Development of FRET-based cap-snatching endonuclease assay

Area of Science:

• Virology and Molecular Biology
• Drug Discovery and Development

Background:

• The order Bunyavirales includes over 300 species of segmented, negative-strand RNA viruses.
• Bunyaviruses cause severe human diseases, with no FDA-approved vaccines or therapeutics currently available.
• The RNA-dependent RNA polymerase (RdRp) of Bunyaviruses possesses a unique N-terminal endonuclease domain crucial for viral transcription via cap-snatching.

Purpose of the Study:

• To develop a sensitive and quantitative in vitro assay for assessing the activity of the Bunyavirus cap-snatching endonuclease.
• To establish a high-throughput screening method for identifying potential antiviral inhibitors targeting the endonuclease domain.
• To provide a critical tool for antiviral drug discovery against Bunyavirales.

Main Methods:

• Bacterial expression and purification of the hantavirus RdRp N-terminal endonuclease domain.
• Development of a fluorescence resonance energy transfer (FRET)-based in vitro assay using a dual-labeled synthetic RNA substrate.
• Quantitative kinetic analysis of the endonuclease activity.

Main Results:

• A sensitive FRET-based assay was successfully established to measure endonuclease activity.
• Cleavage of the FRET-quenched RNA substrate by the purified endonuclease domain yielded a significant dequenched fluorescence signal.
• Kinetic analysis demonstrated a reaction half-life of approximately 3 minutes and a signal-to-background ratio of ~31.

Conclusions:

• The developed FRET assay is a highly sensitive and quantitative tool for evaluating Bunyavirus endonuclease activity.
• This assay is suitable for high-throughput screening of chemical libraries to discover novel antiviral compounds.
• The assay represents a significant advancement for developing therapeutics against Bunyavirales.