CAJ | 학술논문

目的：研究HIV-1 CRF_BC重组亚型逆转录酶（RT）区耐药相关新突变位点I132L、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-1 B亚型感染性克隆PNL4-3 RT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-1 CRF07_BC亚型RT区I132L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[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-I132L、PNL4-3-RT-T139K和PNL4-3-RT-T139R。I132L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，I132L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，I132L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-I132L/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-1 CRF_BC RT区新的耐药相关位点I132L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，I132L在CRF_07BC亚型的感染性克隆上能够降低病毒的复制能力。
목적：연구HIV-1 CRF_BC중조아형역전록매（RT）구내약상관신돌변위점I132L、T139K/R대비핵감류역전록매억제제（NNRTIs）약물민감성화병독복제동력적영향。방법통과점돌변적방법장HIV-1 B아형감염성극륭PNL4-3 RT구적제132위화제139위안기산분별돌변위량안산（L）화소안산（T）/정안산（R），여본과제조이경구건적HIV-1 CRF07_BC아형RT구I132L화T139K/R돌변감염성극륭，공동전염293T세포계진행병독포장병검험병독감염성。검측돌변병독대우NNRTIs적약물[의곡위림（TMC-125)、지랍부정(DLV)、내위랍평(NVP)、의비위륜(EFV）]민감성，급기복제동역학특정。결과통과점돌변적방법성공구건감염성극륭PNL4-3-RT-I132L、PNL4-3-RT-T139K화PNL4-3-RT-T139R。I132L화T139K/R재B아형화CRF07_BC아형중균능강저HIV-1대우NNRTIs적약물민감성，체현위반수유효농도（EC50）적승고。재CRF07_BC아형중，I132L분별사EC50승고2.55、19.35、28.05화6.13배；T139K분별사EC50승고4.67、3.66、7.35、3.30배；T139R분별사EC50승고1.82、4.69、25.12화1.89배；재B아형중，I132L분별사EC50승고3.91、4.61、6.38화3.56배；T139K분별사EC50승고3.13、1.78、2.26화2.10배；T139R분별사EC50승고5.79、3.99、5.78화2.75배。PNL4-3-RT-132L/139K/139R여야독주PNL4-3적복제속도상근，차P24균재제11천체도봉치。단여대조주BC-WT상비，BC-RT-I132L/T139R추지료P24도체봉치적시간，BC-RT-T139R적P24치재제14천체도봉치，BC-RT-I132L재제21천체도봉치，차봉치략저。결론 HIV-1 CRF_BC RT구신적내약상관위점I132L화T139K/R재B아형급CRF07_BC아형감염성극륭상균가강저병독대NNRTIs적약물민감성，I132L재CRF_07BC아형적감염성극륭상능구강저병독적복제능력。
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 CRF07_BC, and the replication ability of CRF_07BC declined by I132L mutation.