Oertle S, Spahr P F
Department of Molecular Biology, University of Geneva, Switzerland.
J Virol. 1990 Dec;64(12):5757-63. doi: 10.1128/JVI.64.12.5757-5763.1990.
Rous sarcoma virus nucleocapsid protein (NC) has been shown by site-directed mutagenesis to be involved in viral RNA packaging and in the subsequent maturation of genomic RNA in the progeny viral particles. To investigate whether NC exerts these activities as a free protein or as a domain of the polyprotein precursor Pr76gag, we have constructed several mutants unable to process Pr76gag and analyzed their properties in a transient-transfection assay of chicken embryo fibroblasts, the natural host of Rous sarcoma virus. A point mutation in the protease (PR) active site completely prevents Pr76gag processing. The full-length Pr76gag polyprotein is still able to package viral RNA, but cannot mature it. A shorter gag precursor polyprotein lacking the C-terminal PR domain, but retaining that of the NC protein, is however, unable even to package viral RNA. This indicates that the NC protein can participate in packaging viral RNA only as part of a full-length Pr76gag and that the PR domain is, indirectly or directly, also involved in RNA packaging. These results also demonstrate that processing of Pr76gag is necessary for viral RNA dimerization.
通过定点诱变已表明，劳氏肉瘤病毒核衣壳蛋白（NC）参与病毒RNA包装以及子代病毒颗粒中基因组RNA的后续成熟过程。为了研究NC是以游离蛋白形式还是作为多蛋白前体Pr76gag的一个结构域发挥这些活性，我们构建了几个无法加工Pr76gag的突变体，并在劳氏肉瘤病毒的天然宿主鸡胚成纤维细胞的瞬时转染试验中分析了它们的特性。蛋白酶（PR）活性位点的一个点突变完全阻止了Pr76gag的加工。全长Pr76gag多蛋白仍能够包装病毒RNA，但无法使其成熟。然而，一个缺少C末端PR结构域但保留NC蛋白结构域的较短gag前体多蛋白甚至无法包装病毒RNA。这表明NC蛋白仅作为全长Pr76gag的一部分才能参与病毒RNA的包装，并且PR结构域也间接或直接参与RNA包装。这些结果还证明，Pr76gag的加工对于病毒RNA二聚化是必需的。
