HIV-1 CRF_BC重组亚型耐药相关新突变位点的复制动力学研究

Replication kinetic properties of HIV-I CRF_BC novel drug resistance associated mutations

目的研究HIV-1CRF_BC重组亚型逆转录酶（RT）区耐药相关新突变位点1132L、T139K／R对非核苷类逆转录酶抑制剂（NNRTIs）药物敏感性和病毒复制动力的影响。方法通过点突变的方法将HIV-1B亚型感染性克隆PNL4—3RT区的第132位和第139位氨基酸分别突变为亮氨酸（L）和苏氨酸（T）／精氨酸（R），与本课题组已经构建的HIV．．CRF07-BC亚型RT区1132L和T139K／R突变感染性克隆，共同转染293T细胞系进行病毒包装并检验病毒感染性。检测突变病毒对于NNRTIs的药物f依曲韦林（TMC-125）、地拉夫定（DLV）、奈韦拉平（NVP）、依非韦伦（EFV）]敏感性，及其复制动力学特征。结果通过点突变的方法成功构建感染性克隆PNL4-3-RT—1132L、PNL4—3-RT．T139K和PNL4—3-RT—T139R。1132L和T139K／R在B亚型和CRF07-BC亚型中均能降低HIV．1对于NNRTIs的药物敏感性，体现为半数有效浓度（EC50）的升高。在CRF07-BC亚型中，1132L分别使EC50升高2．55、19．35、28．05和6．13倍；T139K分别使EC50升高4．67、3．66、7．35、3．30倍；T139R分别使EC50升高1．82、4．69、25．12和1．89倍；在B亚型中，1132L分别使EC50升高3．91、4．61、6．38和3．56倍；T139K分别使EC50升高3．13、1．78、2．26和2．10倍；T139R分别使EC50升高5．79、3．99、5．78和2．75倍。PNL4．3．RT-132L／139K／139R与野毒株PNL4—3的复制速度相近，且P24均在第11天达到峰值。但与对照株BC—wT相比，BC—RT—1132L／T139R推迟了P24到达峰值的时间，BC—RT-T139R的P24值在第14天达到峰值，BC—RT-I132L在第21天达到峰值，且峰值略低。结论HIV-1CRF_BCRT区新的耐药相关位点1132L和T139K／R在B亚型及CRF07-BC亚型感染性克隆上均可降低病毒对NNRTIs的药物敏感性，1132L在CRF_07BC亚型的感染性克隆上能够降低病毒的复制能力。

Objective To study the drug sensitivity and analyze the replication kinetics of HIV-1 B and CRF07_BC subtypes with I132L or T139K/R mutations.Methods The amino acids in position 132 and 139 of reverse transcriptase （RT） region of the infectious clone PNL4-3 （HIV-1 B subtype） were changed to L and T/R through site mutagenesis. Combined with the previously constructed infectious clone of HIV-1 CRF07_BC subtype with I132L and T139K/R mutations in RT region, mutated PNL4-3 infectious clones were transfected into 293T cells. The infection ability of mutated clones was detected. The drug sensitivity to NNRTIs （TMC-125, DLV, NVP, EFV） and the properties of replication kinetics were also evaluated. Results The mutated infectious clones were constructed including PNL4-3-RT-I132L, PNL4-3-RT-T139K and PNL4-3-RT-T139R. The I132L and T139K/R mutations in HIV-1 B and CRF07_BC infectious clones reduced their drug sensitivity to NNRTIs, which accompanied with the increase of EC50 （concentration for 50% of maximal effect）. In subtype CRF07_BC, I132L mutation increased EC50 by 2.55, 19.35, 28.05, 6.13 fold, T139K mutation increased EC50 by 4.67, 3.66, 7.35, 3.30 fold, and T139R mutation increased EC50 by 1.82, 4.69, 25.12, 1.89 fold, respectively. In subtype B, I132L increased EC50 by 3.91, 4.61, 6.38, 3.56 fold, T139K increased EC50 by 3.13, 1.78, 2.26, 2.10 fold, T139R increased EC50 by 5.79, 3.99, 5.78, 2.75 fold, respectively. Similar as wild type PNL4-3, the replication ability of 132L/139K/139R mutated infectious clones reached the peak in day 11. However, compared to wild type BC-WT, I132L/T139R mutations delayed the peak time to day 14 and 21.Conclusion The novel drug resistance associated mutations I132L and T139K/R can reduce the drug sensitivity to NNRTIs in subtype B and CRFO7_BC, and the replication ability of CRF_O7BC declined by I132L mutation.