N-terminal extension of human immunodeficiency virus capsid protein converts the in vitro assembly phenotype from tubular to spherical particles
N-terminal extension of human immunodeficiency virus capsid protein converts the in vitro assembly phenotype from tubular to spherical particles
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DOI：
10.1128/jvi.72.6.4798-4810.1998
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发表时间：
1998-06-01
影响因子：
5.4
通讯作者：
Kräusslich, HG
中科院分区：
文献类型：
--
作者：
Gross, I;Hohenberg, H;Kräusslich, HG
逆转录病毒Gag多聚蛋白的表达足以使具有球形未成熟蛋白壳的病毒样颗粒形态发生。Gag蛋白水解裂解成基质（MA）、衣壳（CA）、核衣壳（NC）和p6结构域（在人类免疫缺陷病毒[HIV]的情况下），导致浓缩成成熟的锥形核心。我们分析了纯化的HIV蛋白体外组装或大肠杆菌细胞内球形或圆柱形颗粒的形成。CA蛋白单独产生的圆柱形颗粒，而所有的N-末端的CA取消圆柱体形成的延伸。相反，观察到具有不均匀直径的球形颗粒或无定形蛋白质聚集体。将CA延伸5个氨基酸足以将组装表型转化为球形颗粒。CA的C-末端序列对于球体形成不是必需的。蛋白水解裂解的N-末端延伸的CA蛋白在体外组装之前导致圆柱形颗粒的形成，而蛋白水解的体外组装产物引起的球体的破坏，但不形成圆柱体。CA和扩展的Ca蛋白在亲环素A（CypA）的存在下，在CA与CypA的摩尔比为10：1的体外组装产生显着更长的圆柱体和异质球，而更高浓度的CypA完全破坏颗粒的形成。我们的结论是，不成熟的HIV颗粒的球形形状是由CA结构域上的N-末端延伸的存在下，并在病毒粒子成熟的核心凝聚需要CA的N末端的解放。
Expression of retroviral Gag polyproteins is sufficient for morphogenesis of virus-like particles with a spherical immature protein shell. Proteolytic cleavage of Gag into the matrix (MA), capsid (CA), nucleocapsid (NC), and p6 domains (in the case of human immunodeficiency virus [HIV]) leads to condensation to the mature cone-shaped core. We have analyzed the formation of spherical or cylindrical particles on in vitro assembly of purified HIV proteins or inside Escherichia coli cells. CA protein alone yielded cylindrical particles, while all N-terminal extensions of CA abolished cylinder formation. Spherical particles with heterogeneous diameters or amorphous protein aggregates were observed instead. Extending CA by 5 amino acids was sufficient to convert the assembly phenotype to spherical particles. Sequences C-terminal of CA were not required for sphere formation. Proteolytic cleavage of N-terminally extended CA proteins prior to in vitro assembly led to the formation of cylindrical particles, while proteolysis of in vitro assembly products caused disruption of spheres but not formation of cylinders. In vitro assembly of CA and extended Ca proteins in the presence of cyclophilin A (CypA) at a CA-to-CypA molar ratio of 10:1 yielded significantly longer cylinders and heterogeneous spheres, while higher concentrations of CypA completely disrupted particle formation. We conclude that the spherical shape of immature HIV particles is determined by the presence of an N-terminal extension on the CA domain and that core condensation during virion maturation requires the liberation of the N terminus of CA.