Scheifele Lisa Z, Kenney Scott P, Cairns Tina M, Craven Rebecca C, Parent Leslie J
Department of Medicine, the Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033, USA.
J Virol. 2007 Oct;81(19):10718-28. doi: 10.1128/JVI.01061-07. Epub 2007 Jul 18.
Nucleocytoplasmic shuttling of the Rous sarcoma virus (RSV) Gag polyprotein is an integral step in virus particle assembly. A nuclear export signal (NES) was previously identified within the p10 domain of RSV Gag. Gag mutants containing deletions of the p10 NES or mutations of critical hydrophobic residues at positions 219, 222, 225, or 229 become trapped within the nucleus and exhibit defects in the efficiency of virus particle release. To investigate other potential roles for Gag nuclear trafficking in RSV replication, we created viruses bearing NES mutant Gag proteins. Viruses carrying p10 mutations produced low levels of particles, as anticipated, and those particles that were released were noninfectious. The p10 mutant viruses contained approximately normal amounts of Gag, Gag-Pol, and Env proteins and genomic viral RNA (vRNA), but several major structural defects were found. Thin-section transmission electron microscopy revealed that the mature particles appeared misshapen, while the viral cores were cylindrical, horseshoe-shaped, or fragmented, with some particles containing multiple small, electron-dense aggregates. Immature virus-like particles produced by the expression of Gag proteins bearing p10 mutations were also aberrant, with both spherical and tubular filamentous particles produced. Interestingly, the secondary structure of the encapsidated vRNA was altered; although dimeric vRNA was predominant, there was an additional high-molecular-weight fraction. Together, these results indicate that the p10 NES domain of Gag is critical for virus replication and that it plays overlapping roles required for the nuclear shuttling of Gag and for the maintenance of proper virion core morphology.
劳氏肉瘤病毒（RSV）Gag多聚蛋白的核质穿梭是病毒粒子组装过程中不可或缺的一步。此前已在RSV Gag的p10结构域内鉴定出一个核输出信号（NES）。含有p10 NES缺失或219、222、225或229位关键疏水残基突变的Gag突变体被困在细胞核内，并在病毒粒子释放效率方面表现出缺陷。为了研究Gag核运输在RSV复制中的其他潜在作用，我们构建了携带NES突变Gag蛋白的病毒。正如预期的那样，携带p10突变的病毒产生的粒子水平较低，并且释放出的那些粒子没有感染性。p10突变病毒含有大致正常量的Gag、Gag-Pol和Env蛋白以及基因组病毒RNA（vRNA），但发现了几个主要的结构缺陷。超薄切片透射电子显微镜显示，成熟粒子形状异常，而病毒核心呈圆柱形、马蹄形或碎片化，一些粒子含有多个小的电子致密聚集体。通过表达携带p10突变的Gag蛋白产生的未成熟病毒样粒子也异常，产生了球形和管状丝状粒子。有趣的是，被包裹的vRNA的二级结构发生了改变；尽管二聚体vRNA占主导，但还有一个额外的高分子量组分。总之，这些结果表明Gag的p10 NES结构域对病毒复制至关重要，并且它在Gag的核穿梭和维持适当的病毒粒子核心形态所需的重叠作用中发挥作用。
