Butan Carmen, Lokhandwala Parvez M, Purdy John G, Cardone Giovanni, Craven Rebecca C, Steven Alasdair C
Laboratory of Structural Biology, National Institute for Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda Maryland 20892, USA.
J Virol. 2010 Jul;84(13):6377-86. doi: 10.1128/JVI.00207-10. Epub 2010 Apr 28.
Retrovirus assembly is driven by polymerization of the Gag polyprotein as nascent virions bud from host cells. Gag is then processed proteolytically, releasing the capsid protein (CA) to assemble de novo inside maturing virions. CA has N-terminal and C-terminal domains (NTDs and CTDs, respectively) whose folds are conserved, although their sequences are divergent except in the 20-residue major homology region (MHR) in the CTD. The MHR is thought to play an important role in assembly, and some mutations affecting it, including the F167Y substitution, are lethal. A temperature-sensitive second-site suppressor mutation in the NTD, A38V, restores infectivity. We have used cryoelectron tomography to investigate the morphotypes of this double mutant. Virions produced at the nonpermissive temperature do not assemble capsids, although Gag is processed normally; moreover, they are more variable in size than the wild type and have fewer glycoprotein spikes. At the permissive temperature, virions are similar in size and spike content as in the wild type and capsid assembly is restored, albeit with altered polymorphisms. The mutation F167Y-A38V (referred to as FY/AV in this paper) produces fewer tubular capsids than wild type and more irregular polyhedra, which tend to be larger than in the wild type, containing approximately 30% more CA subunits. It follows that FY/AV CA assembles more efficiently in situ than in the wild type and has a lower critical concentration, reflecting altered nucleation properties. However, its infectivity is lower than that of the wild type, due to a 4-fold-lower budding efficiency. We conclude that the wild-type CA protein sequence represents an evolutionary compromise between competing requirements for optimization of Gag assembly (of the immature virion) and CA assembly (in the maturing virion).
逆转录病毒的组装是由Gag多蛋白的聚合驱动的，新生病毒粒子从宿主细胞中出芽。然后Gag被蛋白酶水解，释放出衣壳蛋白（CA），在成熟的病毒粒子内重新组装。CA有N端和C端结构域（分别为NTD和CTD），其折叠结构是保守的，尽管它们的序列除了CTD中20个残基的主要同源区域（MHR）外是不同的。MHR被认为在组装中起重要作用，一些影响它的突变，包括F167Y替代，是致死性的。NTD中的一个温度敏感的第二位点抑制突变A38V恢复了感染性。我们使用冷冻电子断层扫描来研究这种双突变体的形态类型。在非允许温度下产生的病毒粒子不组装衣壳，尽管Gag被正常加工；此外，它们的大小比野生型更具变异性，糖蛋白刺突也更少。在允许温度下，病毒粒子的大小和刺突含量与野生型相似，衣壳组装得以恢复，尽管多态性有所改变。F167Y - A38V突变（本文中称为FY/AV）产生的管状衣壳比野生型少，不规则多面体更多，这些多面体往往比野生型更大，含有比野生型多约30%的CA亚基。由此可见，FY/AV CA在原位比野生型更有效地组装，并且具有更低的临界浓度，这反映了成核特性的改变。然而，由于出芽效率降低4倍，其感染性低于野生型。我们得出结论，野生型CA蛋白序列代表了在未成熟病毒粒子的Gag组装优化和成熟病毒粒子的CA组装优化的竞争需求之间的进化折衷。
