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A rapid, quantitative bioassay based on the human immunodeficiency virus trans-activator
J M Hasler1, T F Weighous, T W Pitts
1Division of Cancer and Infectious Diseases Research, Upjohn Company, Kalamazoo, MI 49001.
AIDS Research and Human Retroviruses
|October 1, 1989
Summary
We developed a novel bioassay using a recombinant avian retrovirus (SNV-tat) to detect inhibitors of the human immunodeficiency virus trans-activator (TAT). This system enables rapid, quantitative, and safe screening for potential therapeutic compounds.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- The human immunodeficiency virus (HIV) trans-activator (TAT) protein plays a crucial role in viral replication.
- Developing safe and efficient methods to study TAT function and identify inhibitors is essential for HIV research.
Purpose of the Study:
- To construct a functional avian retroviral expression vector encoding the HIV TAT protein (SNV-tat).
- To establish a quantitative and safe bioassay for screening TAT inhibitors using reporter genes.
Main Methods:
- Constructed a tat cDNA encoding the N-terminal 76 amino acids of HIV TAT.
- Cloned the tat cDNA into a modified spleen necrosis virus (SNV) vector.
- Created recombinant cells stably carrying HIV promoter-driven reporter genes (sCD4 or human preprorenin).
- Infected cells with SNV-tat and measured reporter protein secretion via immunoassays.
Main Results:
- Successfully generated high-titer infectious SNV-tat stocks.
- Demonstrated stable propagation of SNV-tat in dog cells for extended periods.
- Cells infected with SNV-tat exhibited significantly higher secretion of sCD4 and HRN (75-77 ng/10^6 cells/ml) compared to controls (1-10 ng/10^6 cells/ml).
- Reporter protein secretion was constitutive and sustained during subculturing.
Conclusions:
- The TAT protein mediates trans-activation of the HIV reporter gene, leading to increased secretion of sCD4 and HRN.
- The developed system of recombinant cells, SNV-tat, and immunoassays constitutes a rapid, quantitative, and safe bioassay for identifying TAT inhibitors.