High rate of mismatch extension during reverse transcription in a single round of retrovirus replication.
We made spleen necrosis virus-based retroviral vectors with mutations at the 3' end of the primer binding site region to observe the effects of terminal mismatches on retroviral replication. These vectors, when compared to a vector with the wild-type primer binding sequence, allowed us to assay the effects of the mutations on the viral titer during a single cycle of replication. The mutant vectors had titers that were comparable to the wild-type vector, indicating that reverse transcriptase has no trouble extending mismatches of as many as 3 bases under normal in vivo conditions. These results confirm and extend previous in vitro studies [Yu, H. & Goodman, M. (1992) J. Biol. Chem. 15, 10888-10896] that showed that such mismatch extension could occur in a cell-free system at high concentrations of incorrect nucleotides and in the absence of correct nucleotides. We now show that mismatch extension can occur during normal retroviral replication in cells and at normal physiological nucleotide concentrations.
Pulsinelli
G A
GA
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison 53706.
Temin
H M
HM
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers; D004260:DNA Repair; D004261:DNA Replication; D004285:Dogs; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics* / Q000502:physiology; D014162:Transfection; D014407:Tumor Cells, Cultured; D014779:Virus Replication
D017931:DNA Primers; D012194:RNA-Directed DNA Polymerase