Ott D E, Coren L V, Chertova E N, Gagliardi T D, Schubert U
AIDS Vaccine Program, SAIC Frederick, National Cancer Institute, Frederick, Maryland 21702-1201, USA.
Virology. 2000 Dec 5;278(1):111-21. doi: 10.1006/viro.2000.0648.
Our previous biochemical studies of HIV-1 and MuLV virions isolated and identified mature Gag products, HIV-1 p6(Gag) and MuLV p12(Gag), that were conjugated to a single ubiquitin. To study the importance of the monoubiquitination of Gag, a series of lysine to arginine mutants were constructed that eliminated ubiquitination at one or both of the lysines in HIV-1(NL4-3) p6(Gag) and both lysines in Moloney MuLV p12(Gag). HPLC and immunoblot analysis of the HIV-1 mutants demonstrated that either of the lysines in p6(Gag), K27 or K33, could be monoubiquitinated. However, infectivity assays showed that monoubiquitination of HIV-1 p6(Gag) or MuLV p12(Gag) is not required for viral replication in vitro. Pulse-chase radiolabeling of HIV-1-producing cells revealed that monoubiquitination of p6(Gag) does not affect the short-term release of virus from the cell, the maturation of Pr55(Gag), or the sensitivity of these processes to proteasome inhibitors. Experiments with protease-deficient HIV-1 showed that Pr55(Gag) can be monoubiquitinated, suggesting that p6(Gag) is first modified as a domain within Gag. Examination of the proteins inside an HIV-1 mutant found that free ubiquitin was incorporated into the virions in the absence of the lysines in p6(Gag), showing that the ubiquitin inside the virus is not initially brought in as a p6(Gag) conjugate. Although our results establish that monoubiquitination of p6(Gag) and p12(Gag) is not required for viral replication in vitro, this modification may be a by-product of interactions between Gag and cellular proteins during assembly and budding.
我们之前对分离和鉴定出的成熟Gag产物（HIV-1 p6(Gag)和莫洛尼鼠白血病病毒（MuLV）p12(Gag)）的HIV-1和MuLV病毒粒子进行的生化研究表明，它们与单个泛素结合。为了研究Gag单泛素化的重要性，构建了一系列赖氨酸到精氨酸的突变体，这些突变体消除了HIV-1（NL4-3）p6(Gag)中一个或两个赖氨酸以及莫洛尼MuLV p12(Gag)中两个赖氨酸的泛素化。对HIV-1突变体的高效液相色谱（HPLC）和免疫印迹分析表明，p6(Gag)中的赖氨酸K27或K33都可以被单泛素化。然而，感染性测定表明，HIV-1 p6(Gag)或MuLV p12(Gag)的单泛素化对于体外病毒复制不是必需的。对产生HIV-1的细胞进行脉冲追踪放射性标记显示，p6(Gag)的单泛素化不影响病毒从细胞中的短期释放、Pr55(Gag)的成熟或这些过程对蛋白酶体抑制剂的敏感性。对缺乏蛋白酶的HIV-1进行的实验表明，Pr55(Gag)可以被单泛素化，这表明p6(Gag)首先作为Gag中的一个结构域被修饰。对一个HIV-1突变体内的蛋白质进行检查发现，在p6(Gag)中没有赖氨酸的情况下，游离泛素被纳入病毒粒子，这表明病毒内的泛素最初不是作为p6(Gag)共轭物被带入的。尽管我们的结果表明p6(Gag)和p12(Gag)的单泛素化对于体外病毒复制不是必需的，但这种修饰可能是组装和出芽过程中Gag与细胞蛋白相互作用的副产物。
