Gaur M, Leavitt A D
Departments of Laboratory Medicine, University of California, San Francisco, California 94143-0100, USA.
J Virol. 1998 Jun;72(6):4678-85. doi: 10.1128/JVI.72.6.4678-4685.1998.
The core domain of human immunodeficiency virus type 1 (HIV-1) integrase (IN) contains a D,D(35)E motif, named for the phylogenetically conserved glutamic acid and aspartic acid residues and the invariant 35 amino acid spacing between the second and third acidic residues. Each acidic residue of the D,D(35)E motif is independently essential for the 3'-processing and strand transfer activities of purified HIV-1 IN protein. Using a replication-defective viral genome with a hygromycin selectable marker, we recently reported that a mutation at any of the three residues of the D,D(35)E motif produces a 10(3)- to 10(4)-fold reduction in infectious titer compared with virus encoding wild-type IN (A. D. Leavitt et al., J. Virol. 70:721-728. 1996). The infectious titer, as measured by the number of hygromycin-resistant colonies formed following infection of cells in culture, was less than a few hundred colonies per microg of p24. To understand the mechanism by which the mutant virions conferred hygromycin resistance, we characterized the integrated viral DNA in cells infected with virus encoding mutations at each of the three residues of the D,D(35)E motif. We found the integrated viral DNA to be colinear with the incoming viral genome. DNA sequencing of the junctions between integrated viral DNA and host DNA showed that (i) the characteristic 5-bp direct repeat of host DNA flanking the HIV-1 provirus was not maintained, (ii) integration often produced a deletion of host DNA, (iii) integration sometimes occurred without the viral DNA first undergoing 3'-processing, (iv) integration sites showed a strong bias for a G residue immediately adjacent to the conserved viral CA dinucleotide, and (v) mutations at each of the residues of the D,D(35)E motif produced essentially identical phenotypes. We conclude that mutations at any of the three acidic residues of the conserved D,D(35)E motif so severely impair IN activity that most, if not all, integration events by virus encoding such mutations are not IN mediated. IN-independent provirus formation may have implications for anti-IN therapeutic agents that target the IN active site.
1型人类免疫缺陷病毒（HIV-1）整合酶（IN）的核心结构域含有一个D,D(35)E基序，因其在系统发育上保守的谷氨酸和天冬氨酸残基以及第二个和第三个酸性残基之间不变的35个氨基酸间距而得名。D,D(35)E基序的每个酸性残基对于纯化的HIV-1 IN蛋白的3'加工和链转移活性都是独立必需的。使用带有潮霉素选择标记的复制缺陷型病毒基因组，我们最近报道，与编码野生型IN的病毒相比，D,D(35)E基序的三个残基中任何一个发生突变都会使感染性滴度降低10³至10⁴倍（A.D. Leavitt等人，《病毒学杂志》70:721 - 728, 1996）。通过培养细胞感染后形成的潮霉素抗性菌落数量来测量，感染性滴度低于每微克p24几百个菌落。为了了解突变病毒粒子赋予潮霉素抗性的机制，我们对感染了编码D,D(35)E基序三个残基中每个残基突变的病毒的细胞中的整合病毒DNA进行了表征。我们发现整合的病毒DNA与进入的病毒基因组共线性。对整合病毒DNA与宿主DNA之间连接点的DNA测序表明：（i）HIV-1前病毒两侧宿主DNA的特征性5碱基对直接重复序列未得到保留；（ii）整合常常导致宿主DNA缺失；（iii）整合有时在病毒DNA未先进行3'加工的情况下发生；（iv）整合位点对紧邻保守的病毒CA二核苷酸的G残基有强烈偏好；（v）D,D(35)E基序的每个残基发生突变产生的表型基本相同。我们得出结论，保守的D,D(35)E基序的三个酸性残基中任何一个发生突变都会严重损害IN活性，以至于编码此类突变的病毒的大多数（如果不是全部）整合事件都不是由IN介导的。不依赖IN的前病毒形成可能对靶向IN活性位点的抗IN治疗药物有影响。
