ESCRT-III CHMP2A and CHMP3 form variable helical polymers in vitro and act synergistically during HIV-1 budding
ESCRT-III CHMP2A and CHMP3 form variable helical polymers in vitro and act synergistically during HIV-1 budding
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DOI：
10.1111/cmi.12041
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发表时间：
2013-02-01
影响因子：
3.4
通讯作者：
Weissenhorn, Winfried
中科院分区：
文献类型：
--
作者：
Effantin, Gregory;Dordor, Aurelien;Weissenhorn, Winfried
转运蛋白III（ESCRT-III）蛋白所需的内体分选复合物对于某些包膜病毒的出芽、内体处管腔内囊泡的形成以及胞质分裂的终止步骤是必不可少的。ESCRT-III蛋白形成聚合物，收缩膜管，导致分裂。我们已经使用电子低温显微镜来确定多晶型ESCRT-III CHMP 2A-CHMP 3聚合物的分子组织。在22埃分辨率下的三维重建揭示了以头对尾方式组织的CHMP分子细丝的螺旋组织。蛋白酶敏感性实验表明，聚合是通过构象变化，增加了原聚体的稳定性。组合siRNA敲除实验表明，CHMP 3协同地促进HIV-1出芽，并且CHMP 3的贡献与CHMP 2A相比，与CHMP 2B相比，与CHMP 2A相比，CHMP 3的贡献类似于10倍更明显。这与表面等离子体共振亲和力测量结果一致，该测量结果表明连续的CHMP 4 B-CHMP 3-CHMP 2A募集，同时显示CHMP 2A和CHMP 2B都与CHMP 4 B相互作用，与它们在HIV-1出芽中的冗余功能一致。因此，我们的数据表明，在体外观察到的CHMP 2A-CHMP 3聚合物通过在CHMP 4 B聚合物上组装而有助于HIV-1出芽。
The endosomal sorting complex required for transport-III (ESCRT-III) proteins are essential for budding of some enveloped viruses, for the formation of intraluminal vesicles at the endosome and for the abscission step of cytokinesis. ESCRT-III proteins form polymers that constrict membrane tubes, leading to fission. We have used electron cryomicroscopy to determine the molecular organization of pleiomorphic ESCRT-III CHMP2A-CHMP3 polymers. The three-dimensional reconstruction at 22 angstrom resolution reveals a helical organization of filaments of CHMP molecules organized in a head-to-tail fashion. Protease susceptibility experiments indicate that polymerization is achieved via conformational changes that increase the protomer stability. Combinatorial siRNA knockdown experiments indicate that CHMP3 contributes synergistically to HIV-1 budding, and the CHMP3 contribution is similar to 10-fold more pronounced in concert with CHMP2A than with CHMP2B. This is consistent with surface plasmon resonance affinity measurements that suggest sequential CHMP4B-CHMP3-CHMP2A recruitment while showing that both CHMP2A and CHMP2B interact with CHMP4B, in agreement with their redundant functions in HIV-1 budding. Our data thus indicate that the CHMP2A-CHMP3 polymer observed in vitro contributes to HIV-1 budding by assembling on CHMP4B polymers.