Pulsinelli G A, Temin H M
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison 53706.
Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9490-4. doi: 10.1073/pnas.91.20.9490.
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.
我们构建了在引物结合位点区域3'端带有突变的基于脾坏死病毒的逆转录病毒载体，以观察末端错配对逆转录病毒复制的影响。与具有野生型引物结合序列的载体相比，这些载体使我们能够在单个复制周期内测定突变对病毒滴度的影响。突变载体的滴度与野生型载体相当，表明在正常体内条件下，逆转录酶延伸多达3个碱基的错配并无困难。这些结果证实并扩展了先前的体外研究[Yu, H. & Goodman, M. (1992) J. Biol. Chem. 15, 10888 - 10896]，该研究表明这种错配延伸可以在无细胞系统中高浓度错误核苷酸且无正确核苷酸的情况下发生。我们现在表明，错配延伸可以在细胞内正常逆转录病毒复制过程中以及正常生理核苷酸浓度下发生。
