DNA in an infected cell, we characterized the effects of 13 IN mutations on viral replication in cultured cells. Using HIV-1 genomes that express the hygromycin resistance gene and do not express the HIV-1 env gene, we generated stocks of pseudotype virus coated with the murine leukemia virus amphotropic envelope glycoprotein, containing either wild-type or mutant HIV-1 IN. All mutants produced normal amounts of physical particles, as measured by reverse transcriptase activity and capsid protein (p24) concentration, but they formed three groups based on infectious titer and synthesis of viral DNA. Changes at the three highly conserved acidic residues in the IN core domain (D-64, D-116, and E-152) impair provirus formation without affecting viral DNA synthesis or the accumulation of viral DNA in the nucleus of the infected cell, a phenotype predicted by each mutant's lack of in vitro integrase activity. Mutations at positions N-120, R-199, and W-235 minimally affect in vitro integrase activity, but infectious titers are severely reduced, despite normal synthesis of viral DNA, implying a defect during integration in vivo. Mutations in the zinc binding region (H12C, H16V, and H16C), S81R, and a deletion of residues 32 through 275 yield noninfectious particles that synthesize little or no viral DNA following infection, despite wild-type levels of reverse transcriptase activity and viral RNA in the particles. The two latter classes of mutants suggest that IN can affect DNA synthesis or integration during infection in ways that are not appreciated from currently used assays in vitro.
病毒整合酶（IN）蛋白是已知的唯一一种对于将1型人类免疫缺陷病毒（HIV-1）基因组整合到宿主细胞DNA中必不可少的病毒蛋白，这一步骤在病毒生命周期中对于病毒复制至关重要。为了更好地理解体外IN活性与受感染细胞中IN介导的病毒DNA整合之间的关系，我们对13种IN突变对培养细胞中病毒复制的影响进行了表征。使用表达潮霉素抗性基因且不表达HIV-1 env基因的HIV-1基因组，我们制备了包被有鼠白血病病毒嗜异性包膜糖蛋白的假型病毒株，其含有野生型或突变型HIV-1 IN。通过逆转录酶活性和衣壳蛋白（p24）浓度测定，所有突变体产生的物理颗粒数量正常，但根据感染滴度和病毒DNA的合成情况，它们形成了三组。IN核心结构域中三个高度保守的酸性残基（D-64、D-116和E-152）发生变化会损害前病毒的形成，而不影响病毒DNA合成或受感染细胞核中病毒DNA的积累，这一表型由每个突变体缺乏体外整合酶活性所预测。N-120、R-199和W-235位点的突变对体外整合酶活性影响最小，但尽管病毒DNA正常合成，感染滴度却严重降低，这意味着体内整合过程存在缺陷。锌结合区域（H12C、H16V和H16C）、S81R的突变以及32至275位残基的缺失产生了无感染性的颗粒，尽管颗粒中逆转录酶活性和病毒RNA水平为野生型，但感染后合成的病毒DNA很少或没有。后两类突变体表明，IN可以以目前体外检测方法未认识到的方式影响感染期间的DNA合成或整合。
