A succession of mechanisms stimulate efficient reconstituted HIV-1 minus strand strong stop DNA transfer

Donor-acceptor template systems in vitro were designed to test mechanisms of minus strand transfer of human immunodeficiency virus 1 (HIV-1). Donor RNA D199, extending from the 5' end of the HIV-1 genome to the primer binding site (PBS), promoted transfer to only 35% with an acceptor RNA representing the 3' terminal 97 nucleotides, whereas donor RNA D520, including an additional 321 nucleotides 3' of PBS, exhibited 75% transfer. Both donors transferred through an invasion-driven pathway, but transfer was stimulated by the folding structure resulting from the extra segment in D520. In this study, the significance of interaction between the tRNA(lys3) primer and U3 was examined. Measurements utilizing acceptors having or lacking the U3 region complementary with tRNA(lys3) indicated that a tRNA(lys3)-U3 interaction compensated for inefficient acceptor invasion observed with D199. Stimulation presumably occurred because binding to tRNA(lys3) increased the proximity of the acceptor to elongated cDNA, improving transfer to 78% efficiency with D199, and even higher to 85% with D520. The stimulation did not require natural viral sequences but could be achieved by substituting the original U3 sequence with an equal length sequence that binds a different region of tRNA(lys3). Comparison between acceptors sharing the natural region for tRNA(lys3)-U3 interaction but having or lacking the acceptor invasion site demonstrated that tRNA(lys3)-U3 interaction and acceptor invasion cooperate for maximal stimulation. Overall, observations suggest that both proximity and invasion mechanisms are applied successively by HIV-1 for efficient minus strand transfer.