Synthesis of the HIV-1 viral DNA by reverse transcriptase involves two obligatory strand transfer reactions. The second strand transfer corresponds to the annealing of the (-) and (+) DNA copies of the primer binding site (PBS) sequence which is chaperoned by the nucleocapsid protein (NCp7). NCp7 modifies the (+)/(-)PBS annealing mechanism by activating a loop-loop kissing pathway that is negligible without NCp7. To characterize in depth the dynamics of the loop in the NCp7/PBS nucleoprotein complexes, we investigated the time-resolved fluorescence parameters of a (-)PBS derivative containing the fluorescent nucleoside analogue 2-aminopurine at positions 6, 8 or 10. The NCp7-directed switch of (+)/(-)PBS annealing towards the loop pathway was associated to a drastic restriction of the local DNA dynamics, indicating that NCp7 can 'freeze' PBS conformations competent for annealing via the loops. Moreover, the modifications of the PBS loop structure and dynamics that govern the annealing reaction were found strictly dependent on the integrity of the zinc finger hydrophobic platform. Our data suggest that the two NCp7 zinc fingers are required to ensure the specificity and fidelity of the second strand transfer, further underlining the pivotal role played by NCp7 to control the faithful synthesis of viral HIV-1 DNA.