Extended minus-strand DNA as template for R-U5-mediated second-strand transfer in recombinational rescue of primer binding site-modified retroviral vectors.
We have previously demonstrated recombinational rescue of primer binding site (PBS)-impaired Akv murine leukemia virus-based vectors involving initial priming on endogenous viral sequences and template switching during cDNA synthesis to obtain PBS complementarity in second-strand transfer of reverse transcription (Mikkelsen et al., J. Virol. 70:1439-1447, 1996). By use of the same forced recombination system, we have now found recombinant proviruses of different structures, suggesting that PBS knockout vectors may be rescued through initial priming on endogenous virus RNA, read-through of the mutated PBS during minus-strand synthesis, and subsequent second-strand transfer mediated by the R-U5 complementarity of the plus strand and the extended minus-strand DNA acceptor template. Mechanisms for R-U5-mediated second-strand transfer and its possible role in retrovirus replication and evolution are discussed.
Mikkelsen
J G
JG
Department of Molecular and Structural Biology, University of Aarhus, Denmark.
Lund
A H
AH
Dybkaer
K
K
Duch
M
M
Pedersen
F S
FS
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002472:Cell Transformation, Viral; D017931:DNA Primers; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D005822:Genetic Vectors / Q000235:genetics*; D009052:Leukemia Virus, Murine / Q000235:genetics* / Q000502:physiology; D051379:Mice; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D012091:Repetitive Sequences, Nucleic Acid; D017415:Ribonucleoprotein, U5 Small Nuclear / Q000235:genetics*; D013698:Templates, Genetic; D014779:Virus Replication
D017931:DNA Primers; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D017415:Ribonucleoprotein, U5 Small Nuclear