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Development of FRET-based cap-snatching endonuclease assay.

Jeeva Subbiah1, Austin Royster1, Sheema Mir1

  • 1College of Veterinary Medicine, Western University of Health Sciences, Pomona, California, USA.

Microbiology Spectrum
|March 31, 2025
PubMed
Summary
This summary is machine-generated.

Researchers developed a sensitive assay to study the Bunyavirus endonuclease, a key target for antiviral drugs. This assay enables rapid screening for inhibitors to combat dangerous bunyaviruses lacking current treatments.

Keywords:
bunyavirusendonucleasevirus replication

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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.