title: Replication-Competent Influenza Virus and Respiratory Syncytial Virus Luciferase Reporter Strains Engineered for Co-Infections Identify Antiviral Compounds in Combination Screens.
abstract: Myxoviruses such as influenza A virus (IAV) and respiratory syncytial virus (RSV) are major human pathogens, mandating the development of novel therapeutics. To establish a high-throughput screening protocol for the simultaneous identification of pathogen- and host-targeted hit candidates against either pathogen or both, we have attempted co-infection of cells with IAV and RSV. However, viral replication kinetics were incompatible, RSV signal window was low, and an IAV-driven minireplicon reporter assay used in initial screens narrowed the host cell range and restricted the assay to single-cycle infections. To overcome these limitations, we developed an RSV strain carrying firefly luciferase fused to an innovative universal small-molecule assisted shut-off domain, which boosted assay signal window, and a hyperactive fusion protein that synchronized IAV and RSV reporter expression kinetics and suppressed the identification of RSV entry inhibitors sensitive to a recently reported RSV pan-resistance mechanism. Combined with a replication-competent recombinant IAV strain harboring nanoluciferase, the assay performed well on a human respiratory cell line and supports multicycle infections. Miniaturized to 384-well format, the protocol was validated through screening of a set of the National Institutes of Health Clinical Collection (NCC) in quadruplicate. These test screens demonstrated favorable assay parameters and reproducibility. Application to a LOPAC library of bioactive compounds in a proof-of-concept campaign detected licensed antimyxovirus therapeutics, ribavirin and the neuraminidase inhibitor zanamivir, and identified two unexpected RSV-specific hit candidates, Fenretinide and the opioid receptor antagonist BNTX-7. Hits were evaluated in direct and orthogonal dose-response counterscreens using a standard recRSV reporter strain expressing Renilla luciferase.
authors: Institute for Biomedical Sciences, Georgia State University , Atlanta, Georgia 30303-3222, United States.
authors: Dan
authors: D
authors: Yan
authors: Institute for Biomedical Sciences, Georgia State University , Atlanta, Georgia 30303-3222, United States.
authors: Marco
authors: M
authors: Weisshaar
authors: Institute for Biomedical Sciences, Georgia State University , Atlanta, Georgia 30303-3222, United States.
authors: Kristen
authors: K
authors: Lamb
authors: Department of Biology, Stanford University , Stanford, California 94305-5020, United States.
authors: Hokyung K
authors: HK
authors: Chung
authors: Department of Bioengineering, Stanford University , Stanford, California 94305, United States.|Department of Pediatrics, Stanford University , Stanford, California 94305, United States.
authors: Michael Z
authors: MZ
authors: Lin
authors: Institute for Biomedical Sciences, Georgia State University , Atlanta, Georgia 30303-3222, United States.
authors: Richard K
authors: RK
authors: Plemper
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry* / Q000494:pharmacology; D001597:Benzylidene Compounds / Q000494:pharmacology; D002460:Cell Line; D060085:Coinfection; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D005240:Feasibility Studies; D017313:Fenretinide / Q000737:chemistry / Q000494:pharmacology; D017930:Genes, Reporter; D057166:High-Throughput Screening Assays; D006801:Humans; D009980:Influenza A virus / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D049409:Luciferases, Firefly / Q000235:genetics; D049410:Luciferases, Renilla / Q000235:genetics; D009271:Naltrexone / Q000031:analogs & derivatives / Q000494:pharmacology; D009292:Narcotic Antagonists / Q000737:chemistry / Q000494:pharmacology; D009439:Neuraminidase / Q000037:antagonists & inhibitors; D012136:Respiratory Syncytial Viruses / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D012254:Ribavirin / Q000737:chemistry / Q000494:pharmacology; D053586:Virus Internalization / Q000187:drug effects; D014779:Virus Replication; D053243:Zanamivir / Q000737:chemistry / Q000494:pharmacology
chemical_list: D000998:Antiviral Agents; D001597:Benzylidene Compounds; D009292:Narcotic Antagonists; C077358:7-benzylidenenaltrexone; D017313:Fenretinide; D012254:Ribavirin; D009271:Naltrexone; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly; D009439:Neuraminidase; D053243:Zanamivir