To prime reverse transcription, retroviruses require annealing of a transfer RNA molecule to the U5 primer binding site (U5-PBS) region of the viral genome. The residues essential for primer annealing are initially locked in intramolecular interactions; hence, annealing requires the chaperone activity of the retroviral nucleocapsid (NC) protein to facilitate structural rearrangements. Here we show that, unlike classical chaperones, the Moloney murine leukaemia virus NC uses a unique mechanism for remodelling: it specifically targets multiple structured regions in both the U5-PBS and tRNA(Pro) primer that otherwise sequester residues necessary for annealing. This high-specificity and high-affinity binding by NC consequently liberates these sequestered residues--which are exactly complementary--for intermolecular interactions. Furthermore, NC utilizes a step-wise, entropy-driven mechanism to trigger both residue-specific destabilization and residue-specific release. Our structures of NC bound to U5-PBS and tRNA(Pro) reveal the structure-based mechanism for retroviral primer annealing and provide insights as to how ATP-independent chaperones can target specific RNAs amidst the cellular milieu of non-target RNAs.