Human immunodeficiency virus type 1 central DNA flap: Dynamic terminal product of plus-strand displacement DNA synthesis catalyzed by reverse transcriptase assisted by nucleocapsid protein

To terminate the reverse transcription of the human immunodeficiency virus type 1 (HIV-1) genome, a final step occurs within the center of the provira l DNA generating a 99-nucleotide DNA flap (6), This step, catalyzed by reve rse transcriptase (RT), is defined as a discrete strand displacement (SD) s ynthesis between the first nucleotide after the central priming (cPPT) site and the final position of the central termination sequence (CTS) site. Usi ng recombinant HIV-1 RT and a circular single-stranded DNA template harbori ng the cPPT-CTS sequence, we have developed an SD synthesis-directed in vit ro termination assay. Elongation, strand displacement, and complete central flap behavior were analyzed using electrophoresis and electron microscopy approaches. Optimal conditions to obtain complete central flap, which ended at the CTS site, have been defined in using nucleocapsid protein (NCp), th e main accessory protein of the reverse transcription complex. A full-lengt h HIV-1 central DNA flap was then carried out in vitro. Its synthesis appea rs faster in the presence of the HIV-1 NCp or the T4-encoded SSB protein (g p32), Finally, a high frequency of strand transfer was shown during the SD synthesis along the cPPT-CTS site with RT alone. This reveals a local and e fficient 3'-5' branch migration which emphasizes some important structural fluctuations within the flap. These fluctuations may be stabilized by the N Cp chaperone activity, The biological implications of the RT-directed NCp-a ssisted flap synthesis are discussed within the context of reverse transcri ption complexes, assembly of the preintegration complexes, and nuclear impo rt of the HIV-1 proviral DNA to the nucleus toward their chromatin targets.