Insights on the role of nucleic acid/protein interactions in chaperoned nucleic acid rearrangements of HIV-1 reverse transcription

Abstract

HIV-1 reverse transcription requires several nucleic acid rearrangement steps that are "chaperoned" by the nucleocapsid protein (NC), including minus-strand transfer, in which the DNA transactivation response element (TAR) is annealed to the complementary TAR RNA region of the viral genome. These various rearrangement processes occur in NC bound complexes of specific RNA and DNA structures. A major barrier to the investigation of these processes in vitro has been the diversity and heterogeneity of the observed nucleic acid/protein assemblies, ranging from small complexes of only one or two nucleic acid molecules all the way up to large-scale aggregates comprised of thousands of NC and nucleic acid molecules. Herein, we use a flow chamber approach involving rapid NC/nucleic acid mixing to substantially control aggregation for the NC chaperoned irreversible annealing kinetics of a model TAR DNA hairpin sequence to the complementary TAR RNA hairpin, i.e., to form an extended duplex. By combining the flow chamber approach with a broad array of fluorescence single-molecule spectroscopy (SMS) tools (FRET, molecule counting, and correlation spectroscopy), we have unraveled the complex, heterogeneous kinetics that occur during the course of annealing. The SMS results demonstrate that the TAR hairpin reactant is predominantly a single hairpin coated by multiple NCs with a dynamic secondary structure, involving equilibrium between a "Y" shaped conformation and a closed one. The data further indicate that the nucleation of annealing occurs in an encounter complex that is formed by two hairpins with one or both of the hairpins in the "Y" conformation.

Fig. 1.

Structures of various oligonucleotides used in the annealing study. The secondary structures are predicted by the mfold program (www.bioinfo.rpi.edu/applications/mfold/old/dna/).

Fig. 2.

A typical single molecule kinetic measurement of TAR and 2 nM cTAR annealing reaction at 0.2 mM Mg²⁺ and 889 nM NC in buffer solution. (A) The alternating, two-color, laser excitation microscopy setup. (B) The number of molecules in three channels (donor, acceptor, and red) during the course of the annealing reaction. (C) E_A of each single molecule as a function of time, where each colored line corresponds to a single molecule. AOM, acoustic optical modulator; DM, dichroic mirror; F, notch filter; APD, avalanche photodiode; PEG, poly(ethylene glycol). Modulators controlled by two 180° out-of-phase square-wave signals give two-color alternating-laser excitation. After being filtered by a notch filter, the fluorescence is detected by APD1 and -2. Donor and acceptor channel fluorescence detected by APD1 and -2, respectively, is counted by two counters while the green excitation laser is on. Red channel fluorescence is also detected on APD2, except that it is counted by a third counter while red excitation laser is on.

Fig. 3.

E_A(t) histograms constructed at different time after initiation of the NC-induced annealing of Cy3-TAR annealing with Cy5-cTAR.

Fig. 4.

SM-FRET measurements of the annealing kinetics. 〈E_A〉 (A) and %annealed (B) vs. time for the annealing of cTAR with WT and mutant TAR DNA hairpins at 10 nM cTAR, 2 mM Mg²⁺ and 890 nM NC. (C) %Annealed vs. time for annealing of TAR RNA with various immobilized WT and mutant TAR DNA hairpins at 0.2 mM Mg²⁺ and 890 nM NC. For TAR DNA, 10 nM TAR RNA was used, whereas for -L3L4 TAR and -L1L2TAR, 25 nM TAR RNA was used.

Fig. 5.

Representative I(t) curves detecting the emission of Cy5-cTAR DNA. Blue lines are the trajectory of Cy5-cTAR DNA (50 nM) without NC present, exhibiting a continuous signal of ≈160 kHz (detected photons per second). Red lines correspond to Cy5-cTAR DNA (50 nM) with NC (500 nM), revealing intense blips due to aggregation and a decrease in the steady-state signal from monomer Cy5-cTAR DNA to ≈4 kHz. (Inset) FCS curves of cTAR (5 nM) (solid line) and cTAR (5 nM) with NC (500 nM) (dashed line) showing increase of τ_D due to NC binding to cTAR. Here, τ_D is the diffusion time for cTAR or cTAR/NC complexes passing through the focal volume of the laser.

Fig. 6.

The hypothetical kinetic scheme of TAR DNA annealing with its complements chaperoned by HIV-1 NC. Here, T denotes TAR DNA, C denotes complementary cTAR DNA or TAR RNA, and N denotes NC. In this scheme, N bound to T and C leads to a partially melting structure, namely Y form of T (T′) and C (C′). The subscripts, i, j, k, and l are used to describe the number of NC bound to nucleotides. Two partially melting hairpins form an encounter complex that leads to the formation of nucleation complexes. The annealing can go through either zipper nucleation or loop nucleation, therefore, forming zipper nucleation complexes (Z) or loop nucleation complexes (L) that leads to the formation of fully annealed duplexes.