Quan Yudong, Brenner Bluma G, Oliveira Maureen, Wainberg Mark A
McGill University AIDS Centre, Lady Davis Institute-Jewish General Hospital, Montreal, Quebec, Canada.
Antimicrob Agents Chemother. 2003 Feb;47(2):747-54. doi: 10.1128/AAC.47.2.747-754.2003.
The M184V mutation in human immunodeficiency virus (HIV) reverse transcriptase is associated with high-level resistance to both (-)2',3'-dideoxy-3'-thiacytidine (3TC) and (-)2',3'-dideoxy-5-fluoro-3'-thiacytidine as well as low-level resistance to 2',3'-dideoxyinosine, 2',3'-dideoxycytidine, and abacavir. This mutation is also associated with diminished HIV replicative fitness as well as several functional changes in enzyme activity, including diminutions in polymerase processivity, pyrophosphorylysis, and nucleotide primer unblocking. Despite the fact that M184V encodes up to 1,000-fold resistance to 3TC, we asked whether this drug might still display some antiviral effect in regard to viruses containing this mutation. Cell-free assays revealed that high concentrations of 3TC triphosphate (i.e., >100 micro M) could affect chain termination and/or inhibit purified reverse transcriptase containing the M184V substitution. This effect became more pronounced with elongation of reverse transcriptase products. In newly infected cells (i.e., peripheral blood mononuclear cells), we found that the amount of full-length reverse transcriptase product was diminished in the presence of 2 to 10 micro M 3TC, although no decrease in the first product of the reverse transcriptase reaction, i.e., minus strong-stop DNA, was observed. In the presence of two other HIV inhibitors, e.g., nevirapine and indinavir, 3TC exerted additive effects in tissue culture at concentrations only marginally higher than the 50% inhibitory concentration (IC(50)). Reverse transcriptases cloned from clinical isolates harboring M184V in the context of multidrug resistance had similar IC(50) values for 3TC triphosphate compared to reverse transcriptase containing only the M184V mutation. These results suggest that viruses containing M184V can retain a higher degree of sensitivity to 3TC than previously assumed.
人类免疫缺陷病毒(HIV)逆转录酶中的M184V突变与对(-)2',3'-双脱氧-3'-硫代胞苷(3TC)和(-)2',3'-双脱氧-5-氟-3'-硫代胞苷的高水平耐药相关,同时与对2',3'-双脱氧肌苷、2',3'-双脱氧胞苷和阿巴卡韦的低水平耐药相关。该突变还与HIV复制适应性降低以及酶活性的多种功能变化有关,包括聚合酶持续合成能力、焦磷酸解作用和核苷酸引物解封作用的降低。尽管M184V编码了对3TC高达1000倍的耐药性,但我们仍想了解这种药物对含有该突变的病毒是否仍可能具有一些抗病毒作用。无细胞试验表明,高浓度的3TC三磷酸(即>100微摩尔)可影响链终止和/或抑制含有M184V替代的纯化逆转录酶。随着逆转录酶产物的延长,这种作用变得更加明显。在新感染的细胞(即外周血单核细胞)中,我们发现,在存在2至10微摩尔3TC的情况下,全长逆转录酶产物的量减少,尽管未观察到逆转录酶反应的首个产物(即负链强终止DNA)减少。在存在另外两种HIV抑制剂(例如奈韦拉平和茚地那韦)的情况下,3TC在组织培养中以仅略高于50%抑制浓度(IC50)的浓度发挥相加作用。与仅含有M184V突变的逆转录酶相比,从多重耐药背景下携带M184V的临床分离株中克隆的逆转录酶对3TC三磷酸具有相似的IC50值。这些结果表明,含有M184V的病毒对3TC的敏感性可能比先前认为的更高。