Center for AIDS Research, Kumamoto University, Kumamoto, Japan.
PLoS One. 2011;6(11):e27696. doi: 10.1371/journal.pone.0027696. Epub 2011 Nov 15.
Nef is a multifunctional HIV-1 protein that accelerates progression to AIDS, and enhances the infectivity of progeny viruses through a mechanism that is not yet understood. Here, we show that the small molecule compound 2c reduces Nef-mediated viral infectivity enhancement. When added to viral producer cells, 2c did not affect the efficiency of viral production itself. However, the infectivity of the viruses produced in the presence of 2c was significantly lower than that of control viruses. Importantly, an inhibitory effect was observed with Nef(+) wild-type viruses, but not with viruses produced in the absence of Nef or in the presence of proline-rich PxxP motif-disrupted Nef, both of which displayed significantly reduced intrinsic infectivity. Meanwhile, the overexpression of the SH3 domain of the tyrosine kinase Hck, which binds to a PxxP motif in Nef, also reduced viral infectivity. Importantly, 2c inhibited Hck SH3-Nef binding, which was more marked when Nef was pre-incubated with 2c prior to its incubation with Hck, indicating that both Hck SH3 and 2c directly bind to Nef and that their binding sites overlap. These results imply that both 2c and the Hck SH3 domain inhibit the interaction of Nef with an unidentified host protein and thereby reduce Nef-mediated infectivity enhancement. The first inhibitory compound 2c is therefore a valuable chemical probe for revealing the underlying molecular mechanism by which Nef enhances the infectivity of HIV-1.
Nef 是一种多功能的 HIV-1 蛋白，可加速艾滋病的发展，并通过一种尚未完全理解的机制增强病毒的感染力。在这里，我们表明小分子化合物 2c 可降低 Nef 介导的病毒感染力增强。当添加到病毒产生细胞时，2c 本身不会影响病毒产生的效率。然而，在 2c 存在下产生的病毒的感染力明显低于对照病毒。重要的是，观察到对 Nef(+)野生型病毒具有抑制作用，但对缺乏 Nef 或存在富含脯氨酸的 PxxP 基序破坏的 Nef 产生的病毒没有抑制作用，这两种病毒的固有感染力明显降低。同时，酪氨酸激酶 Hck 的 SH3 结构域的过表达，该结构域与 Nef 中的 PxxP 基序结合，也降低了病毒的感染力。重要的是，2c 抑制了 Hck SH3-Nef 结合，当 Nef 在与 Hck 孵育之前先用 2c 预孵育时，这种抑制作用更为明显，表明 Hck SH3 和 2c 均直接与 Nef 结合，并且它们的结合位点重叠。这些结果表明，2c 和 Hck SH3 结构域均抑制了 Nef 与未鉴定的宿主蛋白的相互作用，从而降低了 Nef 介导的感染力增强。因此，第一种抑制性化合物 2c 是揭示 Nef 增强 HIV-1 感染力的潜在分子机制的有价值的化学探针。
