Stable RNA secondary structure in a retroviral vector insert terminates reverse transcriptase elongation in vitro but not in cultured cells.
We wished to test whether an RNA signal that causes termination of elongation by reverse transcriptase in vitro would affect retroviral vector function. A synthetic oligonucleotide containing a sequence capable of forming a very stable RNA secondary structure was subcloned into the retrovirus vector N2. The integration of this sequence into N2 causes termination of elongation by reverse transcriptase in vitro at the precise positions previously reported in a different sequence context. However, no premature termination of DNA synthesis was observed in the unintegrated DNA of vector transduced cells. Likewise, there was no deleterious effect of the sequence insert on vector titer. These results indicate that termination signals defined in in vitro systems cannot be used as predictors of in vivo function and suggest that viral proteins in addition to reverse transcriptase play an important role in transcript initiation and elongation.
Alford
R L
RL
Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX 77030.
Belmont
J W
JW
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000235:genetics; D005822:Genetic Vectors; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D014162:Transfection
D012313:RNA; D004247:DNA; D012194:RNA-Directed DNA Polymerase