Establishment and characterization of plasmid-driven minigenome rescue systems for Nipah virus: RNA polymerase I- and T7-catalyzed generation of functional paramyxoviral RNA.
In this study we report the development and optimization of two minigenome rescue systems for Nipah virus, a member of the Paramyxoviridae family. One is mediated by the T7 RNA polymerase supplied either by a constitutively expressing cell line or by transfection of expression plasmids and is thus independent from infection with a helper virus. The other approach is based on RNA polymerase I-driven transcription, a unique approach for paramyxovirus reverse genetics technology. Minigenome rescue was evaluated by reporter gene activities of (i) the two different minigenome transcription systems, (ii) genomic versus antigenomic-oriented minigenomes, (iii) different ratios of the viral protein expression plasmids, and (iv) time course experiments. The high efficiency and reliability of the established systems allowed for downscaling to 96-well plates. This served as a basis for the development of a high-throughput screening system for potential antivirals that target replication and transcription of Nipah virus without the need of high bio-containment. Using this system we were able to identify two compounds that reduced minigenome activity.
Freiberg
Alexander
A
Department of Pathology, Center for Biodefense and Emerging Infectious Diseases, University of Texas Medical Branch at Galveston, Galveston, TX 77555-0609, USA.
Dolores
Lhia Krista
LK
Enterlein
Sven
S
Flick
Ramon
R
D000998:Antiviral Agents / Q000494:pharmacology; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D016679:Genome, Viral / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D045405:Nipah Virus / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D010252:Paramyxoviridae / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics*; D012318:RNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication / Q000187:drug effects
D000998:Antiviral Agents; D012367:RNA, Viral; D014764:Viral Proteins; D015500:Chloramphenicol O-Acetyltransferase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D012318:RNA Polymerase I