Mattei Simone, Flemming Annica, Anders-Össwein Maria, Kräusslich Hans-Georg, Briggs John A G, Müller Barbara
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Molecular Medicine Partnership Unit, Heidelberg, Germany.
Department of Infectious Diseases, Virology, University Hospital Heidelberg, Heidelberg, Germany.
J Virol. 2015 Oct;89(19):9739-47. doi: 10.1128/JVI.00750-15. Epub 2015 Jul 15.
Human immunodeficiency virus type 1 (HIV-1) is released from infected cells in an immature, noninfectious form in which the structural polyprotein Gag is arranged in a hexameric lattice, forming an incomplete spherical shell. Maturation to the infectious form is mediated by the viral protease, which cleaves Gag at five sites, releasing the CA (capsid) protein, which forms a conical capsid encasing the condensed RNA genome. The pathway of this structural rearrangement is currently not understood, and it is unclear how cone assembly is initiated. RNA represents an integral structural component of retroviruses, and the viral nucleoprotein core has previously been proposed to nucleate mature capsid assembly. We addressed this hypothesis by replacing the RNA-binding NC (nucleocapsid) domain of HIV-1 Gag and the adjacent spacer peptide 2 (SP2) by a leucine zipper (LZ) protein-protein interaction domain [Gag(LZ)] in the viral context. We found that Gag(LZ)-carrying virus [HIV(LZ)] was efficiently released and viral polyproteins were proteolytically processed, though with reduced efficiency. Cryo-electron tomography revealed that the particles lacked a condensed nucleoprotein and contained an increased proportion of aberrant core morphologies caused either by the absence of RNA or by altered Gag processing. Nevertheless, a significant proportion of HIV(LZ) particles contained mature capsids with the wild-type morphology. These results clearly demonstrate that the nucleoprotein complex is dispensable as a nucleator for mature HIV-1 capsid assembly in the viral context.
Formation of a closed conical capsid encasing the viral RNA genome is essential for HIV-1 infectivity. It is currently unclear what viral components initiate and regulate the formation of the capsid during virus morphogenesis, but it has been proposed that the ribonucleoprotein complex plays a role. To test this, we prepared virus-like particles lacking the viral nucleocapsid protein and RNA and analyzed their three-dimensional structure by cryo-electron tomography. While most virions displayed an abnormal morphology under these conditions, some particles showed a normal mature morphology with closed conical capsids. These data demonstrate that the presence of RNA and the nucleocapsid protein is not required for the formation of a mature, cone-shaped HIV-1 capsid.
1型人类免疫缺陷病毒（HIV-1）以不成熟、无感染性的形式从受感染细胞中释放出来，其中结构多聚蛋白Gag以六聚体晶格排列，形成一个不完整的球形外壳。向感染性形式的成熟由病毒蛋白酶介导，该蛋白酶在五个位点切割Gag，释放出衣壳（CA）蛋白，后者形成一个包裹浓缩RNA基因组的锥形衣壳。这种结构重排的途径目前尚不清楚，并且锥形衣壳的组装是如何启动的也不清楚。RNA是逆转录病毒不可或缺的结构成分，并且病毒核蛋白核心此前被认为是成熟衣壳组装的成核物质。我们通过在病毒环境中用亮氨酸拉链（LZ）蛋白-蛋白相互作用结构域[Gag(LZ)]替换HIV-1 Gag的RNA结合核衣壳（NC）结构域和相邻的间隔肽2（SP2）来验证这一假设。我们发现携带Gag(LZ)的病毒[HIV(LZ)]能有效释放，并且病毒多聚蛋白能被蛋白酶水解加工，尽管效率有所降低。冷冻电子断层扫描显示，这些颗粒缺乏浓缩的核蛋白，并且由于RNA的缺失或Gag加工的改变，含有异常核心形态的比例增加。然而，相当一部分HIV(LZ)颗粒含有具有野生型形态的成熟衣壳。这些结果清楚地表明，在病毒环境中，核蛋白复合物作为成熟HIV-1衣壳组装的成核物质是可有可无的。
形成一个包裹病毒RNA基因组的封闭锥形衣壳对于HIV-1的感染性至关重要。目前尚不清楚在病毒形态发生过程中哪些病毒成分启动并调节衣壳的形成，但有人提出核糖核蛋白复合物发挥了作用。为了验证这一点，我们制备了缺乏病毒核衣壳蛋白和RNA的病毒样颗粒，并通过冷冻电子断层扫描分析了它们的三维结构。虽然在这些条件下大多数病毒体呈现出异常形态，但一些颗粒显示出具有封闭锥形衣壳的正常成熟形态。这些数据表明，形成成熟的锥形HIV-1衣壳不需要RNA和核衣壳蛋白的存在。
