Masuda T, Planelles V, Krogstad P, Chen I S
Department of Microbiology & Immunology, UCLA School of Medicine 90095, USA.
J Virol. 1995 Nov;69(11):6687-96. doi: 10.1128/JVI.69.11.6687-6696.1995.
Retroviral integration is the step which leads to establishment of the provirus, cis- and trans-acting regions of the human immunodeficiency type 1 (HIV-1) retrovirus genome, including the attachment site (att) at the ends of the unintegrated viral DNA and the conserved domains within the integrase (IN) protein, have been identified as being important for integration. We investigated the role of each of these regions in the context of an infectious HIV-1 molecular clone through point mutagenesis of the att site and the zinc finger-like and catalytic domains of IN. The effect of each mutation on integration activity was examined by using a single-step infection system with envelope-pseudotype virus. The relative integration efficiency was estimated by monitoring the levels of viral DNA over time in the infected cells. The integration activities of catalytic domain point mutants and att site deletion mutants were estimated to be 0.5 and 5% of wild-type activity, respectively. However, in contrast with previous in vitro cell-free integration studies, alteration of the highly conserved CA dinucleotide resulted in a mutant which still retained 40% of wild-type integration activity. The relative levels of expression of each mutant, as measured by a luciferase reporter gene, correlated with levels of integration. This observation is consistent with those of previous studies indicating that integration is an obligatory step for retroviral gene expression. Interestingly, we found that three different HIV-1 constructs bearing point mutations in the zinc finger-like domain synthesized much lower levels of viral DNA after infection, suggesting impairment of these mutants before or at the initiation of reverse transcription. Western blot (immunoblot) analysis demonstrated wild-type levels of reverse transcriptase within the mutant virions. In vitro endogenous reverse transcription assays indicated that all three mutants in the zinc finger-like domain had wild-type levels of reverse transcriptase activity. These data indicate that in addition to integration, IN may have an effect on the proper course of events in the viral life cycle that precede integration.
逆转录病毒整合是导致前病毒建立的步骤，人类免疫缺陷病毒1型（HIV-1）逆转录病毒基因组的顺式和反式作用区域，包括未整合病毒DNA末端的附着位点（att）和整合酶（IN）蛋白内的保守结构域，已被确定对整合很重要。我们通过对att位点以及IN的锌指样结构域和催化结构域进行点突变，在具有感染性的HIV-1分子克隆背景下研究了这些区域各自的作用。通过使用包膜假型病毒的单步感染系统检查每个突变对整合活性的影响。通过监测感染细胞中病毒DNA随时间的水平来估计相对整合效率。催化结构域点突变体和att位点缺失突变体的整合活性估计分别为野生型活性的0.5%和5%。然而，与先前的体外无细胞整合研究相反，高度保守的CA二核苷酸的改变产生了一个仍保留40%野生型整合活性的突变体。通过荧光素酶报告基因测量的每个突变体的相对表达水平与整合水平相关。这一观察结果与先前的研究一致，表明整合是逆转录病毒基因表达的一个必要步骤。有趣的是，我们发现三个在锌指样结构域带有点突变的不同HIV-1构建体在感染后合成的病毒DNA水平要低得多，这表明这些突变体在逆转录开始之前或之时就受到了损害。蛋白质免疫印迹分析显示突变病毒粒子中逆转录酶的水平与野生型相当。体外内源性逆转录试验表明，锌指样结构域中的所有三个突变体都具有野生型水平的逆转录酶活性。这些数据表明，除了整合之外，IN可能还会对病毒生命周期中整合之前的正常事件进程产生影响。
