Cell-based and cell-free firefly luciferase complementation assay to quantify Human Immunodeficiency Virus type 1...

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.