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Area of Science:

  • Virology
  • Biochemistry
  • Biophysics

Background:

  • The human immunodeficiency virus (HIV) capsid is crucial for viral replication, mediating genetic material delivery.
  • Host cell proteins interact with the capsid at multiple binding sites to regulate viral lifecycle stages.

Purpose of the Study:

  • To develop a sensitive and rapid fluorescence-based assay for screening capsid-binding molecules.
  • To characterize the interactions between HIV capsids and potential host or therapeutic molecules.

Main Methods:

  • Development of a fluorescence fluctuation spectroscopy method utilizing self-assembled HIV capsid particles as bait.
  • Screening of fluorescence-labeled analytes (prey molecules) in solution based on fluorescence intensity spikes.
  • Implementation of a scanning stage for reduced measurement times (10 s) and two-color coincidence detection for quantification.

Main Results:

  • The assay demonstrated high sensitivity in detecting multiple prey molecules binding to the multivalent HIV capsid platform.
  • Reduced measurement times to 10 seconds without compromising assay sensitivity.
  • Enabled quantification of binding levels and determination of binding affinities.

Conclusions:

  • The developed assay provides a powerful tool for discovering and characterizing molecules that interact with the HIV capsid.
  • The method can be adapted to study capsid-host interactions and identify binding interfaces, including those involving defective capsids.
  • The principle is extendable to other multivalent scaffolds for sensitive interaction assays.