Stein C A, Cleary A M, Yakubov L, Lederman S
Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, New York.
Antisense Res Dev. 1993 Spring;3(1):19-31. doi: 10.1089/ard.1993.3.19.
Although having variability in primary sequence, the v3 loop of gp120 in pathogenic strains of human immunodeficiency virus type-1 (HIV-1) is positively charged and known to interact with sulfated polysaccharides. Because the interaction of sulfated polysaccharides with the v3 loop inhibits HIV infection in vitro, we investigated the interaction of the v3 loop with phosphodiester (PO) and phosphorothioate (PS) oligodeoxynucleotides (oligos). In a solid-phase ELISA assay, a PS 28-mer homopolymer of cytidine, SdC28, blocked the binding of the v3 loop-specific monoclonal antibody (mAb) 9284 to rgp120 more potently than did dextran sulfate. In addition, like dextran sulfate, SdC28 appeared to bind specifically to the v3 loop, because neither compound inhibited the binding of other anti-gp120 mAbs. In contrast to PS oligos, PO oligos did not inhibit mAb 9284 binding. The length dependence of the interaction of PS oligos with the v3 loop was studied by using a series of PS oligos. A discrete loss of inhibiting activity occurred as a function of decreasing PS oligo length, which was most marked between PS oligos of 18-mer and 12-mer in length. We further probed the chemical nature of the interaction of oligos with gp120 by measuring the gp120 binding affinities of PS and PO oligos of various lengths. We employed a 5'-32P-labeled alkylating oligo, ClRNH32P-OdT15, and determined that the Km of gp120 binding is 4 microM. We also determined values of competition constant (Kc) for PS competitors of ClRNH32P-OdT15 binding. The binding constant (= 1/Kc) for PS oligos showed a discrete increase in gp120 binding for PS oligos > 12- to 18-mer in length, with no further increment beyond an 18-mer. Given the important role of the v3 loop in HIV-1 pathogenicity, these data suggest that therapeutic trials of PS oligos should be considered.
尽管人类免疫缺陷病毒1型（HIV-1）致病菌株中gp120的v3环一级序列存在变异性，但其带正电荷，且已知可与硫酸化多糖相互作用。由于硫酸化多糖与v3环的相互作用在体外可抑制HIV感染，我们研究了v3环与磷酸二酯（PO）和硫代磷酸酯（PS）寡脱氧核苷酸（寡核苷酸）的相互作用。在固相ELISA分析中，胞苷的PS 28聚体SdC28比硫酸葡聚糖更有效地阻断了v3环特异性单克隆抗体（mAb）9284与rgp120的结合。此外，与硫酸葡聚糖一样，SdC28似乎特异性结合v3环，因为这两种化合物均未抑制其他抗gp120 mAb的结合。与PS寡核苷酸相反，PO寡核苷酸不抑制mAb 9284的结合。通过使用一系列PS寡核苷酸研究了PS寡核苷酸与v3环相互作用的长度依赖性。随着PS寡核苷酸长度的减少，抑制活性出现离散性丧失，这在18聚体和12聚体的PS寡核苷酸之间最为明显。我们通过测量不同长度的PS和PO寡核苷酸与gp120的结合亲和力，进一步探究了寡核苷酸与gp120相互作用的化学性质。我们使用了一种5'-32P标记的烷基化寡核苷酸ClRNH32P-OdT15，并确定gp120结合的Km为4 microM。我们还确定了ClRNH32P-OdT15结合的PS竞争剂的竞争常数（Kc）值。PS寡核苷酸的结合常数（=1/Kc）显示，长度大于12至18聚体的PS寡核苷酸与gp120的结合有离散性增加，超过18聚体后不再增加。鉴于v3环在HIV-1致病性中的重要作用，这些数据表明应考虑对PS寡核苷酸进行治疗试验。
