A highly recombinogenic system for the recovery of infectious Sendai paramyxovirus from cDNA: generation of a novel copy-back nondefective interfering virus.
We have recovered infectious Sendai virus (SeV) from full-length cDNA (FL-3) by transfecting this cDNA and pGEM plasmids expressing the nucleocapsid protein (NP), phosphoprotein and large proteins into cells infected with a vaccinia virus which expresses T7 RNA polymerase. These cells were then injected into chicken eggs, in which SeV grows to very high titers. FL-3 was marked with a BglII site in the leader region and an NsiI site (ATGCAT) in the 5' nontranslated region of the NP gene, creating a new, out-of-frame, 5' proximal AUG. All the virus stocks generated eventually removed this impediment to NP expression, by either point mutation or recombination between FL-3 and pGEM-NP. The recovery system was found to be highly recombinogenic. Even in the absence of selective pressure, one in 20 of the recombinant SeV generated had exchanged the NP gene of FL-3 with that of pGEM-NP. When a fifth plasmid containing a new genomic 3' end without the presumably deleterious BglII site was included as another target for recombination, the new genomic 3' end was found in the recombinant SeV in 12 out of 12 recoveries. Using this approach, a novel copy-back nondefective virus was generated which interferes with wild-type virus replication.
Garcin
D
D
Department of Genetics and Microbiology, University of Geneva Medical School, CMU, Switzerland.
Pelet
T
T
Calain
P
P
Roux
L
L
Curran
J
J
Kolakofsky
D
D
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002645:Chickens; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D010222:Parainfluenza Virus 1, Human / Q000235:genetics* / Q000254:growth & development / Q000302:isolation & purification*; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000235:genetics; D011995:Recombination, Genetic; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D004279:DNA, Viral; D012367:RNA, Viral; D014764:Viral Proteins