The nucleocapsid protein (NC) of HIV-1 exerts critical functions in viral genome replication and virus assembly. Since the recognition of target nucleic acids is required in the initial step of most NC-mediated processes, attempts were made to find small molecules capable of competing with this recognition. In particular, several Trp-rich hexapeptides were recently found to strongly bind RNA sequences targeted by NC. To further validate these peptides as potential anti-NC agents, we studied the ability of Ac-HKWPWW-NH2, taken as a representative, to interfere with the NC chaperone properties required during reverse transcription. Using NMR and steady-state and time-resolved fluorescence spectroscopy, we characterized the structure of Ac-HKWPWW-NH2 as well as its binding to viral sequences such as TAR and PBS involved in the two obligatory strand transfers of reverse transcription. Results show that Ac-HKWPWW-NH2 exhibits an almost symmetric cis-trans equilibrium at the level of the Pro residue where it is structured. The peptide binds both TAR and PBS sequences with low micromolar affinities. The cis-Pro and trans-Pro conformations of the peptide bind with comparable affinities to (-)PBS, mainly through stacking interactions between the Trp residues and the (-)PBS bases. Though all three Trp residues may contribute to the (-)PBS/Ac-HKWPWW-NH2 complex formation, Trp3 and Trp5 residues are the key residues in the complexes with the cis-Pro and trans-Pro conformations, respectively. Moreover, Ac-HKWPWW-NH2 stabilizes cTAR secondary structure and largely inhibits the NC-directed melting of cTAR. This further strengthens the interest of this peptide for deriving modified peptides capable of inhibiting NC and HIV-1 replication.