In vitro selection of mutations in the human immunodeficiency virus type 1 reverse transcriptase that decrease susceptibility to (-)-β-D-dioxolane-guanosine and suppress resistance to 3′-azido-3′-deoxythymidine
In vitro selection of mutations in the human immunodeficiency virus type 1 reverse transcriptase that decrease susceptibility to (-)-β-D-dioxolane-guanosine and suppress resistance to 3′-azido-3′-deoxythymidine
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DOI：
10.1128/aac.44.7.1783-1788.2000
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发表时间：
2000-07-01
影响因子：
4.9
通讯作者：
Mellors, JW
中科院分区：
文献类型：
--
作者：
Bazmi, HZ;Hammond, JL;Mellors, JW
人免疫缺陷病毒1型(HIV-1)对核苷类逆转录酶(RT)抑制剂(-)-β-D-二氧戊环-鸟苷(DXG)的抗性通过连续传代(LAI)进行筛选，经过13代和14代后，分别获得DXG半数有效浓度(EC(50))提高7.3倍和12.2倍的病毒分离株。从第一个耐药株的RT区克隆和DNA测序发现，10个克隆中有10个K65R突变(AAA到AGA)。通过对HIV-1(LAI)的定点突变，证实了该突变在DXG耐药性中的作用。K65R突变对2‘，3’-二脱氧胞苷、2‘，3’-二脱氧肌苷、2‘，3’-二脱氧-3‘-硫代胞苷、9-(2-膦甲氧乙基)腺嘌呤、2-氨基-6-氯嘌呤二氧杂环戊烷、二羟甲基-5-氟胞嘧啶和二氨基嘌呤二氧杂环戊烷的敏感性也有3倍以上的交叉抗性，但对3’-叠氮-3‘-脱氧胸腺嘧啶核苷(AZT)的敏感性影响不大。然而，当将K65R突变引入AZT抗性的遗传背景(D67N、K70R、T215Y、T219Q)时，K65R突变逆转了AZT抗性。对来自第二个耐药株的RT克隆进行DNA测序，发现了L74V突变，此前有报道称L74V突变会导致DDI耐药。当L74V突变引入AZT耐药的遗传背景(D67N、K70R、T215Y、T219Q)时，L74V突变也降低了AZT的耐药性，但程度低于K65R突变。这些发现表明，DXG和某些2‘，3’-双脱氧化合物(例如，DDI)可以选择相同的耐药突变，因此可能不是联合使用的最佳选择。然而，由于K65R和L74V突变对AZT耐药性的抑制作用，AZT与DXG或其口服生物利用型前药(-)-β-D-2，6-二氨基嘌呤-二氧戊烷的联合应用值得探讨。
Human immunodeficiency virus type 1 (HIV-1) isolates resistant to (-)-beta-D-dioxolane-guanosine (DXG), a potent and selective nucleoside analog HIV-1 reverse transcriptase (RT) inhibitor, were selected by serial passage of HIV-1(LAI) in increasing drug concentrations (maximum concentration, 30 mu M) Two independent selection experiments were performed, Viral isolates for which the DXG median effective concentrations (EC(50)s) increased 7.3- and 12.2-fold were isolated after 13 and 14 passages, respectively. Cloning and DNA sequencing of the RT region from the first resistant isolate identified a K65R mutation (AAA to AGA) in 10 of 10 clones. The role of this mutation in DXG resistance was confirmed by site-specific mutagenesis of HIV-1(LAI). The K65R mutation also conferred greater than threefold cross-resistance to 2',3'-dideoxycytidine, 2',3'-dideoxyinosine, 2',3'-dideoxy-3'-thiacytidine, 9-(2-phosphonylmethoxyethyl)adenine, 2-amino-6-chloropurine dioxolane, dioxolanyl-5-fluorocytosine, and diaminopurine dioxolane but had only marginal effects on 3'-azido-3'-deoxthymidine (AZT) susceptibility. However, when introduced into a genetic background for AZT resistance (D67N, K70R, T215Y, T219Q), the K65R mutation reversed the AZT resistance. DNA sequencing of RT clones derived from the second resistant isolate identified the L74V mutation, previously reported to cause ddI resistance. The L74V mutation also decreased the AZT resistance when the mutation was introduced into a genetic background for AZT resistance (D67N, K70R, T215Y, T219Q) but to a lesser degree than the K65R mutation did. These findings indicate that DXG and certain 2',3'-dideoxy compounds (e.g., ddI) can select for the same resistance mutations and thus may not be optimal for use in combination. However, the combination of AZT with DXG or its orally bioavailable prodrug (-)-beta-D-2,6-diaminopurine-dioxolane should be explored because of the suppressive effects of the K65R and L74V mutations on AZT resistance.