An HIV-1 protease screening assay using a non-infectious proviral clone

Area of Science:

• Virology
• Molecular Biology
• Drug Discovery

Background:

• Human Immunodeficiency Virus type 1 (HIV-1) protease is a critical target for antiviral therapy.
• Developing efficient screening assays for HIV-1 protease inhibitors is essential for identifying new therapeutic agents.

Purpose of the Study:

• To establish and validate an in-vitro assay for screening potential HIV-1 protease inhibitors.
• To assess the efficacy of a known protease inhibitor, PD 148310, using the developed assay.

Main Methods:

• Utilized a transiently expressed, non-infectious proviral clone (X19) lacking an envelope gene in COS 7 cells.
• Assessed viral replication via p24 antigen detection in cell supernatants using ELISA.
• Administered PD 148310 post-transfection and analyzed p24 antigen levels and Gag precursor processing via Western blot.

Main Results:

• The assay demonstrated dose-dependent inhibition of p24 antigen release by PD 148310, with an IC50 of 23.6 nM.
• Western blot analysis confirmed the absence of detectable p24 in supernatants treated with 1000 nM PD 148310, indicating accumulation of the unprocessed p55 Gag precursor.
• The assay successfully identified inhibition of viral maturation by a known protease inhibitor.

Conclusions:

• The developed in-vitro assay is a valuable tool for screening and identifying potential HIV-1 protease inhibitors.
• This method allows for the assessment of protease inhibitor efficacy by monitoring p24 antigen release and Gag precursor processing.
• The assay provides a reliable platform for advancing HIV-1 drug discovery efforts.