AU - Soezi, Mahdieh AU - Memarnejadian, Arash AU - Aminzadeh, Saeed AU - Zabihollahi, Rezvan AU - Sadat, Seyed Mehdi AU - Amini, Safieh AU - Hekmat, Soheila AU - Aghasadeghi, Mohammad Reza TI - Toward the Development of a Single-Round Infection Assay Based on EGFP Reporting for Anti-HIV-1 Drug Discovery PT - JOURNAL ARTICLE TA - rbmb JN - rbmb VO - 4 VI - 1 IP - 1 4099 - http://rbmb.net/article-1-65-en.html 4100 - http://rbmb.net/article-1-65-en.pdf SO - rbmb 1 ABĀ  - 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. CP - IRAN IN - Tel: +98 21 66969291; Fax: +98 21 66969291 LG - eng PB - rbmb PG - 1 PT - YR - 2015