University of Pittsburgh School of Medicine, Department of Microbiology and Molecular Genetics, Pittsburgh, PA 15219, USA.
Retrovirology. 2010 Feb 1;7:6. doi: 10.1186/1742-4690-7-6.
The p51 subunit of the HIV-1 reverse transcriptase (RT) p66/p51 heterodimer arises from proteolytic cleavage of the RT p66 subunit C-terminal ribonuclease H (RNH) domain during virus maturation. Our previous work showed that mutations in the RT p51 downward arrowRNH cleavage site resulted in virus with defects in proteolytic processing of RT and significantly attenuated infectivity. In some cases, virus fitness was restored after repeated passage of mutant viruses, due to reversion of the mutated sequences to wild-type. However, in one case, the recovered virus retained the mutated p51 downward arrowRNH cleavage site but also developed an additional mutation, T477A, distal to the cleavage site. In this study we have characterized in detail the impact of the T477A mutation on intravirion processing of RT.
While the T477A mutation arose during serial passage only with the F440V mutant background, introduction of this substitution into a variety of RT p51 downward arrowRNH cleavage site lethal mutant backgrounds was able to restore substantial infectivity and normal RT processing to these mutants. T477A had no phenotypic effect on wild-type HIV-1. We also evaluated the impact of T477A on the kinetics of intravirion Gag-Pol polyprotein processing of p51 downward arrowRNH cleavage site mutants using the protease inhibitor ritonavir. Early processing intermediates accumulated in p51 downward arrowRNH cleavage site mutant viruses, whereas introduction of T477A promoted the completion of processing and formation of the fully processed RT p66/p51 heterodimer.
This work highlights the extraordinary plasticity of HIV-1 in adapting to seemingly lethal mutations that prevent RT heterodimer formation during virion polyprotein maturation. The ability of T477A to restore RT heterodimer formation and thus intravirion stability of the enzyme may arise from increased conformation flexibility in the RT p51 downward arrowRNH cleavage site region, due to loss of a hydrogen bond associated with the normal threonine residue, thereby enabling proteolytic cleavage near the normal RT p51 downward arrowRNH cleavage site.
HIV-1 逆转录酶（RT）p66/p51 异二聚体的 p51 亚基来自于病毒成熟过程中 RT p66 亚基 C 端核糖核酸酶 H（RNH）结构域的蛋白水解切割。我们之前的工作表明，RT p51 向下箭头 RNH 切割位点的突变导致 RT 蛋白水解加工缺陷，并显著降低病毒感染力。在某些情况下，由于突变序列回复为野生型，突变病毒的重复传代会恢复病毒的适应性。然而，在一种情况下，恢复的病毒保留了突变的 p51 向下箭头 RNH 切割位点，但也发展了一个额外的突变，T477A，位于切割位点的远端。在这项研究中，我们详细描述了 T477A 突变对病毒内 RT 加工的影响。
虽然 T477A 突变仅在 F440V 突变背景下的连续传代中出现，但将此取代引入多种 RT p51 向下箭头 RNH 切割位点致死突变背景中，能够恢复这些突变体的大量感染性和正常 RT 加工。T477A 对野生型 HIV-1 没有表型影响。我们还使用蛋白酶抑制剂利托那韦评估了 T477A 对 p51 向下箭头 RNH 切割位点突变体病毒内 Gag-Pol 多蛋白加工动力学的影响。早期加工中间体在 p51 向下箭头 RNH 切割位点突变体病毒中积累，而引入 T477A 则促进了加工的完成和完全加工的 RT p66/p51 异二聚体的形成。
这项工作强调了 HIV-1 在适应似乎致命的突变方面的非凡灵活性，这些突变在病毒多蛋白成熟过程中阻止了 RT 异二聚体的形成。T477A 能够恢复 RT 异二聚体的形成，从而恢复酶的病毒内稳定性，可能是由于 RT p51 向下箭头 RNH 切割位点区域的构象灵活性增加，因为与正常苏氨酸残基相关的氢键丧失，从而使靠近正常 RT p51 向下箭头 RNH 切割位点的蛋白水解切割成为可能。
