Characterization of the in vitro HIV-1 capsid assembly pathway.
Characterization of the in vitro HIV-1 capsid assembly pathway.
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DOI：
10.1016/j.jmb.2009.01.058
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发表时间：
2009-03-27
影响因子：
5.6
通讯作者：
Lopez, Claudia S.
中科院分区：
文献类型：
--
作者：
Barklis, Eric;Alfadhli, Ayna;McQuaw, Carolyn;Yalamuri, Suraj;Still, Amelia;Barklis, Robin Lid;Kukull, Ben;Lopez, Claudia S.
在成熟HIV-1核心的形态发生过程中，病毒衣壳（CA）蛋白在病毒核糖核蛋白复合物周围组装锥形或管状外壳。这个组装步骤在体外通过CA蛋白寡聚形成长管的反应来模拟，这个过程可以被模拟为由一个缓慢的成核期和一个快速的管生长阶段组成。我们开发了一种新的荧光显微镜方法来监测体外组装反应，并将其与电子显微镜分析一起用于表征组装过程。结果表明，温度、盐浓度和pH的变化对管形核和生长步骤有不同的影响。我们还证明了组装可以是单向或双向的，生长可以被限制，蛋白质可以组装到管的表面，产生多壁或嵌套结构。最后，实验表明，体外组装的肽抑制剂也可以拆除预先存在的管，这表明这些试剂可能对病毒组装和脱壳都具有抗病毒作用。我们的研究有助于为理解成熟的HIV-1核心组装机制和抗病毒抑制途径奠定基础。
During morphogenesis of mature HIV-1 cores, the viral capsid (CA) proteins assemble conical or tubular shells around the viral ribonucleoprotein complexes. This assembly step is mimicked in vitro through reactions in which CA proteins oligomerize to form long tubes, and this process can be modeled as consisting of a slow nucleation period followed by a rapid phase of tube growth. We have developed a novel fluorescence microscopy approach to monitor in vitro assembly reactions and have employed it, along with electron microscopy analysis, to characterize the assembly process. Our results indicate that temperature, salt concentration, and pH changes have differential effects on tube nucleation and growth steps. We also demonstrate that assembly can be unidirectional or bidirectional, that growth can be capped, and that proteins can assemble onto the surfaces of tubes, yielding multiwalled or nested structures. Finally, experiments show that a peptide inhibitor of in vitro assembly also can dismantle pre-existing tubes, suggesting that such reagents may possess antiviral effects against both viral assembly and uncoating. Our investigations help establish a basis for understanding the mechanism of mature HIV-1 core assembly, and avenues for antiviral inhibition.
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