AI Article Synopsis
- The HIV-1 transactivator protein (Tat) is crucial for the virus's replication by working with host proteins to enhance RNA production.
- Researchers created an efficient AlphaLISA assay to study the interaction between Tat and a host factor called pTEFb, achieving a strong signal-to-background ratio and validating the assay with known competitive peptides.
- The assay revealed that Tat has a high affinity for pTEFb, and only a small percentage of Tat is functional, aiding in the search for new antiviral treatments against HIV-1.
Article Abstract
The viral transactivator protein (Tat) plays an essential role in the replication of human immunodeficiency type 1 virus (HIV-1) by recruiting the host positive transcription elongation factor (pTEFb) to the RNA polymerase II transcription machinery to enable an efficient HIV-1 RNA elongation process. Blockade of the interaction between Tat and pTEFb represents a novel strategy for developing a new class of antiviral agents. In this study, we developed a homogeneous assay in AlphaLISA (amplified luminescent proximity homogeneous assay) format using His-tagged pTEFb and biotinylated Tat to monitor the interaction between Tat and pTEFb. On optimizing the assay conditions, the signal-to-background ratio was found to be greater than 10-fold. The assay was validated with untagged Tat and peptides known to compete with Tat for pTEFb binding. The Z' of the assay is greater than 0.5, indicating that the assay is robust and can be easily adapted to a high-throughput screening format. Furthermore, the affinity between Tat and pTEFb was determined to be approximately 20 pM, and only 7% of purified Tat was found to be active in forming tertiary complex with pTEFb. Development of this assay should facilitate the discovery of a new class of antiviral agents providing HIV-1 patients with broader treatment choices.
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| http://dx.doi.org/10.1016/j.ab.2014.08.007 | DOI Listing |
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