Abstract
Human immunodeficiency virus type 1 (HIV-1) reporter cell lines are critical tools for drug development. However, one disadvantage of HIV-1 reporter cell lines is that reductions in reporter gene activity need to be normalized to cytotoxicity, i.e., live cell numbers. Here, we developed a dual luciferase assay based on a R. reniformis luciferase (hRLuc)-expressing R5-type HIV-1 (NLAD8-hRLuc) and a CEM cell line expressing CCR5 and firefly luciferase (R5CEM-FiLuc). The NLAD8-hRLuc reporter virus was replication competent in peripheral blood mononuclear cells. The level of hRLuc was correlated with p24 antigen levels (p<0.001, R=0.862). The target cell line, R5CEM-FiLuc, stably expressed the firefly luciferase (FiLuc) reporter gene and allowed the simultaneous monitoring of compound cytotoxicity. The dual reporter assay combining a NLAD8-hRLuc virus with R5CEM-FiLuc cells permitted the accurate determination of drug susceptibility for entry, reverse transcriptase, integrase, and protease inhibitors at different multiplicities of infection. This dual reporter assay provides a rapid and direct method for the simultaneous monitoring of HIV infection and cell viability.
References
Feb 1, 1995·Virology·R I ConnorN R Landau
Sep 11, 1997·The New England Journal of Medicine·S M HammerM A Fischl
Jul 21, 1999·Proceedings of the National Academy of Sciences of the United States of America·K YoshimuraH Mitsuya
Jan 5, 2002·Journal of Virology·Kazuhisa YoshimuraHiroaki Mitsuya
May 23, 2002·Antimicrobial Agents and Chemotherapy·Xiping WeiJohn C Kappes
Aug 7, 2002·Journal of Virology·Olaf KutschDavid N Levy
Jun 7, 2003·Journal of Clinical Microbiology·Hiroshi MiyakeMasanori Baba
Sep 25, 2003·Antimicrobial Agents and Chemotherapy·Yasuhiro KohHiroaki Mitsuya
Jun 3, 2006·The Journal of Infectious Diseases·Rochelle P WalenskyKenneth A Freedberg
Dec 22, 2006·Journal of Virology·Mike WestbyManos Perros
Dec 22, 2006·Journal of Clinical Microbiology·Tomoko Chiba-MizutaniWataru Sugiura
Nov 6, 2007·Journal of Virology·Kazuya ShimuraMasao Matsuoka
May 23, 2008·PLoS Medicine·Gregory P BissonJean B Nachega
Jul 16, 2008·Antiviral Research·Masanori KobayashiTamio Fujiwara
Jan 31, 2009·PLoS Pathogens·Takuya YamamotoJun-ichiro Inoue
Mar 7, 2009·Science·Douglas D RichmanRoger J Pomerantz
Apr 22, 2009·AIDS·Edward M GardnerDavid R Bangsberg
Jan 6, 2010·Current Opinion in HIV and AIDS·Christina Ochsenbauer, John C Kappes
Sep 25, 2010·Virology·Tara G EdmondsChristina Ochsenbauer
Jun 2, 2011·Annals of Internal Medicine·Carl W Dieffenbach, Anthony S Fauci
Jun 4, 2011·Nature Protocols·Dirk DaelemansChristophe Pannecouque
Nov 9, 2012·Journal of Virology·Annette N RatcliffEric J Arts
Dec 3, 2013·Journal of Immunological Methods·Marloes A NaardingJill Gilmour
Mar 13, 2014·Journal of Immunological Methods·Marcella Sarzotti-KelsoeDavid C Montefiori
May 6, 2014·The Journal of General Virology·Kazuhisa YoshimuraShuzo Matsushita