Wang Huating, Norris Kendra M, Mansky Louis M
Molecular, Cellular and Developmental Biology Graduate Program, Ohio State University, Columbus, Ohio 43210, USA.
J Virol. 2003 Sep;77(17):9431-8. doi: 10.1128/jvi.77.17.9431-9438.2003.
The RNA packaging process for retroviruses involves a recognition event of the genome-length viral RNA by the viral Gag polyprotein precursor (PrGag), an important step in particle morphogenesis. The mechanism underlying this genome recognition event for most retroviruses is thought to involve an interaction between the nucleocapsid (NC) domain of PrGag and stable RNA secondary structures that form the RNA packaging signal. Presently, there is limited information regarding PrGag-RNA interactions involved in RNA packaging for the deltaretroviruses, which include bovine leukemia virus (BLV) and human T-cell leukemia virus types 1 and 2 (HTLV-1 and -2, respectively). To address this, alanine-scanning mutagenesis of BLV PrGag was done with a virus-like particle (VLP) system. As predicted, mutagenesis of conserved basic residues as well as residues of the zinc finger domains in the BLV NC domain of PrGag revealed residues that led to a reduction in viral RNA packaging. Interestingly, when conserved basic residues in the BLV MA domain of PrGag were mutated to alanine or glycine, but not when mutated to another basic residue, reductions in viral RNA packaging were also observed. The ability of PrGag to be targeted to the cell membrane was not affected by these mutations in MA, indicating that PrGag membrane targeting was not associated with the reduction in RNA packaging. These observations indicate that these basic residues in the MA domain of PrGag influence RNA packaging, without influencing Gag membrane localization. It was further observed that (i) a MA/NC double mutant had a more severe RNA packaging defect than either mutant alone, and (ii) RNA packaging was not found to be associated with transient localization of Gag in the nucleus. In summary, this report provides the first direct evidence for the involvement of both the BLV MA and NC domains of PrGag in viral RNA packaging.
逆转录病毒的RNA包装过程涉及病毒Gag多蛋白前体（PrGag）对基因组长度的病毒RNA的识别事件，这是颗粒形态发生中的重要一步。大多数逆转录病毒的这种基因组识别事件的潜在机制被认为涉及PrGag的核衣壳（NC）结构域与形成RNA包装信号的稳定RNA二级结构之间的相互作用。目前，关于δ逆转录病毒（包括牛白血病病毒（BLV）和1型及2型人类T细胞白血病病毒（分别为HTLV-1和HTLV-2））RNA包装中涉及的PrGag-RNA相互作用的信息有限。为了解决这个问题，利用病毒样颗粒（VLP）系统对BLV PrGag进行了丙氨酸扫描诱变。如预期的那样，对PrGag的BLV NC结构域中保守的碱性残基以及锌指结构域的残基进行诱变，揭示了导致病毒RNA包装减少的残基。有趣的是，当PrGag的BLV MA结构域中的保守碱性残基突变为丙氨酸或甘氨酸时，但突变为另一个碱性残基时则不会出现这种情况，也观察到了病毒RNA包装的减少。MA中的这些突变并不影响PrGag靶向细胞膜的能力，这表明PrGag膜靶向与RNA包装的减少无关。这些观察结果表明，PrGag的MA结构域中的这些碱性残基影响RNA包装，而不影响Gag膜定位。进一步观察到：（i）MA/NC双突变体比单独的任何一种突变体都有更严重的RNA包装缺陷；（ii）未发现RNA包装与Gag在细胞核中的短暂定位有关。总之，本报告首次直接证明了PrGag的BLV MA和NC结构域都参与了病毒RNA包装。
