Partaledis J A, Yamaguchi K, Tisdale M, Blair E E, Falcione C, Maschera B, Myers R E, Pazhanisamy S, Futer O, Cullinan A B
Vertex Pharmaceuticals Incorporated, Cambridge, Massachusetts 02139-4211, USA.
J Virol. 1995 Sep;69(9):5228-35. doi: 10.1128/JVI.69.9.5228-5235.1995.
Human immunodeficiency virus type 1 (HIV-1) variants with reduced sensitivity to the hydroxyethylamino sulfonamide protease inhibitors VB-11,328 and VX-478 have been selected in vitro by two independent serial passage protocols with HIV-1 in CEM-SS and MT-4 cell lines. Virus populations with greater than 100-fold-increased resistance to both inhibitors compared with the parental virus have been obtained. DNA sequence analyses of the protease genes from VB-11,328- and VX-478-resistant variants reveal a sequential accumulation of point mutations, with similar resistance patterns occurring for the two inhibitors. The deduced amino acid substitutions in the resistant protease are Leu-10-->Phe, Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val. This is the first observation in HIV protease resistance studies of an Ile-50-->Val mutation, a mutation that appears to arise uniquely against the sulfonamide inhibitor class. When the substitutions observed were introduced as single mutations into an HIV-1 infectious clone (HXB2), only the Ile-50-->Val mutant showed reduced sensitivity (two- to threefold) to VB-11,328 and VX-478. A triple protease mutant infectious clone carrying the mutations Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val, however, showed much greater reduction in sensitivity (14- to 20-fold) to VB-11,328 and VX-478. The same mutations were studied in recombinant HIV protease. The mutant protease Ile-50-->Val displays a much lower affinity for the inhibitors than the parent enzyme (< or = 80-fold). The protease triply mutated at Met-46-->Ile, Ile-47-->Val, and Ile-50-->Val shows an even greater decrease in inhibitor binding (< or = 270-fold). The sulfonamide-resistant HIV protease variants remain sensitive to inhibitors from other chemical classes (Ro 31-8959 and L-735,524), suggesting possibilities for clinical use of HIV protease inhibitors in combination or serially.
通过在CEM - SS和MT - 4细胞系中用HIV - 1进行的两种独立连续传代方案，在体外筛选出了对羟乙氨基磺酰胺蛋白酶抑制剂VB - 11328和VX - 478敏感性降低的1型人类免疫缺陷病毒（HIV - 1）变体。已获得对两种抑制剂的抗性比亲代病毒高100倍以上的病毒群体。对来自VB - 11328和VX - 478抗性变体的蛋白酶基因进行DNA序列分析，揭示了点突变的顺序积累，两种抑制剂出现了相似的抗性模式。抗性蛋白酶中推导的氨基酸取代为Leu - 10→Phe、Met - 46→Ile、Ile - 47→Val和Ile - 50→Val。这是HIV蛋白酶抗性研究中首次观察到Ile - 50→Val突变，该突变似乎仅针对磺酰胺抑制剂类出现。当将观察到的取代作为单突变引入HIV - 1感染性克隆（HXB2）时，只有Ile - 50→Val突变体对VB - 11328和VX - 478的敏感性降低（两到三倍）。然而，携带Met - 46→Ile、Ile - 47→Val和Ile - 50→Val突变的三重蛋白酶突变感染性克隆对VB - 11328和VX - 478的敏感性降低幅度更大（14到20倍）。在重组HIV蛋白酶中研究了相同的突变。突变蛋白酶Ile - 50→Val对抑制剂的亲和力比亲本酶低得多（≤80倍）。在Met - 46→Ile、Ile - 47→Val和Ile - 50→Val处三重突变的蛋白酶显示出抑制剂结合能力的更大下降（≤270倍）。对磺酰胺耐药的HIV蛋白酶变体对其他化学类别的抑制剂（Ro 31 - 8959和L - 735524）仍然敏感，这表明HIV蛋白酶抑制剂联合或连续临床使用的可能性。
