The HIV-1 Pharmacological Models Which Resist To Non-nucleoside Reverse-transcriptase Inhibitors

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.