Rescue of measles virus using a replication-deficient vaccinia-T7 vector.
A system which allows the reconstitution of measles virus (MV) from cloned cDNA is described. The severely host cell restricted vaccinia vector MVA-T7 expressing bacteriophage T7 RNA polymerase was used to generate full-length antigenomic MV RNA and simultaneously the mRNAs encoding the viral N, P and L proteins in order to produce replicationally and transcriptionally active nucleocapsids. The functionality of the N, P and L proteins was demonstrated first by their ability to rescue MV specific subgenomic RNAs. Assembly and budding of reconstituted MV was shown by syncytia formation and subsequently by virus isolation. The inability of MVA-T7 to produce progeny virus in most mammalian cells circumvents the necessity to separate the reconstituted MV from the MVA-T7 helper virus. Since all components are expressed transiently, this system is especially suitable for studying the functions of N, P and L. Furthermore, it is useful for investigating later steps in the MV life cycle.
Schneider
H
H
Institut für Molekularbiologie I, Universität Zürich, Switzerland.
Spielhofer
P
P
Kaelin
K
K
Dötsch
C
C
Radecke
F
F
Sutter
G
G
Billeter
M A
MA
D000818:Animals; D017123:Bacteriophage T7 / Q000201:enzymology; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D004279:DNA, Viral / Q000032:analysis; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D003673:Defective Viruses / Q000235:genetics*; D005822:Genetic Vectors; D016679:Genome, Viral; D006367:HeLa Cells; D006801:Humans; D008459:Measles virus / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D008969:Molecular Sequence Data; D014616:Vaccinia virus / Q000235:genetics*; D014709:Vero Cells; D014764:Viral Proteins; D014779:Virus Replication
D004279:DNA, Viral; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases