对非核苷类逆转录酶抑制剂耐药型HIV-1药理模型的建立
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摘要
艾滋病又称获得性免疫缺陷综合症(acquired immune- odeficiency syndrome, AIDS),是由人类免疫缺陷病毒(human immunodeficiency virus, HIV)所引起的致命性慢性传染病。HIV主要侵犯、破坏辅助性T淋巴细胞,导致机体细胞免疫功能受损,最后并发各种严重机会性感染和肿瘤。
艾滋病目前尚无特效治疗方法,早期抗病毒治疗是关键。既能缓解病情,减少机会性感染和肿瘤,又能预防或延缓AIDS相关疾病的发生,延长生存期,提高生活质量。根据HIV生命周期中的主要环节,抗HIV药物分为逆转录酶抑制剂、蛋白酶抑制剂、整合酶抑制剂和融合抑制剂四大类。逆转录酶抑制剂是最早用于临床的抗HIV药物,包括核苷类和非核苷类两种。其中非核苷类逆转录酶抑制剂由于其低毒高效而受到关注,但由于其抗耐药屏障作用较弱,使得这类药物在应用一段时间后很容易发生耐药。因此,研究和开发具有抗耐药作用的新型非核苷类药物成为发展方向,而与之相应的药物筛选方法成为关键环节。
目的:在细胞水平建立三种针对HIV-1复制环节的药物筛选模型,其对现有的非核苷类逆转录酶抑制剂(non-nucleoside reverse- transcriptase inhibitors, NNRTIs)具有特异的耐药性,可为新型NNRTIs研发提供筛选平台。
方法:
(1)应用PCR法扩增含突变位点K103N,K103N/P225H,K103N/G190A的三段目的片段,长度为1.5Kb,介于ApaI与AgeI酶切位点间。
(2)用限制性内切酶ApaI和AgeI分别切割目的片段和HIV-1质粒。
(3) T4连接酶将含相同粘末端的目的片段与HIV-1质粒进行连接,构建新的含突变位点的HIV-1质粒,分别命名为HIV-K103N、HIV-K103N/P225H、HIV-K103N/G190A。
(4)将HIV-K103N、HIV- K103N/P225H、HIV-K103N/G190A分别与水泡性口膜炎病毒的外壳糖蛋白的质粒(Vesicular stomatitis virus glycoprotein, VSV-G)在293细胞中共转染,组装成假病毒模型,分别命名为VSVG/HIV-K103N、VSVG/HIV- K103N/P225H、VSVG/HIV-K103N/G190A,模型携带的荧光素酶报告基因的表达水平可以反映HIV-1的复制程度。
(5)对三种假病毒模型按不同的稀释比(1:10,1:25,1:50,1:100,1:200和1:400)稀释后感染293细胞,用FB15荧光检测器测定细胞中荧光素酶的相对活性(Relative Luciferase activity units, RLUs),并绘制病毒滴度-荧光素酶相对活性曲线。
(6)用核苷类阳性对照药齐多夫定(zidovudine, AZT)和司他夫定(stavudine, d4T);非核苷类阳性对照药奈韦拉平(nevirapine, NVP)和依非韦伦(efavirenz, EFV)分别对三种假病毒模型进行抑制性检测,并用野生株HIV-1假病毒作对照,验证模型的可靠性。
结果:
(1)通过PCR成功扩增出1.5Kb的目的片段,再通过分子克隆方法构建新的HIV-1质粒(HIV-K103N、HIV- K103N/P225H、HIV-K103N/G190A),经过DNA测序分析证实突变引入成功。
(2)对假病毒模型滴度检测证实三种假病毒都具有高度感染能力,VSVG/HIV-K103N和VSVG/HIV- K103N/P225H按1:10稀释后感染293细胞,其RLUs达到3x107,这也是FB15荧光检测器的最大检测值;VSVG/HIV-K103N/G190A按1:10稀释后感染293细胞,其RLUs也达到2x107。病毒滴度-荧光素酶相对活性曲线可以清楚的反应出HIV-1复制水平与荧光素酶报告基因表达水平间的剂量依赖效应。因此,三种假病毒可用于针对HIV-1复制环节的药物筛选模型。
(3) VSVG/HIV-K103N、VSVG/HIV- K103N/P225H和VSVG/HIV- K103N/G190A以及VSVG/HIV-wild可有效的在细胞内进行复制,非核苷类阳性对照药NVP和EFV可有效抑制VSVG/HIV-wild,其IC50分别为0.024±0.006μmol/L和0.83±0.21nmol/L ,与文献中应用其它方法得出的IC50(0.046μmol/L和1.3 nmol/L)基本一致。NVP也可有效抑制VSVG/HIV-K103N、VSVG/HIV-K103N/P225H和VSVG/HIV-K103N/G190A,但IC50明显升高,分别为1.89±0.92μmol/L、5.13±0.58μmol/L和27.61±4.796μmol/L,与文献中应用其它方法得出的IC50(2.21μmol/L、6.12μmol/L和23μmol/L)相一致。EFV也可有效抑制VSVG/HIV-K103N、VSVG/HIV-K103N/P225H和VSVG/HIV- K103N/G190A,IC50也明显升高,分别为16.7±6.0nmol/L、0.1103±0.0328μmol/L和0.1697±0.1147μmol/L,与文献中应用其它方法得出的IC50 (26nmol/L、0.1742μmol/L和0.277μmol/L)相符合。计算三种假病毒对NVP和EFV的耐药倍数(耐药株病毒IC50/野生株病毒IC50),为15～1000倍,说明此三种假病毒模型对NNRTIs产生耐药性。
(4)核苷类阳性对照药AZT和d4T可有效抑制VSVG/HIV-K103N、VSVG/HIV-K103N/P225H和VSVG/HIV- K103N/G190A以及VSVG/HIV-wild,其IC50未有明显变化,约为9.9±1.4nmol/L和0.153±0.034μmol/L,与文献中报道的活病毒IC50,以及实验室应用其它方法得出IC50相符合,说明此三种假病毒模型对NRTIs不产生耐药性。
结论:
HIV-1假病毒模型是针对HIV-1复制环节的细胞水平药理筛选模型,其携带的荧光报告基因的表达水平可以反映HIV-1复制的程度;通过PCR及分子生物学方法将文献报道的针对NNRTIs耐药的K103N、K103N/P225H和K103N/G190A三组突变位点定点引入HIV-1假病毒模型,构建出新的假病毒模型;通过核苷类及非核苷类阳性对照药对模型的验证,证实模型对NNRTIs有特异的耐药性,可为新型非核苷类化合物的研发提供一个安全、高效、稳定、快速的筛选平台。
The AIDS called acquired immunodeficiency syndrome is a fatal chronic infectious disease which caused by human immunodeficiency virus. HIV mainly infringes upon and destroys helper lymphocyte T, leading to damage the cellular immunity function and producing a series of opportunity infection and tumor.
The AIDS has no special method to cure at present, and the antiviral therapy in early phase is the key point. It can not only relieve state of illness and reduce the opportunity infection and tumor, but also can take precautions against or delay diseases which caused by AIDS, prolong a life cycle, and enhance the quality of life. According to the key steps of HIV life cycle, the HIV medicine includes four classes: reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors and fusion inhibitors. The reverse transcriptase inhibitors are used in clinic in the most early stage, including nucleoside and non-nucleoside. The non-nucleoside reverse transcriptase inhibitors are paid attention because their high effect in low dose, but the barrier of resisting drug resistance is weak. So they are easy to develop drug resistance after a short time. The study and development of novel non-nucleoside reverse transcriptase inhibitors which can overcome drug resistance are direction of advancement, and the method of drug screening becomes a key step.
Aim: Establishing three cell-based NNRTIs resistance models for drug screening (to study HIV-1 reproduction, which resist to NNRTIs available now). It can provide a screening platform for new NNRTIs developement.
Method:
(1) Amplified three segments by PCR are 1.5 Kb and span ApaI and AgeI restriction sites, including mutable points (K103N、K103N/P225H or K103N/G190A).
(2) Cut segments and HIV-1 plasmid by ApaI and AgeI restriction enzyme.
(3) Ligate the segment with HIV-1 plasmid which has the same terminal by T4 ligase to construct the new HIV-1 plasmid which have mutable points. We name the new plasmids HIV-K103N、HIV- K103N/P225H and HIV-K103N/G190A, respectively.
(4) HIV-K103N、HIV-K103N/P225H and HIV-K103N/G190A cotran- sfected with Vesicular stomatitis virus glycoprotein(VSV-G) in 293 cells, and composed pseudotyped virus models, which named VSVG/HIV-K103N、VSVG/HIV- K103N/P225H、VSVG/HIV- K103N/G190A, respectively. The expression levels of the luciferase report genes of these models could reflect the levels of HIV-1 reproduction.
(5) Diluted these pseudotyped virions in different proportion(1:10,1:25,1:50,1:100,1:200 and 1:400), then infected 293 cells. Determined Relative Luciferase activity units (RLUs) in 293 cells by using FB15 fluorescence detector, and drew Virus titer-RLUs curve.
(6) Detected the inhibition of these pseudotyped virions with AZT、d4T (nucleoside inhibitors) and NVP、EFV (non- nucleoside inhibitors). In order to check the confidence of these models, wild type of pseudotyped virions were also compared.
Results:
(1) Successfully amplified these 1.5Kb purpose segments by PCR, then construct new HIV-1 plasmids(HIV-K103N、HIV- K103N/P225H and HIV-K103N/G190A) by molecular cloning methods. The mutations were confirmed by DNA sequencing analysis.
(2) It is that these pseudotyped virions all had highly infected abilities by examining the dilutes of the virions. The RLUs of 293 cells which infected by VSVG/HIV-K103N or VSVG/HIV- K103N/P225H (diluted by 1:10) was 3×107, which is the maximal value of the FB15 fluorescence detector. The RLUs of 293 cells infected by VSVG/HIV-K103N/G190A (diluted by 1:10) was 2×107. The Virus titer-RLUs curve could express clearly dose-dependent effect between the level of the viral replication and the expression level of the luciferase report gene. So these pseudotyped virus models could be used to screening the drugs which aimed directly at the reproduction of HIV-1.
(3) VSVG/HIV-K103N、VSVG/HIV-K103N/P225H、VSVG/HIV- K103N/G190A and VSVG/HIV-wild could reproduce effectively in the cells. The NVP and EFV could inhibit the reproduction of VSVG/HIV-wild effectively, and the IC50 were 0.024±0.006μmol/L and 0.83±0.21nmol/L. They were coincidence to the report (0.046μmol/L and 1.3 nmol/L). NVP also could inhibit the reproduction of VSVG/HIV-K103N、VSVG/HIV- K103N/P225H and VSVG/HIV- K103N/G190A effectively, and the IC50 were 1.89±0.92μmol/L、5.13±0.58μmol/L and 27.61±4.796μmol/L, which were higher than that of VSVG/HIV-wild, and were coincidence to the report (2.21μmol/L、6.12μmol/L and 23μmol/L). At the same time, the IC50 of EFV were 16.7±6.0nmol/L、0.1103±0.0328μmol/L and 0.1697±0.1147μmol/L, which were also coincidence to the report (26nmol/L、0.1742μmol/L and 0.277μmol/L). The fold of drug resistance was 15～1000(IC50 of mutable virus / IC50 of wild type virus). Therefore, these pseudotyped virus models resist to the NNRTIs.
(4) AZT and d4T could inhibit the reproduction of VSVG/HIV-K103N、VSVG/HIV-K103N/P225H and VSVG/HIV- K103N/G190A, and the IC50 were 9.9±1.4nmol/L and 0.153±0.034μmol/L, which had not marked change to the VSVG/HIV-wild, and were also coincidence to the report. So these models did not resist to NRTIs.
Conclusion: The HIV-1 pseudotyped virions was the cell-based models for drug screening to study HIV-1 reproduction, and the expression level of the luciferase report genes could reflect the levels of HIV-1 reproduction. Led K103N、K103N/P225H and K103N/G190A to pseudotyped virus model by PCR and molecular biology methods, in order to create the new pseudotyped virus models. Confirmed the confidence of these models by nucleoside and non-nucleoside inhibitors. The pseudotyped virus models which resist to NNRTIs can provide a safe and high performance screening method to screening new types of non-nucleoside compounds.
艾滋病目前尚无特效治疗方法,早期抗病毒治疗是关键。既能缓解病情,减少机会性感染和肿瘤,又能预防或延缓AIDS相关疾病的发生,延长生存期,提高生活质量。根据HIV生命周期中的主要环节,抗HIV药物分为逆转录酶抑制剂、蛋白酶抑制剂、整合酶抑制剂和融合抑制剂四大类。逆转录酶抑制剂是最早用于临床的抗HIV药物,包括核苷类和非核苷类两种。其中非核苷类逆转录酶抑制剂由于其低毒高效而受到关注,但由于其抗耐药屏障作用较弱,使得这类药物在应用一段时间后很容易发生耐药。因此,研究和开发具有抗耐药作用的新型非核苷类药物成为发展方向,而与之相应的药物筛选方法成为关键环节。
目的:在细胞水平建立三种针对HIV-1复制环节的药物筛选模型,其对现有的非核苷类逆转录酶抑制剂(non-nucleoside reverse- transcriptase inhibitors, NNRTIs)具有特异的耐药性,可为新型NNRTIs研发提供筛选平台。
方法:
(1)应用PCR法扩增含突变位点K103N,K103N/P225H,K103N/G190A的三段目的片段,长度为1.5Kb,介于ApaI与AgeI酶切位点间。
(2)用限制性内切酶ApaI和AgeI分别切割目的片段和HIV-1质粒。
(3) T4连接酶将含相同粘末端的目的片段与HIV-1质粒进行连接,构建新的含突变位点的HIV-1质粒,分别命名为HIV-K103N、HIV-K103N/P225H、HIV-K103N/G190A。
(4)将HIV-K103N、HIV- K103N/P225H、HIV-K103N/G190A分别与水泡性口膜炎病毒的外壳糖蛋白的质粒(Vesicular stomatitis virus glycoprotein, VSV-G)在293细胞中共转染,组装成假病毒模型,分别命名为VSVG/HIV-K103N、VSVG/HIV- K103N/P225H、VSVG/HIV-K103N/G190A,模型携带的荧光素酶报告基因的表达水平可以反映HIV-1的复制程度。
(5)对三种假病毒模型按不同的稀释比(1:10,1:25,1:50,1:100,1:200和1:400)稀释后感染293细胞,用FB15荧光检测器测定细胞中荧光素酶的相对活性(Relative Luciferase activity units, RLUs),并绘制病毒滴度-荧光素酶相对活性曲线。
(6)用核苷类阳性对照药齐多夫定(zidovudine, AZT)和司他夫定(stavudine, d4T);非核苷类阳性对照药奈韦拉平(nevirapine, NVP)和依非韦伦(efavirenz, EFV)分别对三种假病毒模型进行抑制性检测,并用野生株HIV-1假病毒作对照,验证模型的可靠性。
结果:
(1)通过PCR成功扩增出1.5Kb的目的片段,再通过分子克隆方法构建新的HIV-1质粒(HIV-K103N、HIV- K103N/P225H、HIV-K103N/G190A),经过DNA测序分析证实突变引入成功。
(2)对假病毒模型滴度检测证实三种假病毒都具有高度感染能力,VSVG/HIV-K103N和VSVG/HIV- K103N/P225H按1:10稀释后感染293细胞,其RLUs达到3x107,这也是FB15荧光检测器的最大检测值;VSVG/HIV-K103N/G190A按1:10稀释后感染293细胞,其RLUs也达到2x107。病毒滴度-荧光素酶相对活性曲线可以清楚的反应出HIV-1复制水平与荧光素酶报告基因表达水平间的剂量依赖效应。因此,三种假病毒可用于针对HIV-1复制环节的药物筛选模型。
(3) VSVG/HIV-K103N、VSVG/HIV- K103N/P225H和VSVG/HIV- K103N/G190A以及VSVG/HIV-wild可有效的在细胞内进行复制,非核苷类阳性对照药NVP和EFV可有效抑制VSVG/HIV-wild,其IC50分别为0.024±0.006μmol/L和0.83±0.21nmol/L ,与文献中应用其它方法得出的IC50(0.046μmol/L和1.3 nmol/L)基本一致。NVP也可有效抑制VSVG/HIV-K103N、VSVG/HIV-K103N/P225H和VSVG/HIV-K103N/G190A,但IC50明显升高,分别为1.89±0.92μmol/L、5.13±0.58μmol/L和27.61±4.796μmol/L,与文献中应用其它方法得出的IC50(2.21μmol/L、6.12μmol/L和23μmol/L)相一致。EFV也可有效抑制VSVG/HIV-K103N、VSVG/HIV-K103N/P225H和VSVG/HIV- K103N/G190A,IC50也明显升高,分别为16.7±6.0nmol/L、0.1103±0.0328μmol/L和0.1697±0.1147μmol/L,与文献中应用其它方法得出的IC50 (26nmol/L、0.1742μmol/L和0.277μmol/L)相符合。计算三种假病毒对NVP和EFV的耐药倍数(耐药株病毒IC50/野生株病毒IC50),为15～1000倍,说明此三种假病毒模型对NNRTIs产生耐药性。
(4)核苷类阳性对照药AZT和d4T可有效抑制VSVG/HIV-K103N、VSVG/HIV-K103N/P225H和VSVG/HIV- K103N/G190A以及VSVG/HIV-wild,其IC50未有明显变化,约为9.9±1.4nmol/L和0.153±0.034μmol/L,与文献中报道的活病毒IC50,以及实验室应用其它方法得出IC50相符合,说明此三种假病毒模型对NRTIs不产生耐药性。
结论:
HIV-1假病毒模型是针对HIV-1复制环节的细胞水平药理筛选模型,其携带的荧光报告基因的表达水平可以反映HIV-1复制的程度;通过PCR及分子生物学方法将文献报道的针对NNRTIs耐药的K103N、K103N/P225H和K103N/G190A三组突变位点定点引入HIV-1假病毒模型,构建出新的假病毒模型;通过核苷类及非核苷类阳性对照药对模型的验证,证实模型对NNRTIs有特异的耐药性,可为新型非核苷类化合物的研发提供一个安全、高效、稳定、快速的筛选平台。
The AIDS called acquired immunodeficiency syndrome is a fatal chronic infectious disease which caused by human immunodeficiency virus. HIV mainly infringes upon and destroys helper lymphocyte T, leading to damage the cellular immunity function and producing a series of opportunity infection and tumor.
The AIDS has no special method to cure at present, and the antiviral therapy in early phase is the key point. It can not only relieve state of illness and reduce the opportunity infection and tumor, but also can take precautions against or delay diseases which caused by AIDS, prolong a life cycle, and enhance the quality of life. According to the key steps of HIV life cycle, the HIV medicine includes four classes: reverse transcriptase inhibitors, protease inhibitors, integrase inhibitors and fusion inhibitors. The reverse transcriptase inhibitors are used in clinic in the most early stage, including nucleoside and non-nucleoside. The non-nucleoside reverse transcriptase inhibitors are paid attention because their high effect in low dose, but the barrier of resisting drug resistance is weak. So they are easy to develop drug resistance after a short time. The study and development of novel non-nucleoside reverse transcriptase inhibitors which can overcome drug resistance are direction of advancement, and the method of drug screening becomes a key step.
Aim: Establishing three cell-based NNRTIs resistance models for drug screening (to study HIV-1 reproduction, which resist to NNRTIs available now). It can provide a screening platform for new NNRTIs developement.
Method:
(1) Amplified three segments by PCR are 1.5 Kb and span ApaI and AgeI restriction sites, including mutable points (K103N、K103N/P225H or K103N/G190A).
(2) Cut segments and HIV-1 plasmid by ApaI and AgeI restriction enzyme.
(3) Ligate the segment with HIV-1 plasmid which has the same terminal by T4 ligase to construct the new HIV-1 plasmid which have mutable points. We name the new plasmids HIV-K103N、HIV- K103N/P225H and HIV-K103N/G190A, respectively.
(4) HIV-K103N、HIV-K103N/P225H and HIV-K103N/G190A cotran- sfected with Vesicular stomatitis virus glycoprotein(VSV-G) in 293 cells, and composed pseudotyped virus models, which named VSVG/HIV-K103N、VSVG/HIV- K103N/P225H、VSVG/HIV- K103N/G190A, respectively. The expression levels of the luciferase report genes of these models could reflect the levels of HIV-1 reproduction.
(5) Diluted these pseudotyped virions in different proportion(1:10,1:25,1:50,1:100,1:200 and 1:400), then infected 293 cells. Determined Relative Luciferase activity units (RLUs) in 293 cells by using FB15 fluorescence detector, and drew Virus titer-RLUs curve.
(6) Detected the inhibition of these pseudotyped virions with AZT、d4T (nucleoside inhibitors) and NVP、EFV (non- nucleoside inhibitors). In order to check the confidence of these models, wild type of pseudotyped virions were also compared.
Results:
(1) Successfully amplified these 1.5Kb purpose segments by PCR, then construct new HIV-1 plasmids(HIV-K103N、HIV- K103N/P225H and HIV-K103N/G190A) by molecular cloning methods. The mutations were confirmed by DNA sequencing analysis.
(2) It is that these pseudotyped virions all had highly infected abilities by examining the dilutes of the virions. The RLUs of 293 cells which infected by VSVG/HIV-K103N or VSVG/HIV- K103N/P225H (diluted by 1:10) was 3×107, which is the maximal value of the FB15 fluorescence detector. The RLUs of 293 cells infected by VSVG/HIV-K103N/G190A (diluted by 1:10) was 2×107. The Virus titer-RLUs curve could express clearly dose-dependent effect between the level of the viral replication and the expression level of the luciferase report gene. So these pseudotyped virus models could be used to screening the drugs which aimed directly at the reproduction of HIV-1.
(3) VSVG/HIV-K103N、VSVG/HIV-K103N/P225H、VSVG/HIV- K103N/G190A and VSVG/HIV-wild could reproduce effectively in the cells. The NVP and EFV could inhibit the reproduction of VSVG/HIV-wild effectively, and the IC50 were 0.024±0.006μmol/L and 0.83±0.21nmol/L. They were coincidence to the report (0.046μmol/L and 1.3 nmol/L). NVP also could inhibit the reproduction of VSVG/HIV-K103N、VSVG/HIV- K103N/P225H and VSVG/HIV- K103N/G190A effectively, and the IC50 were 1.89±0.92μmol/L、5.13±0.58μmol/L and 27.61±4.796μmol/L, which were higher than that of VSVG/HIV-wild, and were coincidence to the report (2.21μmol/L、6.12μmol/L and 23μmol/L). At the same time, the IC50 of EFV were 16.7±6.0nmol/L、0.1103±0.0328μmol/L and 0.1697±0.1147μmol/L, which were also coincidence to the report (26nmol/L、0.1742μmol/L and 0.277μmol/L). The fold of drug resistance was 15～1000(IC50 of mutable virus / IC50 of wild type virus). Therefore, these pseudotyped virus models resist to the NNRTIs.
(4) AZT and d4T could inhibit the reproduction of VSVG/HIV-K103N、VSVG/HIV-K103N/P225H and VSVG/HIV- K103N/G190A, and the IC50 were 9.9±1.4nmol/L and 0.153±0.034μmol/L, which had not marked change to the VSVG/HIV-wild, and were also coincidence to the report. So these models did not resist to NRTIs.
Conclusion: The HIV-1 pseudotyped virions was the cell-based models for drug screening to study HIV-1 reproduction, and the expression level of the luciferase report genes could reflect the levels of HIV-1 reproduction. Led K103N、K103N/P225H and K103N/G190A to pseudotyped virus model by PCR and molecular biology methods, in order to create the new pseudotyped virus models. Confirmed the confidence of these models by nucleoside and non-nucleoside inhibitors. The pseudotyped virus models which resist to NNRTIs can provide a safe and high performance screening method to screening new types of non-nucleoside compounds.
引文
1 The Joint United Nations Programme on HIV/AIDS (UNAIDS) and World Health Organization (WHO):2007 AIDS epidemic update. Geneva: UNAIDS and WHO, 2007
2 Van Hal SJ, Muthiah K, Matthews G, et al. Declining incidence of intestinal microsporidiosis and reduction in AIDS-related mortality following introduction of HAART in Sydney, Australia. Trans R Soc Trop Med Hyg, 2007, 101: 1096-1100
3 Ilina T, Parniak MA. Inhibitiors of HIV-1 reverse transcriptase. Adv Pharmacol, 2008, 56: 121-167
4 ZHOU Ting, XIE Lan. Progress in HIV non-nucleoside reverse transcriptase inhibitors (NNRTIs). Acta Pharm Sin (药学学报), 2004, 39:666-672
5 Boone LR. Next generation HIV-1 non-nucleoside reverse transcriptase inhibitors. Curr Opin Investig Drugs, 2006, 7:128-135
6 Blair WS, Isaacson JS, Li X, et al. A novel HIV-1 antiviral high throughput screening approach for the discovery of HIV-1 inhibitors. Antiviral Res, 2005, 65: 107-116
7 He J, Choe S, Walker R, et al. Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity. J Virol, 1995, 69:6705-6711
8 Connor RI, Chen BK, Choe S, et al. Vpr is required for efficient replication of human immunodeficiency virus type-1in mononuclear phagocytes. Virology, 1995, 206:935-944
9 Vivet-Boudou V, Didierjean J, Isel C, et al. Nucleoside and nucleotide inhibitors of HIV-1 replication. Cell Mol Life Sci, 2006, 63:163-186
10 Cao J, Isaacson J, Patick AK, et al. High-throughput human immunodeficiency virus type1 (HIV-1) full replication assay that includes HIV-1 Vif as an antiviral target. Antimicrob Agents Chemother, 2005, 49:3833-3841
11 Domaoal RA, Demeter LM. Structural and biochemical effects of human immunodeficiency virus mutant resistant to non-nucleoside reverse transcriptase inhibitors. J Biochem Cell Biol, 2004, 36:1735-1751
12 Petropoulos CJ, Parkin NT, Limoli KL, et al. A novel phenotypic drug susceptibility assay for human immuno- deficiency virus type 1. Antimicrob Agents Chemother, 2000, 44:920-928
13 Jackson JB, Becker-Pergola G, Guay LA, et al. Identification of the K103N resistance mutation in Ugandan women receiving nevirapine to prevent HIV-1 vertical transmission. AIDS, 2000, 14:111-115
14 Romano S, Gabriella DM, Giuseppe LR, et al. Novel Indoly-laryl sulfones active against HIV-1 carrying NNRTI resistance mutation:synthesis and SAR studies. J Med Chem, 2003, 46:248-252
15 Hsiou Y, Ding J, Das K, et al. The Lys103Asn mutation of HIV-1 RT: a novel mechanism of drug resistance. J Mol Biol, 2001, 309:437-45
1 The Joint United Nations Programme on HIV/AIDS(UNAIDS) and World Health Organization(WHO): 2006 Report on the global AIDS epidemic. Geneva: UNAIDS and WHO, 2006
2 Palella FJ Jr, Delaney KM, Moorman AC, et al. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. N Engl J Med, 1998, 338: 853-60
3 Richman D, Bozette S, Morton S, et al. The prevalence of antiretroviral drug resistance in the US. In: Program and abstracts of the Interscience Conference on Antimicrobial Agents and Chemotherapy. Chicago, December 16–19, 2001, Washington, D.C.: American Society for Microbiology, 2001. abstract
4 Little SJ, Holte S, Routy J.P, et al. Antiretroviral-drug resistance among patients recently infected with HIV. N Engl J Med, 2002, 347:385-94
5 Shafer RW, Winters MA, Palmer S, et al. Multiple concurrent reverse transcriptase and protease mutations and multidrug resistance of HIV-1 isolates from heavily treated patients. Ann Intern Med, 1998, 128:906-11
6 Hertogs K, Bloor S, Kemp SD, et al. Phenotypic and genotypic analysis of clinical HIV-1 isolates reveals extensive protease inhibitor cross-resistance: a survey of over 6000 samples. AIDS, 2000, 14:1203-1210
7 Esnouf RM, Ren J, Hopkins AL, et al. Unique features in the structure of the complex between HIV-1 reverse transcriptase and the bis(heteroaryl)piperazine (BHAP) U-90152 explain resistance mutations for this nonnucleoside inhibitor. Proc Natl Acad Sci USA , 1997, 94:3984-9
8 Coffin JM. HIV population dynamics in vivo: implications for genetic variation, pathogenesis and therapy. Science, 1995, 267:483-489
9 Ho DD, Neumann AU, Perelson AS, et al. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature, 1995, 373:123-6
10 Perelson AS, Neumann AU, Markowitz M, et al. HIV-1 dynamics in vivo: virion clearance rate, infected cell lifespan, and viral generation time. Science, 1996, 271:1582-6
11 Larder BA, Darby G, Richman DD, et al. HIV with reduced sensitivity to zidovudine(AZT) isolated during prolonged therapy. Science, 1989,243: 1731-1734
12 Sagir A, Oette M, Kaiser R, et al. Trends of prevalence of primary HIV drug resistance in Germany. J Antimicrob Chemother, 2007, 60(4):843-8
13 Gatanaga H, Ibe S, Matsuda M, et al. Drug-resistant HIV-1 prevalence in patients newly diagnosed with HIV/AIDS in Japan. Antiviral Res. 2007, 75(1):75-82
14 Poveda E, de Mendoza C, Martin-Carbonero L, et al. Prevalence of darunavir resistance mutations in HIV-1 infected patients failing other protease inhibitors. JAntimicrob Chemother, 2007, 60(4):885-8
15 Sluis-Cremer, Arion D, Parikh U, et al. The 3'-azido group is not the primary determinant of 3'-azido-3'-deoxythy- midine(AZT) responsible for the excision phenoltype of AZT-resistant HIV-1. J Biol Chem, 2005, 280(32):29047-52
16 Parikh UM, Zelina S, Suis-Cremer N, et al. Molecular mechanisms of bidirectional antagonism between K65R and thymidine analog mutations in HIV-1 reverse transcriptase. AIDS, 2007,21(11):1405-14
17 Brehm JH, Koontz D, Meteer JD, et al. Selection of mutations in the connection and RNase H domains of human immunodeficiency virus type1 reverse transcriptase that increase resistance to 3'-azido-3'-dideoxythymidine. J Virol, 2007, 81(15):7852-9
18 Perez-Bercoff D, Wurtzer S, Compain S, et al. Human immunodeficiency virus type1: resistance to nucleoside analogues and replicative capacity in primary human macrophages. J Virol, 2007, 81(9):4540-50
19 Richman DD. Susceptibility to nucleoside analogues of zidovudine-resistant isolates of human immunodeficiency virus. Am J Med, 1990, 88:8S-10S
20 Gregory M, Lucas MD. Treatment of antiretroviral-exper- ienced patients and immune-based therapy. Hopkins HIV Repert Archive, 2002
21 Naeger LK, Margot NA, Miller MD. ATP-dependent removal of nucleoside reverse transcriptase inhibitors by humanimmunodeficiency virus type1 reverse transcriptase. Antimicrob Agents Chemother, 2002, 46:2179-2184
22 Rigourd M, Ehresmann C, Parniak MA, et al. Primer unblocking and rescue of DNA synthesis by AZT-resistant HIV-1 reverse transcriptase: comparison between initiation and elongation of reverse transcription between (-) and (+) strand DNA synthesis. J Biol Chem, 2002, 277:18611-18
23 Meyer PR, Lennerstrand J, Matsuura SE, et al. Effects of dipeptide insertions between codons 69 and 70 of human immunodeficiency virus type1 reverse transcriptase on primer unblocking, deoxynucleoside triphosphate inhibition, and DNA chain elongation. J Virol, 2003, 77:3871-3877
24 Nikolenko GN, Palmer S, Maldarelli F, et al. Mechanism for nucleoside analog-mediated abrogation of HIV-1 replication: balance between RNaseH activity and nucleotide excision. Proc Natl Acad Sci USA, 2005, 102:2093-98
25 Odriozola L, Cruchaga C, Dolle V, et al. Nonnucleoside inhibitors of HIV-1 reverse transcriptase inhibit phosphor- olysis and resensitize the AZT-resistant polymerase to AZTTP. J Biol Chem, 2003, 278:1163-1172
26 Selmi B, Deval J, Alvarez K, et al. The Y181C substitution in AZT-resistant human immunodeficiency virus type 1 reverse transcriptase suppresses the ATP-mediated repair of the AZTMP-terminated primer. J Biol Chem, 2003, 278:40464- 40470
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30 Rhee SY, Gonzales MJ, Kantor R, et al. Human immune- eodeficiency virus reverse transcriptase and protease sequence database. Nucleic Acids Res, 2003, 31:298-303
31 Sarafianos SG, Das K, Clark AD, et al. Lamivudine (3TC) resistance in HIV-1 reverse transcriptase involves steric hindrance with beta-branched amino acids. Proc Natl Acad Sci USA, 1999, 96:10027-10032
32 Feng JY and Anderson KS. Mechanistic studies examining the efficiency and fidelity of DNA synthesis by the
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33 Nicastri E, Sarmati L, Ettorre G, et al. High prevalence of M184 mutation among patients with viroimmunologic discordant responses to highly active antiretroviral therapyand outcomes after change of therapy guided by genotypic analysis. J Clin Microbiol, 2003, 41(7):3007-12
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2 Van Hal SJ, Muthiah K, Matthews G, et al. Declining incidence of intestinal microsporidiosis and reduction in AIDS-related mortality following introduction of HAART in Sydney, Australia. Trans R Soc Trop Med Hyg, 2007, 101: 1096-1100
3 Ilina T, Parniak MA. Inhibitiors of HIV-1 reverse transcriptase. Adv Pharmacol, 2008, 56: 121-167
4 ZHOU Ting, XIE Lan. Progress in HIV non-nucleoside reverse transcriptase inhibitors (NNRTIs). Acta Pharm Sin (药学学报), 2004, 39:666-672
5 Boone LR. Next generation HIV-1 non-nucleoside reverse transcriptase inhibitors. Curr Opin Investig Drugs, 2006, 7:128-135
6 Blair WS, Isaacson JS, Li X, et al. A novel HIV-1 antiviral high throughput screening approach for the discovery of HIV-1 inhibitors. Antiviral Res, 2005, 65: 107-116
7 He J, Choe S, Walker R, et al. Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activity. J Virol, 1995, 69:6705-6711
8 Connor RI, Chen BK, Choe S, et al. Vpr is required for efficient replication of human immunodeficiency virus type-1in mononuclear phagocytes. Virology, 1995, 206:935-944
9 Vivet-Boudou V, Didierjean J, Isel C, et al. Nucleoside and nucleotide inhibitors of HIV-1 replication. Cell Mol Life Sci, 2006, 63:163-186
10 Cao J, Isaacson J, Patick AK, et al. High-throughput human immunodeficiency virus type1 (HIV-1) full replication assay that includes HIV-1 Vif as an antiviral target. Antimicrob Agents Chemother, 2005, 49:3833-3841
11 Domaoal RA, Demeter LM. Structural and biochemical effects of human immunodeficiency virus mutant resistant to non-nucleoside reverse transcriptase inhibitors. J Biochem Cell Biol, 2004, 36:1735-1751
12 Petropoulos CJ, Parkin NT, Limoli KL, et al. A novel phenotypic drug susceptibility assay for human immuno- deficiency virus type 1. Antimicrob Agents Chemother, 2000, 44:920-928
13 Jackson JB, Becker-Pergola G, Guay LA, et al. Identification of the K103N resistance mutation in Ugandan women receiving nevirapine to prevent HIV-1 vertical transmission. AIDS, 2000, 14:111-115
14 Romano S, Gabriella DM, Giuseppe LR, et al. Novel Indoly-laryl sulfones active against HIV-1 carrying NNRTI resistance mutation:synthesis and SAR studies. J Med Chem, 2003, 46:248-252
15 Hsiou Y, Ding J, Das K, et al. The Lys103Asn mutation of HIV-1 RT: a novel mechanism of drug resistance. J Mol Biol, 2001, 309:437-45
1 The Joint United Nations Programme on HIV/AIDS(UNAIDS) and World Health Organization(WHO): 2006 Report on the global AIDS epidemic. Geneva: UNAIDS and WHO, 2006
2 Palella FJ Jr, Delaney KM, Moorman AC, et al. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. N Engl J Med, 1998, 338: 853-60
3 Richman D, Bozette S, Morton S, et al. The prevalence of antiretroviral drug resistance in the US. In: Program and abstracts of the Interscience Conference on Antimicrobial Agents and Chemotherapy. Chicago, December 16–19, 2001, Washington, D.C.: American Society for Microbiology, 2001. abstract
4 Little SJ, Holte S, Routy J.P, et al. Antiretroviral-drug resistance among patients recently infected with HIV. N Engl J Med, 2002, 347:385-94
5 Shafer RW, Winters MA, Palmer S, et al. Multiple concurrent reverse transcriptase and protease mutations and multidrug resistance of HIV-1 isolates from heavily treated patients. Ann Intern Med, 1998, 128:906-11
6 Hertogs K, Bloor S, Kemp SD, et al. Phenotypic and genotypic analysis of clinical HIV-1 isolates reveals extensive protease inhibitor cross-resistance: a survey of over 6000 samples. AIDS, 2000, 14:1203-1210
7 Esnouf RM, Ren J, Hopkins AL, et al. Unique features in the structure of the complex between HIV-1 reverse transcriptase and the bis(heteroaryl)piperazine (BHAP) U-90152 explain resistance mutations for this nonnucleoside inhibitor. Proc Natl Acad Sci USA , 1997, 94:3984-9
8 Coffin JM. HIV population dynamics in vivo: implications for genetic variation, pathogenesis and therapy. Science, 1995, 267:483-489
9 Ho DD, Neumann AU, Perelson AS, et al. Rapid turnover of plasma virions and CD4 lymphocytes in HIV-1 infection. Nature, 1995, 373:123-6
10 Perelson AS, Neumann AU, Markowitz M, et al. HIV-1 dynamics in vivo: virion clearance rate, infected cell lifespan, and viral generation time. Science, 1996, 271:1582-6
11 Larder BA, Darby G, Richman DD, et al. HIV with reduced sensitivity to zidovudine(AZT) isolated during prolonged therapy. Science, 1989,243: 1731-1734
12 Sagir A, Oette M, Kaiser R, et al. Trends of prevalence of primary HIV drug resistance in Germany. J Antimicrob Chemother, 2007, 60(4):843-8
13 Gatanaga H, Ibe S, Matsuda M, et al. Drug-resistant HIV-1 prevalence in patients newly diagnosed with HIV/AIDS in Japan. Antiviral Res. 2007, 75(1):75-82
14 Poveda E, de Mendoza C, Martin-Carbonero L, et al. Prevalence of darunavir resistance mutations in HIV-1 infected patients failing other protease inhibitors. JAntimicrob Chemother, 2007, 60(4):885-8
15 Sluis-Cremer, Arion D, Parikh U, et al. The 3'-azido group is not the primary determinant of 3'-azido-3'-deoxythy- midine(AZT) responsible for the excision phenoltype of AZT-resistant HIV-1. J Biol Chem, 2005, 280(32):29047-52
16 Parikh UM, Zelina S, Suis-Cremer N, et al. Molecular mechanisms of bidirectional antagonism between K65R and thymidine analog mutations in HIV-1 reverse transcriptase. AIDS, 2007,21(11):1405-14
17 Brehm JH, Koontz D, Meteer JD, et al. Selection of mutations in the connection and RNase H domains of human immunodeficiency virus type1 reverse transcriptase that increase resistance to 3'-azido-3'-dideoxythymidine. J Virol, 2007, 81(15):7852-9
18 Perez-Bercoff D, Wurtzer S, Compain S, et al. Human immunodeficiency virus type1: resistance to nucleoside analogues and replicative capacity in primary human macrophages. J Virol, 2007, 81(9):4540-50
19 Richman DD. Susceptibility to nucleoside analogues of zidovudine-resistant isolates of human immunodeficiency virus. Am J Med, 1990, 88:8S-10S
20 Gregory M, Lucas MD. Treatment of antiretroviral-exper- ienced patients and immune-based therapy. Hopkins HIV Repert Archive, 2002
21 Naeger LK, Margot NA, Miller MD. ATP-dependent removal of nucleoside reverse transcriptase inhibitors by humanimmunodeficiency virus type1 reverse transcriptase. Antimicrob Agents Chemother, 2002, 46:2179-2184
22 Rigourd M, Ehresmann C, Parniak MA, et al. Primer unblocking and rescue of DNA synthesis by AZT-resistant HIV-1 reverse transcriptase: comparison between initiation and elongation of reverse transcription between (-) and (+) strand DNA synthesis. J Biol Chem, 2002, 277:18611-18
23 Meyer PR, Lennerstrand J, Matsuura SE, et al. Effects of dipeptide insertions between codons 69 and 70 of human immunodeficiency virus type1 reverse transcriptase on primer unblocking, deoxynucleoside triphosphate inhibition, and DNA chain elongation. J Virol, 2003, 77:3871-3877
24 Nikolenko GN, Palmer S, Maldarelli F, et al. Mechanism for nucleoside analog-mediated abrogation of HIV-1 replication: balance between RNaseH activity and nucleotide excision. Proc Natl Acad Sci USA, 2005, 102:2093-98
25 Odriozola L, Cruchaga C, Dolle V, et al. Nonnucleoside inhibitors of HIV-1 reverse transcriptase inhibit phosphor- olysis and resensitize the AZT-resistant polymerase to AZTTP. J Biol Chem, 2003, 278:1163-1172
26 Selmi B, Deval J, Alvarez K, et al. The Y181C substitution in AZT-resistant human immunodeficiency virus type 1 reverse transcriptase suppresses the ATP-mediated repair of the AZTMP-terminated primer. J Biol Chem, 2003, 278:40464- 40470
27 Boyer PL, Julias JG, Ambrose Z, et al. The NucleosideAnalogs 4’C-Methyl Thymidine and 4’C-Ethyl Thymidine Block DNA Synthesis by Wild-type HIV-1 RT and Excision Proficient NRTI Resistant RT Variants. J Mol Biol, 2007, 371 (4):873-82
28 Huang H, Chopra R, Verdine G, et al. Structure of a covalently trapped catalytic complex of HIV-1 reverse transcriptase: implications for drug resistance. Science, 1998, 282:1669- 1675
29 Whitcomb JM, Parkin NT, Chappey C, et al.Broad nucleoside reverse-transcriptase inhibitor cross-resistance in human immunodeficiency virus type1 clinical isolates. J Infect Dis, 2003, 188:992-1000
30 Rhee SY, Gonzales MJ, Kantor R, et al. Human immune- eodeficiency virus reverse transcriptase and protease sequence database. Nucleic Acids Res, 2003, 31:298-303
31 Sarafianos SG, Das K, Clark AD, et al. Lamivudine (3TC) resistance in HIV-1 reverse transcriptase involves steric hindrance with beta-branched amino acids. Proc Natl Acad Sci USA, 1999, 96:10027-10032
32 Feng JY and Anderson KS. Mechanistic studies examining the efficiency and fidelity of DNA synthesis by the
3TC-resistant mutant (184V) of HIV-1 reverse transcriptase. Biochemistry, 1999, 38:9440-9448
33 Nicastri E, Sarmati L, Ettorre G, et al. High prevalence of M184 mutation among patients with viroimmunologic discordant responses to highly active antiretroviral therapyand outcomes after change of therapy guided by genotypic analysis. J Clin Microbiol, 2003, 41(7):3007-12
34 Bouchonnet F, Dam E, Mammano F, et al. Quantification of the effects on viral DNA synthesis of reverse transcriptase mutations conferring human immunode- ficiency virus type1 resistance to nucleoside analogues. J Virol, 2005,79: 812-822
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