HIV-1前体蛋白早成熟化激活剂筛选模型的建立及应用

Establishment and application of a high-throughput screening assay for premature activation of HIV-1 precursors

摘要:
HIV-1蛋白酶 (PR) 活性的严格调控对于病毒的生存至关重要。在病毒蛋白表达及病毒颗粒装配过程中, 处于病毒前体蛋白Gag-Pol中的蛋白酶必须以无活性状态存在, 避免前体蛋白Gag-Pol和Gag被提前酶切加工 (前体蛋白早成熟化)。干扰HIV-1蛋白酶活性的调控机制, 特异性的激活前体蛋白中的蛋白酶, 诱导前体蛋白早成熟化, 就可以直接抑制病毒的复制。根据这一设想, 运用生物发光共振能量转移技术, 建立细胞水平的HIV-1前体蛋白早成熟化激活剂筛选模型, 并通过3 000个化合物的试验性筛选对筛选模型进行评价。研究结果表明该筛选方法灵敏可靠, 特异性高, 重复性好 (Z' 因子为0.905)。

Abstract:
Strict regulation of HIV-1 PR function is critical for efficient production of mature viral particles. During viral protein expression and viral assembly, HIV-1 PR located within Gag-Pol precursor must be inactive to prevent premature cytoplasmic processing of the viral Gag and Gag-Pol precursors. Premature activation of HIV-1 precursors leads to major defects in viral assembly and production of viral particles. A cell-level premature activation of HIV-1 precursors assay using bioluminescence resonance energy transfer (BRET) was established. Three thousand compounds were screened to evaluate this assay. The results showed that the assay is sensitive, specific and stable (Z' factor is 0.905).