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 and Omega E was studied. The efficiency of transduction of the neo marker 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 60 transduced proviruses analyzed harbored a primer binding site sequence matching a glutamine tRNA primer. Sequence analysis of the regions flanking the glutamine tRNA primer binding site revealed a distinct pattern 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 recombination partner RNA was found in virions from Psi-2 cells as detected by analysis of glutamine tRNA-initiated cDNA and by sequence analysis of regions at or around the glutamine tRNA primer binding site. We propose that the forced recombination of primer binding site mutants involves initial priming on endogenous viral sequences and requires template switching during minus-strand synthesis in the region between the neo gene and the mutated primer binding site to allow correct second-strand transfer in reverse transcription. The system thereby selects for a reverse transcriptase-mediated recombination event in the 5' untranslated region. A panel of sequence differences between the recombination partners in this region has allowed mapping of the site of recombination for each transduction event. Interestingly, the majority of the recombination events were clustered within a narrow, 33-nucleotide region thought to be involved in genomic RNA dimerization.