Department of Antiviral Research, Merck Research Laboratories, 770 Sumneytown Pike, WP26A-3000, West Point, PA 19486, USA.
Methods. 2009 Apr;47(4):277-82. doi: 10.1016/j.ymeth.2009.02.025. Epub 2009 Mar 13.
This report describes methods for the selection and analysis of antiretroviral resistance to HIV integrase strand transfer inhibitors (InSTIs) in cell culture. The method involves the serial passage of HIV-1 in the presence of increasing concentrations of test inhibitors, followed by the cloning and sequencing of the integrase coding region from the selected viruses. The identified mutations are subsequently re-engineered into a reference wild-type molecular clone, and the resulting replication capacity and level of drug resistance are determined relative to the wild-type virus. Here we describe examples of selection and analysis of InSTI-resistant viruses using four integrase inhibitors from three structurally distinct chemical classes; a diketo acid, two naphthyridines, and a pyrimidinecarboxamide. Each inhibitor selected an independent route to resistance. Interestingly, the shift in the IC50 required to suppress the re-engineered resistant mutant viruses closely matched the concentration of compound used during the selection of drug resistance.
本报告介绍了在细胞培养中选择和分析 HIV 整合酶链转移抑制剂（INSTIs）抗药性的方法。该方法涉及在逐渐增加的测试抑制剂浓度下连续传代 HIV-1，然后从选定的病毒中克隆和测序整合酶编码区。随后将鉴定出的突变重新设计到参考野生型分子克隆中，并相对于野生型病毒确定产生的复制能力和耐药水平。在这里，我们描述了使用来自三个结构不同的化学类别（二酮酸、两种萘啶和嘧啶甲酰胺）的四种整合酶抑制剂选择和分析 INSTI 耐药病毒的示例。每种抑制剂都选择了独立的耐药途径。有趣的是，抑制重新设计的耐药突变病毒所需的 IC50 变化与耐药选择过程中使用的化合物浓度非常匹配。
