Effect of the murine leukemia virus extended packaging signal on the ratesand locations of retroviral recombination

Reverse transcriptase (RT) switches templates frequently during DNA synthes is; the acceptor template can be the same RNA (intramolecular) or the copac kaged RNA (intermolecular). Previous results indicated that intramolecular template switching occurred far more frequently than intermolecular templat e switching. We hypothesized that intermolecular template-switching events (recombination) occurred at a lower efficiency because the copackaged RNA w as not accessible to the RT. To test our hypothesis, the murine leukemia vi rus (MLV) extended packaging signal (Psi(+)) containing a dimer linkage str ucture (DLS) was relocated from the 5' untranslated region (UTR) to between selectable markers, allowing the two viral RNAs to interact closely in thi s region. It was found that the overall maximum recombination rates of vect ors with yr in the 5' UTR or Psi(+) between selectable markers were not dra stically different. However, vectors with Psi(+) located between selectable markers reached a plateau of recombination rate at a shorter distance. Thi s suggested a limited enhancement of recombination by Psi(+). The locations of the recombination events were also examined by using restriction enzyme markers. Recombination occurred in all four regions between the selectable markers; the region containing 5' Psi(+) including DLS did not undergo mor e recombination than expected from the size of the region. These experiment s indicated that although the accessibility of the copackaged RNA was impor tant in recombination, other factors existed to limit the number of viruses that were capable of undergoing intermolecular template switching. In addi tion, recombinants with multiple template switches were observed at a frequ ency much higher than expected, indicating the presence of high negative in terference in the MLV-based system. This extends our observation with the s pleen necrosis virus system and suggests that high negative interference ma y he a common phenomenon in retroviral recombination.