Toward the Development of a Single-Round Infection Assay Based on EGFP Reporting for Anti-HIV-۱ Drug Discovery

Background: The rapid increase of HIV-۱ strains resistant to current antiretroviral drugs is a challenge for successful AIDS therapy. This necessitates the development of novel drugs, and to this end, availability of screening systems for in vitro drug discovery is a priority. Herein, we report the modification of a previously developed system for increased sensitivity, ease of use, and cost-efficiency, based on the application of the EGFP marker. Methods: A PCR-amplified gfp gene (gfp) was cloned into pmzNL۴-۳, the plasmid already designed to produce single-cycle replicable virions, in frame with the reverse-transcriptase gene to construct the pmzNL۴-۳/GFP plasmid. GFP-mzNL۴-۳ pseudo-typed virions, as the first progeny viruses, were recovered from the culture supernatant of HEK۲۹۳T cells co-transfected with pmzNL۴-۳/GFP and the helper plasmids pSPAX۲ and pMD۲G, which respectively encode HIV-۱ Gag-Pol and vesicular stomatitis virus glycoprotein. Single-cycle replication and virion production were assessed by syncytia formation, p۲۴ antigen assays, and electron and fluorescence microscopy. Results: The incorporation of EGFP into the viral particles allowed their quantification by fluorometry, flow-cytometry, and fluorescence microscopy; however, this modification did not affect the single-round infectivity or production rate of the GFP fluorescence-emitting virions. Conclusions: Our results certify the development of a rapid, inexpensive, and safe GFP-reporting single-cycle replicable system for anti-HIV drug discovery. Further experiments are needed to measure the validity and robustness of the assay.

Keywords:

Drug discovery , EGFP , Fluorometry , HIV-۱ , Single-round infection