Comparative analysis of RNA/protein dynamics for the arginine-rich-binding motif and zinc-finger-binding motif proteins encoded by HIV-1.

We report a comparative study in which a single-molecule fluorescence resonance energy transfer approach was used to examine how the binding of two families of HIV-1 viral proteins to viral RNA hairpins locally changes the RNA secondary structures. The single-molecule fluorescence resonance energy transfer results indicate that the zinc finger protein (nucleocapsid) locally melts the TAR RNA and RRE-IIB RNA hairpins, whereas arginine-rich motif proteins (Tat and Rev) may strengthen the hairpin structures through specific binding interactions. Competition experiments show that Tat and Rev can effectively inhibit the nucleocapsid-chaperoned annealing of complementary DNA oligonucleotides to the TAR and RRE-IIB RNA hairpins, respectively. The competition binding data presented here suggest that the specific nucleic acid binding interactions of Tat and Rev can effectively compete with the general nucleic acid binding/chaperone functions of the nucleocapsid protein, and thus may in principle help regulate critical events during the HIV life cycle.

Single-molecule study of the inhibition of HIV-1 transactivation response region DNA/DNA annealing by argininamide.

Single-molecule spectroscopy was used to examine how a model inhibitor of HIV-1, argininamide, modulates the nucleic acid chaperone activity of the nucleocapsid protein (NC) in the minus-strand transfer step of HIV-1 reverse transcription, in vitro. In minus-strand transfer, the transactivation response region (TAR) RNA of the genome is annealed to the complementary "TAR DNA" generated during minus-strand strong-stop DNA synthesis. Argininamide and its analogs are known to bind to the hairpin bulge region of TAR RNA as well as to various DNA loop structures, but its ability to inhibit the strand transfer process has only been implied. Here, we explore how argininamide modulates the annealing kinetics and secondary structure of TAR DNA. The studies reveal that the argininamide inhibitory mechanism involves a shift of the secondary structure of TAR, away from the NC-induced "Y" form, an intermediate in reverse transcription, and toward the free closed or "C" form. In addition, more potent inhibition of the loop-mediated annealing pathway than stem-mediated annealing is observed. Taken together, these data suggest a molecular mechanism wherein argininamide inhibits NC-facilitated TAR RNA/DNA annealing in vitro by interfering with the formation of key annealing intermediates.

Probing nucleation, reverse annealing, and chaperone function along the reaction path of HIV-1 single-strand transfer.

Reverse transcription of the HIV-1 genome involves several nucleic acid rearrangement steps that are catalyzed (chaperoned) by the nucleocapsid protein (NC), including the annealing of the transactivation response region (TAR) RNA of the genome to the complementary sequence (TAR DNA) in minus-strand strong-stop DNA. It has been extremely challenging to obtain unambiguous mechanistic details on the annealing process at the molecular level because of the kinetic involvement of a complex and heterogeneous set of nucleic acid/protein complexes of variable structure and variable composition. Here, we investigate the in vitro annealing mechanism using a multistep single-molecule spectroscopy kinetic method. In this approach, an immobilized hairpin is exposed to a multistep programmed concentration sequence of NC, model complementary targeted-oligonucleotides, and buffer-only solutions. The sequence controllably "drags" single immobilized TAR hairpins among the kinetic stable states of the reaction mechanism; i.e., reactants, intermediates, and products. This single-molecule spectroscopy method directly probes kinetic reversibility and the chaperone (catalytic) role of NC at various stages along the reaction sequence, giving access to previously inaccessible kinetic processes and rate constants. By employing target oligonucleotides for specific TAR regions, we kinetically trap and investigate structural models for putative nucleation complexes for the annealing process. The new results lead to a more complete and detailed understanding of the ability of NC to promote nucleic acid/nucleic acid rearrangement processes. This includes information on the ability of NC to chaperone "reverse annealing" in single-strand transfer and the first observation of partially annealed, conformational substates in the annealing mechanism.