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en Toward the Development of a Single-Round Infection Assay Based on EGFP Reporting for Anti-HIV-1 Drug Discovery میکروب شناسی Microbiology 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. Drug discovery, EGFP, Fluorometry, HIV-1, Single-round infection 1 9 http://rbmb.net/browse.php?a_code=A-10-1-44&slc_lang=en&sid=1 Mahdieh Soezi 10031947532846006077 10031947532846006077 No Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran - National Institute of Genetic Engineering and Biotechnology, Tehran, Iran Arash Memarnejadian memarnejad@pasteur.ac.ir, mrasadeghi@pasteur.ac.ir 10031947532846006078 10031947532846006078 Yes Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran Saeed Aminzadeh 10031947532846006079 10031947532846006079 No Rezvan Zabihollahi 10031947532846006080 10031947532846006080 No Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran Seyed Mehdi Sadat 10031947532846006081 10031947532846006081 No Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran Safieh Amini 10031947532846006082 10031947532846006082 No Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran Soheila Hekmat 10031947532846006083 10031947532846006083 No Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran Mohammad Reza Aghasadeghi 10031947532846006084 10031947532846006084 No Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran