Development of a rapid cell-fusion-based phenotypic HIV-1 tropism assay

Area of Science:

• Virology
• Molecular Biology
• Assay Development

Background:

• A novel phenotypic tropism assay (PTA) for HIV-1 infection, termed DSP-Pheno, was developed using a dual split reporter protein system (DSP).
• The DSP system combines Renilla luciferase (RL) and green fluorescent protein (GFP) split into two constructs (DSP1-7 and DSP8-11).

Purpose of the Study:

• To develop and validate a rapid, sensitive, and safe phenotypic tropism assay for HIV-1 using the DSP system.
• To evaluate the DSP-Pheno assay's performance in determining HIV-1 co-receptor usage (R5, X4, or dual tropic).

Main Methods:

• Stable expression of DSP1-7 in NP-2 cell lines engineered to express CD4/CXCR4 (N4X4) or CD4/CCR5 (N4R5).
• Construction of an expression vector (pRE11) for DSP8-11, into which HIV-1 envelope genes were subcloned (pRE11-env).
• Transfection of 293FT cells with pRE11-env and incubation with indicator cell lines (N4R5-DSP1-7, N4X4-DSP1-7) to assess tropism via RL or GFP activity.

Main Results:

• The DSP-Pheno assay demonstrated concordant results with expected co-receptor usage based on reference HIV-1 strains.
• Assay sensitivity using RL activity was comparable to established pseudovirus-based phenotypic assays.
• The assay provided results within 5 days of plasma acquisition, with all clinical samples showing positive RL signals on R5 indicator cells.
• Higher median RLU values on X4 indicator cells in the low CD4 group suggested increased prevalence of dual or X4 tropic viruses.

Conclusions:

• A new cell-fusion-based, high-throughput PTA (DSP-Pheno) for HIV-1 was successfully developed, suitable for in-house studies.
• The DSP-Pheno assay is sensitive, offers a short turnaround time, and provides a safe system without using infectious viruses.
• DSP-Pheno is a potentially valuable laboratory tool, especially for studying non-B subtype HIV-1 tropism, pending further validation.