HIV-1 gp41 six-helix bundle formation occurs rapidly after the engagement of gp120 by CXCR4 in the HIV-1 Env-mediated fusion process
HIV-1 gp41 six-helix bundle formation occurs rapidly after the engagement of gp120 by CXCR4 in the HIV-1 Env-mediated fusion process
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DOI：
10.1021/bi0155596
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发表时间：
2001-10-16
期刊：
影响因子：
2.9
通讯作者：
Blumenthal, R
中科院分区：
文献类型：
--
作者：
Gallo, SA;Puri, A;Blumenthal, R
由HIV-1 IIIB Env介导的细胞融合开始之前有15-20分钟的滞后期。由CD 4非依赖性HIV-1 Env 8x介导的融合能够直接与CXCR 4相互作用，滞后期大大减少。我们探索了HIV-1 IIIB Env介导的融合中滞后期的中间步骤，Leu 3-a是gp 120与CD 4连接的抑制剂，AMD 3100是gp 120与CXCR 4连接的抑制剂，C34是干扰gp 41向融合活性状态转变的合成肽。C34和AMD 3100对融合的抑制作用随时间的变化是等效的，表明CXCR 4与gp 120的结合和gp 41六螺旋束的形成遵循相似的动力学。由CD 4非依赖性Env 8x介导的融合的初始步骤太快，这些抑制剂无法干扰。然而，当在37 ℃孵育之前，在存在AMD 3100或C34的情况下，将8x Env表达细胞与靶细胞在25 ℃孵育时，这些抑制剂能够抑制8x。Env介导的融合。为了进一步检测CXCR 4与gp 120的结合以及C34结合位点的暴露，我们通过向培养基中加入细胞松弛素B在37 ℃可逆地抑制了融合反应。我们发现，CXCR 4的参与和六螺旋束的形成只发生在细胞松弛素释放后。停滞，表明需要高度的协同性来触发HIV-1 Env介导的融合的初始步骤。
The onset of cell fusion mediated by HIV-1 IIIB Env is preceded by a lag phase of 15-20 min. Fusion mediated by the CD4-independent HIV-1 Env 8x, which is capable, of interacting directly with CXCR4, proceeds with a greatly reduced lag phase. We probed the intermediate steps during the lag phase in HIV-1 IIIB Env-mediated fusion with Leu3-a, an inhibitor of attachment of gp120 to CD4, AMD3100, an inhibitor of attachment of gp120 to CXCR4, and C34, a synthetic peptide that interferes with the transition of gp41 to the fusion active state. Inhibitions of fusion as a function of time of addition of C34 and of AMD3100 were equivalent, indicating that engagement of gp120 by CXCR4 and formation of the gp41 six-helix bundle follow similar kinetics. The initial steps in fusion mediated by the CD4-independent Env 8x are too rapid for these inhibitors to interfere with. However, when 8x Env-expressing cells were incubated with target cells at 25 degreesC in the presence of AMD3100 or C34, prior to incubation at 37 degreesC, these inhibitors were capable of inhibiting 8x. Env-mediated fusion. To further examine engagement of gp120 by CXCR4 and exposure of binding sites for C34, we have reversibly arrested the fusion reaction at 37 degreesC by adding cytochalasin B to the medium. We show that CXCR4 engagement and six-helix bundle formation only occur after the release of the cytochalasin. arrest, indicating that a high degree of cooperativity is required to trigger the initial steps in HIV-1 Env-mediated fusion.