Lund A H, Duch M, Lovmand J, Jørgensen P, Pedersen F S
Department of Molecular and Structural Biology, University of Aarhus, Denmark.
J Virol. 1997 Feb;71(2):1191-5. doi: 10.1128/JVI.71.2.1191-1195.1997.
Reverse transcription of retroviral genomes is primed by a tRNA annealed to an 18-nucleotide primer binding site. Here, we present a primer complementation system to study molecular interaction of the replication machinery with the primer and primer binding site in vivo. Introduction of eight base substitutions into the primer binding site of a murine leukemia virus-based vector allowed efficient RNA encapsidation but resulted in severely reduced vector replication capacity. Replication was restored upon complementation with a synthetic gene designed to encode a complementary tRNA-like primer, but not with a noncomplementary tRNA-like molecule. The engineered primer was shown to be involved in both the initiation of first-strand synthesis and second-strand transfer. These results provide an in vivo demonstration that the retroviral replication machinery may recognize sequence complementarity rather than actual primer binding site and 3' primer sequences. Use of mutated primer binding site vectors replicating via engineered primers may add additional control features to retroviral gene transfer technology.
逆转录病毒基因组的逆转录由退火至18个核苷酸引物结合位点的tRNA引发。在此，我们提出一种引物互补系统，用于在体内研究复制机制与引物及引物结合位点的分子相互作用。在基于鼠白血病病毒的载体的引物结合位点引入八个碱基替换，可实现高效的RNA包装，但导致载体复制能力严重降低。用设计编码互补tRNA样引物的合成基因进行互补后，复制得以恢复，但用非互补tRNA样分子则不能。工程化引物被证明参与了第一链合成的起始和第二链转移。这些结果提供了一个体内证据，即逆转录病毒复制机制可能识别序列互补性而非实际的引物结合位点和3'引物序列。使用通过工程化引物复制的突变引物结合位点载体可能会为逆转录病毒基因转移技术增加额外的控制特性。
