The process of identifying novel human immunodeficiency virus 1 (HIV-1) inhibitors presents a challenge for industrial and scientific research.Virus-cell-based screening approaches offer some advantages in the quest f...The process of identifying novel human immunodeficiency virus 1 (HIV-1) inhibitors presents a challenge for industrial and scientific research.Virus-cell-based screening approaches offer some advantages in the quest for novel inhibitors because they include multiple targets in a single screen and in some cases reveal targets not captured in biochemical assays.In this study,a high-throughput screening (HTS) system for HIV-1 inhibitors was developed,which allows the simultaneous screening of all the HIV-1 targets required for replication in the cell culture.HeLa/cluster of differentiation 4 (CD4)/long terminal repeat (LTR) indicator cells,which stably expressed high levels of HIV receptor CD4 and contained the firefly luciferase reporter gene under the control of the HIV-1 LTR promoter,were generated.The expression of CD4 and LTR function in this cell line was validated by Western blot and luciferase assay.MT2 cells,a human T-cell leukemia cell line that support high levels of HIV-1 replication,were infected with HIV-1,and then the infected MT2 cells were co-cultured with HeLa/CD4/LTR cells.In the optimized assay conditions,HIV-1 replication occurs rapidly in the MT2 cells,resulting in the infection of the HeLa/CD4/LTR cells and a significant induction of luciferase signals through LTR,which is activated by the expression of HIV-1 tat gene.The luciferase signals of HeLa/CD4/LTR cells co-cultured with HIV-1 infected MT2 cells were significantly stronger than the signals of noninfected HeLa/CD4/LTR cells (P < 0.001).The inhibitory effects of HIV-1 inhibitors (3'-Azido-3'-deoxythymidine [AZT],efavirenz [EFV],and nevirapine [NVP]) were evaluated with this assay,and the inhibitory concentration 50% (IC50) values of the above three inhibitors were 58,1.4,and 85 nmol/L,respectively,indicating that the assay provides the necessary sensitivity for identifying antiviral molecules.The Z' factor had a value of 0.563,indicating this is a very robust assay.These results suggested that HIV-1 infection assay represents a novel approach to HIV-1 antiviral screening that allows for the effective execution of HTS campaigns.展开更多
文摘The process of identifying novel human immunodeficiency virus 1 (HIV-1) inhibitors presents a challenge for industrial and scientific research.Virus-cell-based screening approaches offer some advantages in the quest for novel inhibitors because they include multiple targets in a single screen and in some cases reveal targets not captured in biochemical assays.In this study,a high-throughput screening (HTS) system for HIV-1 inhibitors was developed,which allows the simultaneous screening of all the HIV-1 targets required for replication in the cell culture.HeLa/cluster of differentiation 4 (CD4)/long terminal repeat (LTR) indicator cells,which stably expressed high levels of HIV receptor CD4 and contained the firefly luciferase reporter gene under the control of the HIV-1 LTR promoter,were generated.The expression of CD4 and LTR function in this cell line was validated by Western blot and luciferase assay.MT2 cells,a human T-cell leukemia cell line that support high levels of HIV-1 replication,were infected with HIV-1,and then the infected MT2 cells were co-cultured with HeLa/CD4/LTR cells.In the optimized assay conditions,HIV-1 replication occurs rapidly in the MT2 cells,resulting in the infection of the HeLa/CD4/LTR cells and a significant induction of luciferase signals through LTR,which is activated by the expression of HIV-1 tat gene.The luciferase signals of HeLa/CD4/LTR cells co-cultured with HIV-1 infected MT2 cells were significantly stronger than the signals of noninfected HeLa/CD4/LTR cells (P < 0.001).The inhibitory effects of HIV-1 inhibitors (3'-Azido-3'-deoxythymidine [AZT],efavirenz [EFV],and nevirapine [NVP]) were evaluated with this assay,and the inhibitory concentration 50% (IC50) values of the above three inhibitors were 58,1.4,and 85 nmol/L,respectively,indicating that the assay provides the necessary sensitivity for identifying antiviral molecules.The Z' factor had a value of 0.563,indicating this is a very robust assay.These results suggested that HIV-1 infection assay represents a novel approach to HIV-1 antiviral screening that allows for the effective execution of HTS campaigns.
