Establishment and characterization of plasmid-driven minigenome rescue systems for Nipah virus: RNA polymerase I- and T7-catalyzed generation of functional paramyxoviral RNA.

Abstract

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.

• Authors: Alexander Freiberg, Lhia Krista Dolores, Sven Enterlein, Ramon Flick
• Journal: Virology (Virology) Vol. 370 Issue 1 Pg. 33-44 (Jan 05 2008) ISSN: 0042-6822 [Print] United States
• PMID: 17904180 (Publication Type: Evaluation Study, Journal Article, Research Support, N.I.H., Extramural)

Chemical References

• Antiviral Agents
• RNA, Viral
• Viral Proteins
• Chloramphenicol O-Acetyltransferase
• bacteriophage T7 RNA polymerase
• DNA-Directed RNA Polymerases
• RNA Polymerase I

Topics

• Antiviral Agents (pharmacology)
• Cell Line
• Chloramphenicol O-Acetyltransferase (metabolism)
• DNA-Directed RNA Polymerases (genetics, metabolism)
• Genome, Viral (genetics)
• HeLa Cells
• Humans
• Microbial Sensitivity Tests (methods)
• Nipah Virus (drug effects, genetics, physiology)
• Paramyxoviridae (genetics, metabolism)
• Plasmids (genetics)
• RNA Polymerase I (genetics, metabolism)
• RNA, Viral (drug effects, genetics, metabolism)
• Transcription, Genetic (drug effects)
• Transfection
• Viral Proteins (genetics, metabolism)
• Virus Replication (drug effects)