title: Toward the development of a single-round infection assay based on EGFP reporting for anti-HIV-1 drug discovery.
abstract: 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.
authors.affiliation: Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran; National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
authors.forename: Mahdieh
authors.initials: M
authors.lastname: Soezi
authors.affiliation: Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Arash
authors.initials: A
authors.lastname: Memarnejadian
authors.affiliation: National Institute of Genetic Engineering and Biotechnology, Tehran, Iran.
authors.forename: Saeed
authors.initials: S
authors.lastname: Aminzadeh
authors.affiliation: Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Rezvan
authors.initials: R
authors.lastname: Zabihollahi
authors.affiliation: Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Seyed Mehdi
authors.initials: SM
authors.lastname: Sadat
authors.affiliation: Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Safieh
authors.initials: S
authors.lastname: Amini
authors.affiliation: Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Soheila
authors.initials: S
authors.lastname: Hekmat
authors.affiliation: Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Mohammad Reza
authors.initials: MR
authors.lastname: Aghasadeghi
keywords: Drug discovery; EGFP; Fluorometry; HIV-1; Single-round infection