Kinetics of HIV-1 Latency Reversal Quantified on the Single-Cell Level Using a Novel Flow-Based Technique

Area of Science:

• Virology
• Immunology
• Cell Biology

Background:

• Human Immunodeficiency Virus type 1 (HIV-1) establishes a latent reservoir early in infection, evading immune detection.
• Current HIV-1 cure strategies focus on latency reversal and eliminating these reservoirs.
• Kinetics of viral reactivation and subsequent cellular changes remain poorly understood.

Purpose of the Study:

• To develop and validate a novel single-cell flow cytometry approach for quantifying HIV-1 mRNA and protein dynamics.
• To characterize phenotypic changes in HIV-1 infected cells during latency reversal and de novo infection.
• To provide insights into the kinetics of HIV-1 reactivation for therapeutic development.

Main Methods:

• Adaptation of flow cytometry for simultaneous detection of gagpol mRNA, p24 Gag protein, and cell surface markers.
• Stimulation of latently HIV-1 infected J89 cells with tumor necrosis factor alpha (TNF-α)/romidepsin (RMD).
• Infection of primary CD4(+) T cells with HIV-1 to study de novo infection kinetics.

Main Results:

• Quantification of gagpol mRNA in J89 cells as early as 3 hours (TNF-α) and 12 hours (RMD) post-stimulation.
• Detection of p24 Gag protein starting at 18 hours post-stimulation.
• Early downregulation of CD4, BST-2, and HLA class I on HIV-1 infected primary CD4(+) T cells preceding Gag protein detection.

Conclusions:

• A novel flow cytometry method enables precise quantification of HIV-1 mRNA and protein synthesis kinetics at the single-cell level.
• This technique allows for detailed phenotypic characterization of infected cells across different viral life cycle stages.
• Findings provide critical data for understanding HIV-1 reactivation and developing targeted therapies.