Specific packaging of spliced retroviral vector transcripts lacking the Psi-region.
The effect of a cryptic splice acceptor (cSA) site located at the end of the extended packaging signal in murine leukemia virus (MLV)-based vectors was investigated. Although this cSA is also present in wild type MLV, it was found to result in a smaller transcript in which the packaging signal (Psi) had been removed by splicing only in MLV-derived vectors. Splicing occurs both in packaging cells producing the MLV-vectors as well as in the infected target cells. Transcripts lacking the Psi-sequence (Psi(-)) are packaged relatively efficiently into virus particles, even in the presence of wild type Psi(+)-vector transcripts. The Psi(-)-viral RNA is reverse transcribed in vector transduced cells as is any other retroviral genome. The titer obtained from the Psi(-)-vector was only 1000-fold reduced in comparison to the same Psi(+)-vector. These results suggest that Psi(-)-transcripts may be packaged more frequently than previously supposed and that splicing patterns should be carefully analysed on an individual basis for retroviral vectors used in gene therapy.
Pambalk
Konstanze
K
Institute of Virology, University of Veterinary Medicine, Veterinaerplatz 1, A-1210 Vienna, Austria.
Hohenadl
Christine
C
Salmons
Brian
B
Günzburg
Walter H
WH
Renner
Matthias
M
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D017931:DNA Primers; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D009052:Leukemia Virus, Murine / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D012190:Retroviridae / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic
D017931:DNA Primers; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins