title: A novel dual luciferase assay for the simultaneous monitoring of HIV infection and cell viability.
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.
authors.affiliation: Department of Immunology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo, Japan; AIDS Research Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640, Japan; Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mt. Sinai, One Gustave Levy Place, Box 1090, New York, NY 10029, USA.
authors.forename: Yu-Ya
authors.initials: YY
authors.lastname: Mitsuki
authors.affiliation: Laboratory of Adjuvant Innovation, National Institute of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan; Laboratory of Vaccine Science, World Premier International Immunology Frontier Research Center (IFReC), Osaka University, 6F IFReC Research Building, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan; Center for AIDS Research, Kumamoto University, 2-2-1 Honjo, Chuo, Kumamoto 860-0811, Japan.
authors.forename: Takuya
authors.initials: T
authors.lastname: Yamamoto
authors.affiliation: Department of Immunology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo, Japan.
authors.forename: Fuminori
authors.initials: F
authors.lastname: Mizukoshi
authors.affiliation: Laboratory of Adjuvant Innovation, National Institute of Biomedical Innovation, Health and Nutrition, 7-6-8 Saito-Asagi, Ibaraki, Osaka 567-0085, Japan; Laboratory of Vaccine Science, World Premier International Immunology Frontier Research Center (IFReC), Osaka University, 6F IFReC Research Building, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
authors.forename: Masatoshi
authors.initials: M
authors.lastname: Momota
authors.affiliation: Department of Immunology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo, Japan.
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Terahara
authors.affiliation: AIDS Research Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640, Japan; Center for AIDS Research, Kumamoto University, 2-2-1 Honjo, Chuo, Kumamoto 860-0811, Japan.
authors.forename: Kazuhisa
authors.initials: K
authors.lastname: Yoshimura
authors.affiliation: AIDS Research Center, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo 162-8640, Japan. Electronic address: shigeh@nih.go.jp.
authors.forename: Shigeyoshi
authors.initials: S
authors.lastname: Harada
authors.affiliation: Department of Immunology, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo, Japan. Electronic address: yyokota@nih.go.jp.
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Tsunetsugu-Yokota
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology*; D002460:Cell Line; D002470:Cell Survival; D017930:Genes, Reporter; D015497:HIV-1 / Q000187:drug effects / Q000254:growth & development*; D049409:Luciferases, Firefly / Q000032:analysis* / Q000235:genetics; D049410:Luciferases, Renilla / Q000032:analysis* / Q000235:genetics
chemical_list: D019380:Anti-HIV Agents; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly
keywords: Firefly luciferase; HIV-1; Renilla luciferase; Reporter cell; Reporter virus