A PREFERRED REGION FOR RECOMBINATIONAL PATCH REPAIR IN THE 5'-UNTRANSLATED REGION OF PRIMER BINDING SITE-IMPAIRED MURINE LEUKEMIA-VIRUS VECTORS

Transduction of primer binding site-impaired Akv murine leukemia virus -based retroviral vectors from the murine packaging cell lines Psi-2 a nd Omega E was studied. The efficiency of transduction of the neo mark er of all mutated constructs was found to decrease by 5 to 6 orders of magnitude compared with that of the wild-type vector. Thirty-two of 6 0 transduced proviruses analyzed harbored a primer binding site sequen ce matching a glutamine tRNA primer. Sequence analysis of the regions flanking the glutamine tRNA primer binding site revealed a distinct pa ttern of nucleotide differences from the Akv-based vector, suggesting the involvement of a specific endogenous virus-like sequence in patch repair rescue of the primer binding site mutants. The putative recombi nation partner RNA was found in virions from Psi-2 cells as detected b y analysis of glutamine tRNA-initiated cDNA and by sequence analysis o f regions at or around the glutamine tRNA primer binding site. We prop ose that the forced recombination of primer binding site mutants invol ves initial priming on endogenous viral sequences and requires templat e switching during minus-strand synthesis in the region between the ne o gene and the mutated primer binding site to allow correct second-str and transfer in reverse transcription. The system thereby selects for a reverse transcriptase-mediated recombination event in the 5' untrans lated region. A panel of sequence differences between the recombinatio n partners in this region has allowed mapping of the site of recombina tion for each transduction event. Interestingly, the majority of the r ecombination events were clustered within a narrow, 33-nucleotide regi on thought to be involved in genomic RNA dimerization.