Toward the development of a single-round infection assay based on EGFP reporting for anti-HIV-1 drug discovery

Background: the rapid increase of hiv-1 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 pmznl4-3, the plasmid already designed to produce single-cycle replicable virions, in frame with the reverse-transcriptase gene to construct the pmznl4-3/gfp plasmid. gfp-mznl4-3 pseudo-typed virions, as the first progeny viruses, were recovered from the culture supernatant of hek293t cells co-transfected with pmznl4-3/gfp and the helper plasmids pspax2 and pmd2g, which respectively encode hiv-1 gag-pol and vesicular stomatitis virus glycoprotein. single-cycle replication and virion production were assessed by syncytia formation, p24 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.