title: Recovery of murine norovirus and feline calicivirus from plasmids encoding EMCV IRES in stable cell lines expressing T7 polymerase.
abstract: Reverse genetics systems constitute one of the most important and powerful tools to study the molecular biology of viruses. We developed a new strategy for the recovery of murine norovirus from a single plasmid in which a bacteriophage T7 RNA polymerase (T7pol) promoter for transcription and an EMCV IRES for efficient translation were engineered immediately upstream of the viral genome. Infectious noroviruses were recovered following transfection of the newly designed plasmid into nonpermissive BHK-21 and HEK293T cell lines that were engineered to express T7pol constitutively. Recovery of the virus did not require the presence of a ribozyme at the 3'-end of the virus genome. The strategy worked also for the efficient recovery of feline calicivirus in these normally nonpermissive cell types. This simplified reverse genetics approach may be broadly applicable to other caliciviruses.
authors.affiliation: Caliciviruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, DHHS, Bethesda, MD, USA.
authors.forename: Carlos
authors.initials: C
authors.lastname: Sandoval-Jaime
authors.affiliation: Caliciviruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, DHHS, Bethesda, MD, USA.
authors.forename: Kim Y
authors.initials: KY
authors.lastname: Green
authors.affiliation: Caliciviruses Section, Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, DHHS, Bethesda, MD, USA. Electronic address: ss216m@nih.gov.
authors.forename: Stanislav V
authors.initials: SV
authors.lastname: Sosnovtsev
mesh_terms: D000818:Animals; D017927:Calicivirus, Feline / Q000235:genetics / Q000502:physiology*; D002460:Cell Line; D006224:Cricetinae; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000378:metabolism*; D004686:Encephalomyocarditis virus / Q000235:genetics*; D006801:Humans; D000067556:Internal Ribosome Entry Sites; D029322:Norovirus / Q000235:genetics / Q000502:physiology*; D010957:Plasmids; D014176:Protein Biosynthesis; D059386:Reverse Genetics / Q000379:methods*; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000067556:Internal Ribosome Entry Sites; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases
keywords: EMCV IRES; Feline calicivirus; Murine norovirus; Reverse genetics system; T7 RNA polymerase