HIV-1 nucleocapsid protein zinc finger structures induce tRNA(Lys,3) structural changes but are not critical for primer/template annealing

Retroviral reverse transcriptases use host cellular tRNAs as primers to ini tiate reverse transcription. In the case of human immunodeficiency virus ty pe 1 (HIV-1), the 3' 18 nucleotides of human tRNA(Lys,3) are annealed to a complementary sequence on the RNA genome known as the primer binding site ( PBS). The HIV-1 nucleocapsid protein (NC) facilitates this annealing. To un derstand the structural changes that are, induced upon NC binding to the tR NA alone, we employed a chemical probing method using the lanthanide metal terbium. At low concentrations of NC, the strong terbium cleavage observed in the core region of the tRNA is significantly attenuated. Thus, NC bindin g first results in disruption of the tRNA's metal binding pockets, includin g those that stabilize the D-T PC tertiary interaction. When NC concentrati ons approach the amount needed for complete primer/template annealing, NC f urther destabilizes the tRNA acceptor-T psiC stem minihelix, as evidenced b y increased terbium cleavage in this domain. A mutant form of NC (SSHS NC), which lacks the zinc finger structures, is able to anneal tRNA(Lys,3) effi ciently to the PBS, and to destabilize the tRNA tertiary core, albeit less effectively than wild-type NC. This mutant form of NC does not affect cleav age significantly in the helical regions, even when bound at high concentra tions. These results, as well as experiments conducted in the presence of p olyLys, suggest that in the absence of the zinc finger structures, NC acts as a polycation, neutralizing the highly negative phosphodiester backbone. The presence of an effective multivalent cationic peptide is sufficient for efficient tRNA primer annealing to the PBS. (C) 2001 Academic Press.