FLARE: A Flow Cytometry-Based Fluorescent Assay for Measuring HSV-1 Nuclear Egress

Area of Science:

• Virology
• Cell Biology
• Molecular Biology

Background:

• Herpesvirus replication involves nuclear egress, a process where viral capsids move from the nucleus to the cytosol.
• Traditional methods like microscopy for measuring nuclear egress are labor-intensive and may introduce bias due to limited cell examination.

Purpose of the Study:

• To develop a quantitative, high-throughput flow cytometry-based assay for measuring HSV-1 nuclear egress.
• To provide a more reliable alternative to traditional imaging techniques for studying viral nuclear egress.

Main Methods:

• Developed FLARE (FLow cytometry-based Assay of nucleaR Egress), a double fluorescent reporter system.
• Utilized HSV-1-tdTomato to identify infected cells and an Alexa Fluor-488-conjugated antibody for cytosolic capsids.
• Employed flow cytometry for quantitative analysis of nuclear egress.

Main Results:

• FLARE accurately quantifies HSV-1 nuclear egress.
• The assay enables large-scale, high-throughput screening, suitable for applications like CRISPR/Cas9 screens.
• Demonstrated potential for adaptation to other herpesviruses with appropriate antibodies.

Conclusions:

• FLARE offers a significant advancement for studying herpesvirus nuclear egress.
• The assay provides quantitative, high-throughput analysis, overcoming limitations of traditional methods.
• FLARE is adaptable for broader applications in herpesvirus research and drug discovery.