Enhanced genetic rescue of negative-strand RNA viruses: use of an MVA-T7 RNA polymerase vector and DNA replication inhibitors.
A modified cDNA rescue system that improves recovery of recombinant nonsegmented, negative-strand RNA viruses from cloned DNAs is described. Rescue systems based on vaccinia virus-T7 RNA polymerase vectors have been used to derive many negative-strand viruses; however, some strains can be recalcitrant to rescue possibly because of the simultaneous replication of the vaccinia virus-T7 vector. Our goal was to engineer a system where replication of the vaccinia virus-T7 vector could be blocked, yet allow for sufficient T7 RNA polymerase expression to enable genetic rescue. To that end, a recombinant modified vaccinia virus Ankara (MVA) was engineered that contained the bacteriophage T7 gene-1 under the control of a strong early promoter that would enable T7 RNA polymerase expression in the absence of MVA DNA replication. The new T7 helper, MVAGKT7, was then utilized successfully for the genetic rescue of a measles virus minigenome and full-length cDNAs, in the presence of DNA synthesis inhibitors. In addition to blocking completely MVAGKT7 replication, AraC treatment was found to enhance minigenome-encoded gene expression and the efficiency of measles virus rescue.
Kovacs
Gerald R
GR
Wyeth Research, Viral Vaccine Discovery, 401 North Middletown Road, Pearl River, NY 10965, USA. gkovacs@niaid.nih.gov
Parks
Christopher L
CL
Vasilakis
Nikos
N
Udem
Stephen A
SA
D000818:Animals; D017123:Bacteriophage T7 / Q000235:genetics*; D002460:Cell Line; D004261:DNA Replication / Q000187:drug effects; D018076:DNA, Complementary / Q000235:genetics; D004279:DNA, Viral / Q000096:biosynthesis*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D005814:Genes, Viral; D005822:Genetic Vectors; D016679:Genome, Viral; D008459:Measles virus / Q000235:genetics / Q000502:physiology; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011401:Promoter Regions, Genetic; D011995:Recombination, Genetic; D014162:Transfection / Q000379:methods; D014616:Vaccinia virus / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins; D014779:Virus Replication
D018076:DNA, Complementary; D004279:DNA, Viral; D019384:Nucleic Acid Synthesis Inhibitors; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases