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Updated: Aug 28, 2025
Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry
Published on: June 26, 2019
Cell-based and cell-free firefly luciferase complementation assay to quantify Human Immunodeficiency Virus type 1
Tucker Hansen1, Jodie Baris1, Min Zhao1
1Department of Internal Medicine, Section of Infectious Diseases, Yale School of Medicine, New Haven, CT, 06520, United States.
Insights
Researchers developed new assays to measure Human Immunodeficiency Virus type 1 (HIV) Rev protein interactions. These assays can rapidly screen for compounds that inhibit HIV replication by targeting Rev function.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Rev is a critical Human Immunodeficiency Virus type 1 (HIV) regulatory protein.
- Rev facilitates the export of viral RNA, essential for HIV replication.
- Quantifying Rev-Rev interactions is key to understanding its function and developing inhibitors.
Purpose of the Study:
- To develop robust cell-intact and cell-free assays for quantifying Rev-Rev interactions.
- To validate these assays using mutant Rev proteins and known inhibitors.
- To establish a high-throughput screening method for identifying compounds that inhibit Rev function.
Main Methods:
- Utilized a firefly split-luciferase complementation system for Rev-Rev interaction assays.
- Developed both cell-based and cell-free assay formats.
- Tested various Rev mutants, Crm1, nanobodies, RNAse, HIV RRE RNA, and purified GST-Rev protein.
Main Results:
- Both cell-intact and cell-free assays reliably quantified Rev-Rev interactions.
- Assay results correlated well with a functional Rev trans-complementation infectivity assay.
- A camelid nanobody known to inhibit Rev function enhanced Rev-Rev interaction.
- The cell-free assay demonstrated rapid interaction kinetics and high Z'-factor (∼0.85).
Conclusions:
- Developed reliable, rapid, and reproducible assays for quantifying Rev-Rev interactions.
- The cell-free assay is particularly suitable for high-throughput screening of potential HIV inhibitors.
- These assays provide a valuable tool for advancing HIV research and drug discovery.
Abstract:
Rev is an essential regulatory protein of Human Immunodeficiency Virus type 1 (HIV) that is found in the nucleus of infected cells. Rev multimerizes on the Rev-response element (RRE) of HIV RNA to facilitate the export of intron-containing HIV mRNAs from the nucleus to the cytoplasm, and, as such, HIV cannot replicate in the absence of Rev. We have developed cell-intact and cell-free assays based upon a robust firefly split-luciferase complementation system, both of which quantify Rev-Rev interaction. Using the cell-based system we show that additional Crm1 did not impact the interaction, whereas excess Rev reduced it. Furthermore, when a series of mutant Revs were tested, there was a strong correlation between the results of the cell-based assay and the results of a functional Rev trans-complementation infectivity assay. Of interest, a camelid nanobody (NB) that was known to inhibit Rev function enhanced Rev-Rev interaction in the cell-based system. We observed a similar increase in Rev-Rev interaction in a cell-free system, when cell lysates expressing Rev-NLUC or CLUC-Rev were simply mixed. In the cell-free system Rev-Rev interaction occurred within minutes and was inhibited by excess Rev. The levels of interaction between the mutant Revs tested varied by mutant type. Treatment of Rev lysates with RNAse minimally reduced the degree of interaction whereas addition of HIV RRE RNA enhanced the interaction. Purified GST-Rev protein inhibited the interaction. The Z-factor (Z') for the cell-free system was ∼0.85 when tested in 96-well format, and the anti-Rev NB enhanced the interaction in the cell-free system. Thus, we have developed both cell-intact and cell-free systems that can reliably, rapidly, and reproducibly quantify Rev-Rev interaction. These assays, particularly the cell-free one, may be useful in screening and identifying compounds that inhibit Rev function on a high throughput basis.
