Carrillo A, Stewart K D, Sham H L, Norbeck D W, Kohlbrenner W E, Leonard J M, Kempf D J, Molla A
Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, Illinois 60064, USA.
J Virol. 1998 Sep;72(9):7532-41. doi: 10.1128/JVI.72.9.7532-7541.1998.
ABT-378, a new human immunodeficiency virus type 1 (HIV-1) protease inhibitor which is significantly more active than ritonavir in cell culture, is currently under investigation for the treatment of AIDS. Development of viral resistance to ABT-378 in vitro was studied by serial passage of HIV-1 (pNL4-3) in MT-4 cells. Selection of viral variants with increasing concentrations of ABT-378 revealed a sequential appearance of mutations in the protease gene: I84V-L10F-M46I-T91S-V32I-I47V. Further selection at a 3.0 microM inhibitor concentration resulted in an additional change at residue 47 (V47A), as well as reversion at residue 32 back to the wild-type sequence. The 50% effective concentration of ABT-378 against passaged virus containing these additional changes was 338-fold higher than that against wild-type virus. In addition to changes in the protease gene, sequence analysis of passaged virus revealed mutations in the p1/p6 (P1' residue Leu to Phe) and p7/p1 (P2 residue Ala to Val) gag proteolytic processing sites. The p1/p6 mutation appeared in several clones derived from early passages and was present in all clones obtained from passage P11 (0.42 microM ABT-378) onward. The p7/p1 mutation appeared very late during the selection process and was strongly associated with the emergence of the additional change at residue 47 (V47A) and the reversion at residue 32 back to the wild-type sequence. Furthermore, this p7/p1 mutation was present in all clones obtained from passage P17 (3.0 microM ABT-378) onward and always occurred in conjunction with the p1/p6 mutation. Full-length molecular clones containing protease mutations observed very late during the selection process were constructed and found to be viable only in the presence of both the p7/p1 and p1/p6 cleavage-site mutations. This suggests that mutation of these gag proteolytic cleavage sites is required for the growth of highly resistant HIV-1 selected by ABT-378 and supports recent work demonstrating that mutations in the p7/p1/p6 region play an important role in conferring resistance to protease inhibitors (L. Doyon et al., J. Virol. 70:3763-3769, 1996; Y. M. Zhang et al., J. Virol. 71:6662-6670, 1997).
ABT - 378是一种新型的1型人类免疫缺陷病毒（HIV - 1）蛋白酶抑制剂，在细胞培养中其活性显著高于利托那韦，目前正处于治疗艾滋病的研究阶段。通过在MT - 4细胞中对HIV - 1（pNL4 - 3）进行连续传代，研究了体外对ABT - 378的病毒耐药性发展情况。用浓度递增的ABT - 378筛选病毒变体，结果显示蛋白酶基因中突变依次出现：I84V - L10F - M46I - T91S - V32I - I47V。在3.0微摩尔抑制剂浓度下进一步筛选，导致47位残基出现额外变化（V47A），同时32位残基又回复到野生型序列。ABT - 378对含有这些额外变化的传代病毒的50%有效浓度比对野生型病毒的高338倍。除了蛋白酶基因的变化外，传代病毒的序列分析还揭示了p1/p6（P1'残基从亮氨酸变为苯丙氨酸）和p7/p1（P2残基从丙氨酸变为缬氨酸）gag蛋白水解加工位点的突变。p1/p6突变出现在几个早期传代衍生的克隆中，并且在从第11代传代（0.42微摩尔ABT - 378）获得的所有克隆中都存在。p7/p1突变在筛选过程中出现得非常晚，并且与47位残基的额外变化（V47A）以及32位残基回复到野生型序列的出现密切相关。此外，这个p7/p1突变在从第17代传代（3.0微摩尔ABT - 378）获得的所有克隆中都存在，并且总是与p1/p6突变一起出现。构建了在筛选过程后期观察到含有蛋白酶突变的全长分子克隆，发现只有在同时存在p7/p1和p1/p6切割位点突变的情况下它们才能存活。这表明这些gag蛋白水解切割位点的突变是ABT - 378选择的高度耐药HIV - 1生长所必需的，并且支持了最近的研究工作，即p7/p1/p6区域的突变在赋予对蛋白酶抑制剂的耐药性方面起重要作用（L. 多永等人，《病毒学杂志》70:3763 - 3769，1996；Y.M. 张等人，《病毒学杂志》71:6662 - 6670，1997）。
