Efficient insertion from an internal long terminal repeat (LTR)-LTR sequence on a reticuloendotheliosis virus vector is imprecise and cell specific.
To examine the fidelity and efficiency of integration from a covalently closed long terminal repeat (LTR)-LTR sequence in vivo, we isolated individual spleen necrosis virus proviruses that arose following infection of chicken embryo fibroblasts (CEFs) and sequenced the provirus-cell DNA junctions. Some but not all CEF preparations allowed efficient insertion from the internal sequence. Moreover, in contrast to integration from the normal ends of the viral DNA, which occurs with precision with respect to the viral DNA, insertion from the internal sequence was not precise. In particular, there were short deletions of variable size from the viral DNA and these proviruses were not flanked by short direct repeats. Although this imprecise insertion can be efficient in CEFs, such integration is very inefficient in two other cell types (D17 and QT47) that support the replication of reticuloendotheliosis viruses. Thus, it is possible that there is a cell-specific factor(s) in CEFs required for efficient but imprecise insertion or, alternatively, D17 and QT47 cells contain a factor that abrogates integration from an internal LTR-LTR junction. Virus particles released from CEFs do not efficiently use the LTR-LTR junction following infection of D17 cells. Therefore, if there is a CEF-specific factor required for insertion, it does not appear to be transferred through particles.
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
A T
AT
Panganiban
K J
KJ
Talbot
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D002642:Chick Embryo; D004279:DNA, Viral / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology; D005822:Genetic Vectors / Q000235:genetics*; D008969:Molecular Sequence Data; D011533:Proviruses / Q000235:genetics* / Q000302:isolation & purification; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics*; D001356:Reticuloendotheliosis virus / Q000235:genetics*; D013045:Species Specificity; D016662:Virus Integration / Q000235:genetics*
D004279:DNA, Viral