Establishment of a high-throughput screening system for universal anti-HIV targets

Abstract

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% (IC 50 ) 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. HIV-1, high-throughput screening, inhibitor, virus-cell-based assayCitation: Yin Q, Zhuang D M, Jiang Y Q, et al. Establishment of a high-throughput screening system for universal anti-HIV targets. Chinese Sci Bull, 2010, 55: 937−942,

Calculation of the intracellular elastic modulus based on an atomic force microscope micro-cutting system

“…These results demonstrate that this micro-cutting method can be used to measure intracellular mechanical properties, which could in turn provide a more accurate experimental basis for the development of novel methods for the diagnosis and treatment of various diseases. The cytoskeleton has a major effect on the responsiveness of cells to external stimuli [1][2][3][4][5] and is composed of a variety of protein filaments of different sizes and degrees of stiffness [6][7][8][9][10]. Cells can be cut into sequential layers using an atomic force microscope (AFM) tip, which allows the elastic modulus of the cytoskeleton and the organelles to be determined.…”