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.
