Tramontano Enzo, Onidi Loredana, Esposito Francesca, Badas Roberta, La Colla Paolo
Department of Sciences and Biomedical Technologies, University of Cagliari, Cittadella Universitaria SS554, 09142 Monserrato, Cagliari, Italy.
Biochem Pharmacol. 2004 May 1;67(9):1751-61. doi: 10.1016/j.bcp.2004.01.015.
Human Immunodeficiency Virus type 1 (HIV-1) integrase (IN) is an attractive target for the development of new antiviral therapies. Recently, several HIV-1 recombinant IN (rIN) in vitro inhibitors have been described. However, the great majority of them failed to block the virus replication in cell-based assays, suggesting the inadequacy of the in vitro assay systems used for inhibitor screening. To improve these systems, we designed a 40(mer) duplex DNA reaction substrate consisting of recognition sequences from both U3 and U5 HIV-1 long terminal repeat (LTR) termini. The HIV-1 rIN was able to catalyze its enzyme activities recognizing both ends of the 40(mer) dsDNA. Using this substrate we assayed the effects on rIN catalysis of different classes of compounds which inhibit the HIV-1 rIN in vitro when the reaction substrate is the standard 21(mer) U5 dsDNA, and that are either active or inactive on the HIV-1 replication. We also compared the efficacy of these compounds when added to the reaction before or after the formation of the rIN-dsDNA complex. In this system, the enzyme preincubation with the two-ended 40(mer) dsDNA before the addition of the compounds allowed a strong correlation between the effects of hydroxylated aromatics derivatives on rIN activity in cell-free assays and their effects on viral replication in cell-culture assays. This increase in drug selectivity of the rIN in vitro assay was explored by investigating whether it was due to the length of the 40(mer), longer than the standard 21(mer), or to presence of both viral ends, versus only one viral end. To this purpose we designed four 40(mer) oligonucleotides containing either only one viral end or two-repetitive ends, finding that the architecture of the rIN-dsDNA complex and its compound susceptibility is significantly influenced by the sequence of the dsDNA substrate.
1型人类免疫缺陷病毒（HIV-1）整合酶（IN）是开发新型抗病毒疗法的一个有吸引力的靶点。最近，已有几种HIV-1重组整合酶（rIN）的体外抑制剂被报道。然而，其中绝大多数在基于细胞的试验中未能阻断病毒复制，这表明用于抑制剂筛选的体外试验系统存在不足。为了改进这些系统，我们设计了一种40（聚体）双链DNA反应底物，它由HIV-1长末端重复序列（LTR）U3和U5末端的识别序列组成。HIV-1 rIN能够识别40（聚体）双链DNA的两端并催化其酶活性。使用该底物，我们检测了不同类别的化合物对rIN催化作用的影响，这些化合物在反应底物为标准的21（聚体）U5双链DNA时可在体外抑制HIV-1 rIN，并且对HIV-1复制有活性或无活性。我们还比较了这些化合物在rIN - dsDNA复合物形成之前或之后添加到反应中时的效果。在这个系统中，在添加化合物之前将酶与两端的40（聚体）双链DNA预孵育，使得羟基化芳香族衍生物在无细胞试验中对rIN活性的影响与其在细胞培养试验中对病毒复制的影响之间具有很强的相关性。通过研究这种rIN体外试验中药物选择性的提高是由于40（聚体）的长度比标准的21（聚体）长，还是由于存在病毒的两端，而不是仅一个病毒末端，来探讨这种现象。为此，我们设计了四种40（聚体）寡核苷酸，它们要么只包含一个病毒末端，要么包含两个重复末端，结果发现dsDNA底物的序列对rIN - dsDNA复合物的结构及其对化合物的敏感性有显著影响。
