Mellors J W, Bazmi H Z, Schinazi R F, Roy B M, Hsiou Y, Arnold E, Weir J, Mayers D L
Department of Medicine, University of Pittsburg School of Medicine, PA 15261, USA.
Antimicrob Agents Chemother. 1995 May;39(5):1087-92. doi: 10.1128/AAC.39.5.1087.
Foscarnet (phosphonoformic acid) is a pyrophosphate analog that inhibits the replication of human immunodeficiency virus type 1 (HIV-1) in vitro and in patients with AIDS. HIV-1 resistance to foscarnet has not been reported despite long-term foscarnet therapy of AIDS patients with cytomegalovirus disease. We therefore attempted to select foscarnet-resistant HIV-1 in vitro by serial endpoint passage of virus in 400 microM foscarnet. After 13 cycles of passage in MT-2 cells, virus exhibiting > or = 8.5-fold foscarnet resistance was isolated. The reverse transcriptase (RT) from resistant virions exhibited a similar level of foscarnet resistance in enzyme inhibition assays (approximately 10-fold resistance). Foscarnet-resistant virus showed increased susceptibility to 3'-azido-3'-deoxythymidine (90-fold) and to the HIV-1-specific RT inhibitors TIBO R82150 (30-fold) and nevirapine (20-fold). DNA sequence analysis of RT clones from resistant virus revealed the coexistence of two mutations in all clones: Gln-161 to Leu (CAA to CTA) and His-208 to Tyr (CAT to TAT). Sequence analysis of six clinical HIV-1 isolates showing reduced susceptibility to foscarnet revealed the Tyr-208 mutation in two, the Leu-161 mutation in one, and a Trp-88-to-Ser or -Gly mutation in four isolates. Site-specific mutagenesis and production of mutant recombinant viruses demonstrated that the Leu-161, Ser-88, and Tyr-208 mutations reduced HIV-1 susceptibility to foscarnet 10.5-, 4.3-, and 2.4-fold, respectively, in MT-2 cells. In the crystal structure of HIV-1 RT, the Gln-161 residue lies in the alpha E helix beneath the putative deoxynucleoside triphosphate (dNTP) binding site. The Gln-161-to-Leu mutation may affect the structure of the dNTP binding site and its affinity for foscarnet. The location of the Trp-88 residue in the Beta5a strand of HIV-1 RT suggest that the Ser-88 mutation affects template-primer binding, as do several mutations that affect RT susceptibility to nucleoside analogs.
膦甲酸钠（磷酰甲酸）是一种焦磷酸盐类似物，在体外和艾滋病患者体内可抑制1型人类免疫缺陷病毒（HIV-1）的复制。尽管对患有巨细胞病毒病的艾滋病患者进行了长期膦甲酸钠治疗，但尚未报道HIV-1对膦甲酸钠产生耐药性。因此，我们试图通过在400微摩尔膦甲酸钠中对病毒进行连续终点传代，在体外筛选出对膦甲酸钠耐药的HIV-1。在MT-2细胞中传代13次后，分离出对膦甲酸钠耐药性提高≥8.5倍的病毒。在酶抑制试验中，耐药病毒粒子的逆转录酶（RT）表现出相似水平的膦甲酸钠耐药性（约10倍耐药）。对膦甲酸钠耐药的病毒对3'-叠氮-3'-脱氧胸苷（90倍）、HIV-1特异性RT抑制剂TIBO R82150（30倍）和奈韦拉平（20倍）的敏感性增加。对耐药病毒的RT克隆进行DNA序列分析发现，所有克隆中均存在两种突变共存：Gln-161突变为Leu（CAA突变为CTA）和His-208突变为Tyr（CAT突变为TAT）。对6株对膦甲酸钠敏感性降低的临床HIV-1分离株进行序列分析发现，其中2株存在Tyr-208突变，1株存在Leu-161突变，4株存在Trp-88突变为Ser或Gly的突变。位点特异性诱变和突变重组病毒的产生表明，在MT-细胞中，Leu-161、Ser-88和Tyr-208突变分别使HIV-1对膦甲酸钠的敏感性降低10.5倍、4.3倍和2.4倍。在HIV-1 RT的晶体结构中，Gln-161残基位于假定的脱氧核苷三磷酸（dNTP）结合位点下方的αE螺旋中。Gln-161突变为Leu可能影响dNTP结合位点的结构及其对膦甲酸钠的亲和力。HIV-1 RT的β5a链中Trp-88残基的位置表明，Ser-88突变影响模板-引物结合，影响RT对核苷类似物敏感性的几种突变也是如此。
