Wills J W, Cameron C E, Wilson C B, Xiang Y, Bennett R P, Leis J
Department of Microbiology and Immunology, Pennsylvania State University College of Medicine, Hershey 17033.
J Virol. 1994 Oct;68(10):6605-18. doi: 10.1128/JVI.68.10.6605-6618.1994.
The Gag protein of Rous sarcoma virus has the ability to direct particle assembly at the plasma membrane in the absence of all the other virus-encoded components. An extensive deletion analysis has revealed that very large regions of this protein can be deleted without impairing budding and has suggested that the essential functions map to three discrete regions. In the studies reported here, we establish the location of assembly domain 2 (AD2) within the proline-rich p2b sequence of this Gag protein. AD2 mutants lacking the p2b sequence were completely defective for particle release even though their Gag proteins were tightly associated with the membrane fraction and exhibited high levels of protease activity. Mutations that inactivate the viral protease did not restore budding to wild-type levels for these mutants, indicating that the defect is not due simply to a loss of protease regulation. AD2 mutants could be rescued into dense particles in genetic complementation assays, indicating that their defect is not due to a gross alteration of the overall conformation of the protein and that the assembly function is not needed on every Gag molecule in the population. Several mutants with amino acid substitutions in the p2b sequence were found to have an intermediate capacity for budding. Inactivation of the protease of these mutants stabilized the Gag polyprotein within the cells and allowed an increase in particle release; however, the rate of budding remained slow. We favor the idea that AD2 is a dynamic region of movement, perhaps serving as a molecular hinge to allow the particle to emerge from the surface of the cell during budding.
劳氏肉瘤病毒的Gag蛋白能够在没有所有其他病毒编码成分的情况下，指导在质膜处进行病毒粒子组装。广泛的缺失分析表明，该蛋白的很大区域可以被删除而不影响出芽，这表明其基本功能定位于三个离散区域。在本文报道的研究中，我们确定了组装结构域2（AD2）在该Gag蛋白富含脯氨酸的p2b序列中的位置。缺乏p2b序列的AD2突变体在病毒粒子释放方面完全存在缺陷，尽管它们的Gag蛋白与膜部分紧密结合并表现出高水平的蛋白酶活性。使病毒蛋白酶失活的突变并不能将这些突变体的出芽恢复到野生型水平，这表明缺陷并非仅仅由于蛋白酶调节的丧失。在基因互补试验中，AD2突变体可以被挽救成致密颗粒，这表明它们的缺陷并非由于蛋白质整体构象的严重改变，并且组装功能并非群体中每个Gag分子都需要。在p2b序列中有氨基酸取代的几个突变体被发现具有中等出芽能力。这些突变体的蛋白酶失活使细胞内的Gag多蛋白稳定，并使病毒粒子释放增加；然而，出芽速率仍然很慢。我们倾向于认为AD2是一个动态的移动区域，可能作为一个分子铰链，使病毒粒子在出芽过程中能够从细胞表面出现。
