Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Bethesda, Maryland 20892, USA.
Biochemistry. 2010 May 4;49(17):3715-22. doi: 10.1021/bi100130f.
Resistance to raltegravir (RAL), the first HIV-1 integrase (IN) inhibitor approved by the FDA, involves three genetic pathways: IN mutations N155H, Q148H/R/K, and Y143H/R/C. Those mutations are generally associated with secondary point mutations. The resulting mutant viruses show a high degree of resistance against RAL but somehow are affected in their replication capacity. Clinical and virological data indicate the high relevance of the combination G140S + Q148H because of its limited impact on HIV replication and very high resistance to RAL. Here, we report how mutations at the amino acid residues 140, 148, and 155 affect IN enzymatic activity and RAL resistance. We show that single mutations at position 140 have limited impact on 3'-processing (3'-P) but severely inactivate strand transfer (ST). On the other hand, single mutations at position 148 have a more profound effect and inactivate both 3'-P and ST. By examining systematically all of the double mutants at the 140 and 148 positions, we demonstrate that only the combination G140S + Q148H is able to restore the catalytic properties of IN. This rescue only operates in cis when both the 140S and 148H mutations are in the same IN polypeptide flexible loop. Finally, we show that the G140S-Q148H double mutant exhibits the highest resistance to RAL. It also confers cross-resistance to elvitegravir but less to G-quadraduplex inhibitors such as zintevir. Our results demonstrate that IN mutations at positions 140 and 148 in the IN flexible loop can account for the phenotype of RAL-resistant viruses.
对拉替拉韦（RAL）的耐药性，RAL 是 FDA 批准的首个 HIV-1 整合酶（IN）抑制剂，涉及三种遗传途径：IN 突变 N155H、Q148H/R/K 和 Y143H/R/C。这些突变通常与次要的点突变相关。由此产生的突变病毒对 RAL 表现出高度的耐药性，但在复制能力上受到一定影响。临床和病毒学数据表明，G140S+Q148H 的组合具有高度相关性，因为它对 HIV 复制的影响有限，而且对 RAL 的耐药性非常高。在这里，我们报告氨基酸残基 140、148 和 155 上的突变如何影响 IN 的酶活性和 RAL 的耐药性。我们表明，位置 140 上的单个突变对 3'-加工（3'-P）的影响有限，但严重失活链转移（ST）。另一方面，位置 148 上的单个突变具有更深远的影响，使 3'-P 和 ST 都失活。通过系统地检查 140 和 148 位置的所有双突变体，我们证明只有 G140S+Q148H 组合能够恢复 IN 的催化特性。这种挽救仅在顺式操作，即当 140S 和 148H 突变都在同一个 IN 多肽柔性环中时。最后，我们表明 G140S-Q148H 双突变体对 RAL 表现出最高的耐药性。它还赋予对艾维雷韦的交叉耐药性，但对 G-四链体抑制剂如齐特维的耐药性较低。我们的结果表明，IN 柔性环中位置 140 和 148 的 IN 突变可以解释 RAL 耐药病毒的表型。
