We have developed a reconstituted system which models the events associated with human immunodeficiency virus type 1 (HIV-1) plus-strand transfer. These events include synthesis of plus-strand strong-stop DNA [(+) SSDNA] from a minus-strand DNA donor template covalently attached to human tRNA3Lys, tRNA primer removal, and annealing of (+) SSDNA to the minus-strand DNA acceptor template. Termination of (+) SSDNA synthesis at the methyl A (nucleotide 58) near the 3' end of tRNA3Lys reconstitutes the 18-nucleotide primer binding site (PBS). Analysis of (+) SSDNA synthesis in vitro and in HIV-1 endogenous reactions indicated another major termination site: the pseudouridine at nucleotide 55. In certain HIV-1 strains, complementarity between nucleotides 56 to 58 and the first three bases downstream of the PBS could allow all of the (+) SSDNA products to be productively transferred. Undermodification of the tRNA may be responsible for termination beyond the methyl A. In studies of tRNA removal, we find that initial cleavage of the 3' rA by RNase H is not sufficient to achieve successful strand transfer. The RNA-DNA hybrid formed by the penultimate 17 bases of tRNA still annealed to (+) SSDNA must also be destabilized. This can occur by removal of additional 3'-terminal bases by RNase H (added either in cis or trans). Alternatively, the nucleic acid chaperone activity of nucleocapsid protein (NC) can catalyze this destabilization. NC stimulates annealing of the complementary PBS sequences in (+) SSDNA and the acceptor DNA template. Reverse transcriptase also promotes annealing but to a lesser extent than NC.