Goobar-Larsson L, Bäckbro K, Unge T, Bhikhabhai R, Vrang L, Zhang H, Orvell C, Strandberg B, Oberg B
Department of Virology, Karolinska Institute, Stockholm, Sweden.
Virology. 1993 Oct;196(2):731-8. doi: 10.1006/viro.1993.1530.
The conserved aspartic acid residue 488 in the RNase H domain of HIV-1 reverse transcriptase (RT) was mutated to alanine. RT was expressed in Escherichia coli alone or with the entire pol-gene polyprotein consisting of proteinase, RT, and integrase and processed by the HIV-1 proteinase in the bacterial cell. Expression of mutant RT together with the proteinase resulted in an overproduction of RT p51 vs p66. The mutation also altered the conformation of the RT p66/p51 heterodimer as shown by the loss of binding of monoclonal antibodies to mutant RT in ELISA. Crystallographic data shows that a salt bridge exists between Asp 488 and Lys 465 of RNase H which stabilizes the uncleavable form of RT p66, and that substitution of Asp for Ala would prevent the formation of this salt bridge. Our results indicate that disruption of this salt bridge through mutation of Asp 488 interferes with the conformational changes that regulate the limited processing of p66 to 51 by the virus proteinase. Homology data suggest that such a bridge may be present in other lentiviruses. The mutation introduced caused a moderate decrease in both the RNase H activity and the polymerase activity of RT, indicating that the proper folding of the RNase H domain of RT is necessary to achieve full polymerase activity.
将 HIV-1 逆转录酶（RT）核糖核酸酶 H 结构域中保守的天冬氨酸残基 488 突变为丙氨酸。RT 可单独在大肠杆菌中表达，也可与由蛋白酶、RT 和整合酶组成的完整 pol 基因多聚蛋白一起表达，并在细菌细胞中由 HIV-1 蛋白酶进行加工处理。突变型 RT 与蛋白酶一起表达导致 RT p51 相对于 p66 的过量产生。如 ELISA 中所示，该突变还改变了 RT p66/p51 异二聚体的构象，单克隆抗体与突变型 RT 的结合丧失。晶体学数据表明，核糖核酸酶 H 的天冬氨酸 488 与赖氨酸 465 之间存在盐桥，该盐桥稳定了 RT p66 的不可切割形式，用丙氨酸替代天冬氨酸会阻止该盐桥的形成。我们的结果表明，通过天冬氨酸 488 突变破坏该盐桥会干扰由病毒蛋白酶将 p66 有限加工为 51 所调控的构象变化。同源性数据表明，其他慢病毒中可能也存在这样的桥。引入的突变导致 RT 的核糖核酸酶 H 活性和聚合酶活性均适度降低，这表明 RT 的核糖核酸酶 H 结构域的正确折叠对于实现完全的聚合酶活性是必要的。
