Long-term replication of Sendai virus defective interfering particle nucleocapsids in stable helper cell lines.
An essential prerequisite for generating a stable helper cell line, which constitutively expresses functional Sendai virus RNA-dependent RNA polymerase, is the expression of all three Sendai virus nucleocapsid (NC) proteins, NP, P, and L, simulataneously. Generating a stable helper cell line was accomplished by cotransfecting cell line 293 with all three corresponding viral genes under the control of cytomegalovirus promoter-enhancer elements. Cotransfection with a dominant selectable marker enabled selection for stably transfected cells. The levels of the expressed P and NP proteins reached up to 1/10th and 1/20th of the protein levels in Sendai virus-infected cells, respectively. The Sendai virus polymerase activity of the coexpressed proteins was demonstrated by an in vivo polymerase assay. The cell clone H29 gave the strongest signal and produced DI genomes continuously for at least 3 months. This result demonstrates that it is possible to stably express adequate levels of all three viral NC proteins to form Sendai virus polymerase activity, thereby performing the replication and encapsidation of viral RNA, essential prerequisites for a helper cell line to be competent in producing recombinant viruses.
Willenbrink
W
W
Abteilung für Virusforschung, Max-Planck-Institut für Biochemie, Martinsried, Germany.
Neubert
W J
WJ
D001483:Base Sequence; D002213:Capsid / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification; D002460:Cell Line; D002999:Clone Cells; D003587:Cytomegalovirus / Q000235:genetics; D017931:DNA Primers; D003673:Defective Viruses / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D006801:Humans; D008969:Molecular Sequence Data; D010222:Parainfluenza Virus 1, Human / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012324:RNA-Dependent RNA Polymerase / Q000096:biosynthesis / Q000378:metabolism; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000302:isolation & purification; D006377:T-Lymphocytes, Helper-Inducer; D014162:Transfection; D014758:Viral Core Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000302:isolation & purification
D017931:DNA Primers; D011994:Recombinant Proteins; D014758:Viral Core Proteins; D012324:RNA-Dependent RNA Polymerase