SUPPRESSION OF HIV-1 IN VITRO BY PUTATIVE TRIPLE HELIX

本报告描述了嘧啶寡核苷酸（ON）， 与HIV-1前病毒基因组上的特定靶标互补，并形成 “三螺旋”结构，可通过阻断 目标双链DNA的大沟。 我们设计了几个 与HIV-1基因组内富含嘌呤的区域互补的ON， 抑制艾滋病毒感染的目标。 这些靶向的HIV基因 ON包括gag、pol、达特、nef和vif。 我们测试了这些 ON进入细胞并抑制H9的HIV-1（MN）感染 淋巴细胞系 将ON和病毒以100 ° C直接加入到细胞培养基中。 同时还研究与讨论 ONs存在于处理细胞的细胞核中， 10分钟的初始治疗。 用10至50 μ g/ml的 任何ON的μ M浓度显著降低病毒水平 如以下所示：1）细胞外p24抗原减少50- 90 通过p24捕获ELISA测量，2）同时减少 通过免疫荧光标记和FACSCAN检测细胞内p24 3）病毒DNA的量的类似减少（通过DNA检测）， 细胞裂解物的PCR测定）相对于未处理的感染细胞的相对值。 细胞 在这些低ON浓度下，生长和活力不受影响。 在最初的研究中，用50 μ M GAG特异性抗体处理的感染细胞， ON产生的p24抗原比用50 μ M处理的感染细胞少85 的非特异性，嘧啶ON与类似的基础组成（对照）。 对非特异性ON的进一步研究正在进行中。 初步 对原代人外周血淋巴细胞的研究也表明， 通过HIV特异性三螺旋ON抑制病毒。 这些ON显示 作为抗艾滋病药物的前景。 正在进行的研究涉及：1） ON治疗对病毒转录物的影响，2）ON的持续性 在细胞和细胞核中，以及3）ON形成三螺旋的能力 与HIV序列的复合物。

This report describes pyrimidine oligonucleotides (ONs) designed to complement a specific target on the HIV-1 proviral genome and form a "triple helix" structure which could affect virus activity by blocking the major groove of the target double-stranded DNA. We designed several ONs complementary to purine- rich regions within the HIV-1 genome with the goal of supressing HIV infection. The HIV genes targeted by these ONs include gag, pol, tat, nef and vif. We tested the ability of these ONs to enter the cells and suppress HIV-1 (MN) infection of the H9 lymphocyte cell line. ON and virus were added directly to cell media at the same time. ONs were present in the nuclei of treated cells within 10 mins of initial treatment. Treatment of infected cells with 10 to 50 uM concentrations of any of the ONs significantly reduced virus levels as demonstrated by: 1) a 50-90% decrease in extracellular p24 antigen as measured by p24 capture ELISA, 2) a concomitant decrease in intracellular p24 as detected by immunofluorescence labeling and FACSCAN analysis, 3) a similar reduction in amounts of viral DNA (detected by DNA PCR assay of cell lysates) relative to untreated infected cells. Cell growth and viability was not affected at these low ON concentrations. In an initial study, infected cells treated with 50uM of GAG specific ON produced 85% less p24 antigen than infected cells treated with 50 uM of a nonspecific, pyrimidine ON with similar base composition (control). Futher studies with nonspecific ONs aare in progress. Preliminary studies with primary human peripheral blood lymphocytes also demonstrated suppression of virus by HIV-specific triple helix ONs. These ONs show promise as anti-HIV agents. Ongoing studies are addressing 1) the effects of ON treatment on viral transcripts, 2) the persistence of ON in cells and nuclei, and 3) the ability of the ONs to form triple helix complexes with HIV sequences.