Gao Q, Gu Z X, Parniak M A, Li X G, Wainberg M A
Lady Davis Institute-Jewish General Hospital, Chemin Cote Ste-Catherine, Quebec, Canada.
J Virol. 1992 Jan;66(1):12-9. doi: 10.1128/JVI.66.1.12-19.1992.
Drug-resistant variants of human immunodeficiency virus type 1 (HIV-1) have been isolated by in vitro selection. MT-4 cells were infected with either a laboratory strain (HIV-IIIB) or a clinical isolate (no. 187) of HIV-1 and maintained in medium containing subeffective concentrations of the drugs 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxyinosine (ddI). By gradually increasing the drug concentration in the culture medium during propagation of the virus on fresh MT-4 cells, we were able to isolate variants of HIV-IIIB and clinical isolate 187 which showed up to 100-fold increases in resistance to the drugs. The drug resistance phenotypes remained stable after propagation of the variants in the absence of drug pressure for over 2 months. However, variants resistant to one drug showed little or no cross-resistance to the other, suggesting that the genetic bases for resistance to the compounds differed. Genotypic analysis of these nucleoside-resistant variants by polymerase chain reaction (PCR) with primer pairs previously shown to correspond to mutations responsible for resistance to AZT was also carried out. A heterogeneity of genotypes was observed, with known mutations at pol codons 70 and 215 occurring in most of the AZT-resistant variants generated from either HIV-IIIB or clinical strain 187. However, mutations in codons 67 and 219 were less frequently detected, and none of these changes were observed in each of four variants resistant to ddI. Cloning and sequencing studies of the reverse transcriptase coding region of two of the isolates were also performed and confirmed the PCR data that had been obtained. In addition to previously described mutation sites responsible for resistance to AZT, an HIV-IIIB-resistant variant was shown to be mutated at positions 108 (Val----Ala) and 135 (Ile----Thr), while a resistant variant of strain 187 was mutated at positions 50 (Ile----Val) and 135 (Ile----Val).
通过体外筛选已分离出1型人类免疫缺陷病毒（HIV-1）的耐药变异株。用HIV-1的实验室毒株（HIV-IIIB）或临床分离株（187号）感染MT-4细胞，并将其置于含有亚有效浓度药物3'-叠氮-3'-脱氧胸苷（AZT）和2'，3'-双脱氧肌苷（ddI）的培养基中培养。在病毒于新鲜MT-4细胞上增殖期间，通过逐渐提高培养基中的药物浓度，我们得以分离出HIV-IIIB和临床分离株187的变异株，这些变异株对药物的耐药性提高了高达100倍。在无药物压力的情况下，这些变异株传代培养2个多月后，耐药表型仍保持稳定。然而，对一种药物耐药的变异株对另一种药物几乎没有或完全没有交叉耐药性，这表明对这两种化合物耐药的遗传基础不同。还通过聚合酶链反应（PCR）对这些核苷耐药变异株进行了基因分型分析，所用引物对先前已证明与导致对AZT耐药的突变相对应。观察到基因型存在异质性，在由HIV-IIIB或临床毒株187产生的大多数AZT耐药变异株中，pol密码子70和215处出现了已知突变。然而，密码子67和219处的突变较少被检测到，并且在对ddI耐药的四个变异株中的每一个中均未观察到这些变化。还对其中两个分离株的逆转录酶编码区进行了克隆和测序研究，证实了所获得的PCR数据。除了先前描述的导致对AZT耐药的突变位点外，一个HIV-IIIB耐药变异株在第108位（缬氨酸→丙氨酸）和第135位（异亮氨酸→苏氨酸）发生了突变，而毒株187的一个耐药变异株在第50位（异亮氨酸→缬氨酸）和第135位（异亮氨酸→缬氨酸）发生了突变。
