COMPLEMENTATION OF A PRIMER BINDING SITE-IMPAIRED MURINE LEUKEMIA VIRUS-DERIVED RETROVIRAL VECTOR BY A GENETICALLY-ENGINEERED TRANSFER-RNA-LIKE PRIMER

Reverse transcription of retroviral genomes is primed by a tRNA anneal ed to an 18-nucleotide primer binding site. Here, we present a primer complementation system to study molecular interaction of the replicati on machinery with the primer and primer binding site in vivo, Introduc tion of eight base substitutions into the primer binding site of a mur ine leukemia virus-based vector allowed efficient RNA encapsidation bu t resulted in severely reduced vector replication capacity. Replicatio n was restored upon complementation with a synthetic gene designed to encode a complementary tRNA-like primer, but not with a noncomplementa ry tRNA-like molecule. The engineered primer was shown to be involved in both the initiation of first-strand synthesis and second-strand tra nsfer. These results provide an in vivo demonstration that the retrovi ral replication machinery may recognize sequence complementarity rathe r than actual primer binding site and 3' primer sequences. Use of muta ted primer binding site vectors replicating via engineered primers may add additional control features to retroviral gene transfer technolog y.