Gu Z, Arts E J, Parniak M A, Wainberg M A
McGill University AIDS Center, Lady Davis Institute-Jewish General Hospital, Montreal, QC, Canada.
Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2760-4. doi: 10.1073/pnas.92.7.2760.
A lysine-to-arginine substitution at amino acid 65 (K65R) in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is associated with resistance to 2',3'-dideoxycytidine (ddC), 2',3'-dideoxyinosine (ddI), and the (-) enantiomer of 2',3'-dideoxy-3'-thiacytidine (3TC). To further characterize the molecular basis of such resistance, we expressed the pp6/p51 heterodimer of wild-type RT, K65R mutated RT, and a doubly mutated (K65R/M184V) RT in Escherichia coli and assessed the characteristics of nucleotide incorporation and chain termination in cell-free reverse transcription reactions in the presence and absence of various nucleoside triphosphate analogs. These reactions employed a HIV RNA template (HIV-PBS) that contained the primer binding sequence (PBS) and the U5 and R regions of HIV-1 genomic RNA and an oligodeoxynucleotide (dPR) complementary to the HIV-1 PBS as primer. The K65R and K65R/M184V RTs showed significantly decreased chain-termination effects during polymerization with the 5'-triphosphates of ddC, 3TC, 2',3'-dideoxyadenosine, and AZT (3'-azido-3'-deoxythymidine) in comparison with wild-type RT. Detailed analysis with ddCTP and wild-type RT revealed that chain termination occurred at all guanines in the RNA template. However, the frequency of dideoxynucleoside triphosphate (ddNTP)-induced chain termination was decreased at certain guanines but not others in reactions catalyzed by K65R RT. Both the K65R mutant RT and wild-type RT had similar processive activity. These results indicate that decreased chain termination of K65R RT in the presence of ddNTPs is consistent with data obtained in viral replication assays.
人类免疫缺陷病毒1型（HIV-1）逆转录酶（RT）中第65位氨基酸（K65R）的赖氨酸到精氨酸替换与对2'，3'-双脱氧胞苷（ddC）、2'，3'-双脱氧肌苷（ddI）以及2'，3'-双脱氧-3'-硫代胞苷（3TC）的（-）对映体的耐药性相关。为了进一步阐明这种耐药性的分子基础，我们在大肠杆菌中表达了野生型RT、K65R突变型RT和双突变（K65R/M184V）RT的pp6/p51异二聚体，并在有无各种核苷三磷酸类似物存在的情况下，评估了无细胞逆转录反应中核苷酸掺入和链终止的特性。这些反应使用了一个HIV RNA模板（HIV-PBS），它包含引物结合序列（PBS）以及HIV-1基因组RNA的U5和R区域，还有一个与HIV-1 PBS互补的寡脱氧核苷酸（dPR）作为引物。与野生型RT相比，K65R和K65R/M184V RT在与ddC、3TC、2'，3'-双脱氧腺苷和叠氮胸苷（3'-叠氮-3'-脱氧胸苷，AZT）的5'-三磷酸进行聚合反应时，链终止效应显著降低。用ddCTP和野生型RT进行的详细分析表明，链终止发生在RNA模板中的所有鸟嘌呤处。然而，在K65R RT催化的反应中，双脱氧核苷三磷酸（ddNTP）诱导的链终止频率在某些鸟嘌呤处降低，但在其他鸟嘌呤处未降低。K65R突变型RT和野生型RT具有相似的持续合成活性。这些结果表明，在ddNTP存在的情况下，K65R RT链终止的减少与病毒复制试验中获得的数据一致。
