Kitasato Institute for Life Sciences and Graduate School of Infection Control, Kitasato University, Shirokane 5-9-1, Minato-ku, Tokyo 108-8641, Japan.
Antimicrob Agents Chemother. 2011 Sep;55(9):4251-60. doi: 10.1128/AAC.00299-11. Epub 2011 Jul 11.
Human immunodeficiency virus (HIV) Gag protein targets to the plasma membrane and assembles into viral particles. In the next round of infection, the mature Gag capsids disassemble during viral entry. Thus, Gag plays a central role in the HIV life cycle. Using a yeast membrane-associated two-hybrid assay based on the SOS-RAS signaling system, we developed a system to measure the Gag-Gag interaction and isolated 6 candidates for Gag assembly inhibitors from a chemical library composed of 20,000 small molecules. When tested in the human MT-4 cell line and primary peripheral blood mononuclear cells, one of the candidates, 2-(benzothiazol-2-ylmethylthio)-4-methylpyrimidine (BMMP), displayed an inhibitory effect on HIV replication, although a considerably high dose was required. Unexpectedly, neither particle production nor maturation was inhibited by BMMP. Confocal microscopy confirmed that BMMP did not block Gag plasma membrane targeting. Single-round infection assays with envelope-pseudotyped and luciferase-expressing viruses revealed that BMMP inhibited HIV replication postentry but not simian immunodeficiency virus (SIV) or murine leukemia virus infection. Studies with HIV/SIV Gag chimeras indicated that the Gag capsid (CA) domain was responsible for the BMMP-mediated HIV postentry block. In vitro studies indicated that BMMP accelerated disassembly of HIV cores and, conversely, inhibited assembly of purified CA protein in a dose-dependent manner. Collectively, our data suggest that BMMP primarily targets the HIV CA domain and disrupts viral infection postentry, possibly through inducing premature disassembly of HIV cores. We suggest that BMMP is a potential lead compound to develop antiretroviral drugs bearing novel mechanisms of action.
人类免疫缺陷病毒（HIV）Gag 蛋白靶向质膜并组装成病毒颗粒。在下一轮感染中，成熟的 Gag 衣壳在病毒进入时解体。因此，Gag 在 HIV 生命周期中发挥着核心作用。我们使用基于 SOS-RAS 信号系统的酵母膜相关双杂交测定法开发了一种测量 Gag-Gag 相互作用的系统，并从由 20000 个小分子组成的化学文库中分离出 6 种 Gag 组装抑制剂候选物。在人 MT-4 细胞系和原代外周血单核细胞中进行测试时，候选物之一 2-(苯并噻唑-2-基甲基硫代)-4-甲基嘧啶（BMMP）显示出抑制 HIV 复制的作用，尽管需要相当高的剂量。出乎意料的是，BMMP 既不抑制颗粒产生也不抑制成熟。共聚焦显微镜证实 BMMP 不阻断 Gag 质膜靶向。用包膜假型和荧光素酶表达病毒进行的单轮感染实验表明，BMMP 抑制 HIV 复制，但不抑制猴免疫缺陷病毒（SIV）或鼠白血病病毒感染。用 HIV/SIV Gag 嵌合体进行的研究表明，Gag 衣壳（CA）结构域负责 BMMP 介导的 HIV 进入后阻断。体外研究表明，BMMP 加速了 HIV 核心的解体，相反，以剂量依赖的方式抑制纯化 CA 蛋白的组装。总的来说，我们的数据表明，BMMP 主要靶向 HIV CA 结构域并破坏病毒进入后的感染，可能是通过诱导 HIV 核心的过早解体。我们建议 BMMP 是一种潜在的先导化合物，可以开发具有新型作用机制的抗逆转录病毒药物。
