Molecular determinants of HIV-1 NCp7 chaperone activity in maturation of the HIV-1 dimerization initiation site

Abstract

Human immunodeficiency virus genome dimerization is initiated through an RNA-RNA kissing interaction formed via the dimerization initiation site (DIS) loop sequence, which has been proposed to be converted to a more thermodynamically stable linkage by the viral p7 form of the nucleocapsid protein (NC). Here, we systematically probed the role of specific amino acids of NCp7 in its chaperone activity in the DIS conversion using 2-aminopurine (2-AP) fluorescence and nuclear magnetic resonance spectroscopy. Through comparative analysis of NCp7 mutants, the presence of positively charged residues in the N-terminus was found to be essential for both helix destabilization and strand transfer functions. It was also observed that the presence and type of the Zn finger is important for NCp7 chaperone activity, but not the order of the Zn fingers. Swapping single aromatic residues between Zn fingers had a significant effect on NCp7 activity; however, these mutants did not exhibit the same activity as mutants in which the order of the Zn fingers was changed, indicating a functional role for other flanking residues. RNA chaperone activity is further correlated with NCp7 structure and interaction with RNA through comparative analysis of nuclear magnetic resonance spectra of NCp7 variants, and complexes of these proteins with the DIS dimer.

Figure 1.

(A) NCp7 wild-type protein sequence. Zn coordinating and basic residues in the N-terminus are shown in bold. The aromatic residues Phe16 and Trp37 are in bold and circled. (B) SL1 hairpin constructs. The residues changed from the wild-type sequence in the DIS loop region are bolded, and the adenines that are replaced by 2-AP in the two fluorescent constructs [DIS24(GA)-4ap and DIS24(GA)-12ap] are circled. The DIS24(UC) contains a complementary bulged uracil that forms a base-pair on maturation and the exchanged stem in the DIS23(HxUC), which disfavors formation of the duplex via strand exchange, are boxed. The DIS23(GA) hairpin used in the NMR experiments is the same sequence as the DIS24(GA) constructs with the exception that the bulged adenosine at position 4 is deleted. (C) Representative plot of the DIS kissing complex to extended duplex conversion measured by 2-AP fluorescence emission quenching as a function of time after the addition of wild-type NCp7 to the DIS24(GA)-4ap•DIS24(UC) kissing complex at physiological pH (225 nM NCp7 protein was added to 100 nM of RNA kissing complex). (Inset) Schematic of the DIS kissing complex to extended duplex conversion is shown with the 2-AP position circled.

Figure 2.

(A) Comparison of the time dependent 2-AP fluorescence emission quenching plots of the wild-type NCp7 and the Zn finger positional variants of NCp7 (Table 1). NC 1/1 (green); NC 2/2 (purple); NC 2/1 (blue). (B) Comparison of the time dependent 2-AP fluorescence emission quenching plots of the wild-type NCp7 and the Zn finger coordination variants of NCp7: H23C (green), H44C (blue) and H23C/H44C (purple), and SSHS/SSHS (black). In both panels, the NCp7 wild-type curve (red) is plotted as a reference for comparison. In each of these assays, 225 nM of the mutant NCp7 protein was added to 100 nM of RNA kissing complex. The 2-AP quenching curves were fit to an exponential equation to obtain the rates of conversion of kissing complex to extended duplex (Table 2).

Figure 3.

(A) Comparison of the time dependent 2-AP fluorescence emission quenching plots of the wild-type NCp7 and the aromatic amino acid point mutants of NCp7. Time-dependent fluorescence quenching plots of the wild-type NCp7 (red) and single point alanine mutants of the aromatic residues, F16A (green), W37A (blue) and the double mutant F16A/W37A, named here as FAWA (dark green). (B) Fluorescence decay curves of the aromatic amino acid residue swap mutants: F16W (green), W37F (blue) and FWWF (purple). In both panels, the NCp7 wild-type curve (red circles) is plotted as a reference for comparison. In each of these assays, 225 nM of the mutant NCp7 protein was added to 100 nM of RNA kissing complex. The 2-AP quenching curves were fit to an exponential equation to obtain the rates of conversion of kissing complex to extended duplex (Table 2).

Figure 4.

Comparison of the time dependent fluorescence decay plots of the wild-type NC (black) and the N-terminal mutant of NCp7 (gray). In this assay, 225 nM of N-terminal mutant of NCp7 protein was added to 100 nM of RNA kissing complex to again be consistent with the wild-type NCp7 2-AP fluorescence assay.

Figure 5.

Structural characterization of wild-type NCp7 binding to the DIS kissing complex. (A) An overlay of the ¹⁵N-¹H HSQC spectrum of the wild-type NCp7 (blue) and its complex with the DIS kissing complex (red). The backbone amide resonances are labeled as well as the side chain indole NH group (He1) of W37 and the side chain amino resonances (NH₂) of N17 are shown with a solid line connecting them. Resonances that are broadened beyond detection on binding the RNA are circled, and shifted resonances are indicated with solid lines. The secondary structure of NCp7 is drawn with residues showing significant chemical shift perturbation in bold. The N-terminal residues for which amide resonances are not observed in the HSQC spectrum are boxed. (B) An overlay of the ¹⁵N-¹H HSQC spectrum of the wild-type NCp7 bound to the DIS kissing complex (red) and the same complex after addition of 40 µM of MgCl₂ (blue). The residues whose amide proton resonances either reappear or sharpen on addition of 40 µM of MgCl₂ are labeled.

Figure 6.

(A) An overlay of ¹⁵N-¹H HSQC spectrum of N-terminal NCp7 (blue) and its complex with the DIS kissing complex (red). The residues that are mutated to alanine are highlighted in red in the structure of NCp7. The backbone amide resonances are labeled besides the side chain indole NH group (He1) of W37, and the side chain amino resonances of N17 (NH₂) are shown with a solid line connecting them. The N-terminal residues for which amide resonances are not observed in the HSQC spectrum are boxed. (B) Overlay of ¹⁵N-¹H HSQC spectrum of N-terminal NCp7 in complex with the DIS kissing complex (red) and the same complex in the presence of 40 µM of MgCl₂ (blue).

Figure 7.

An NMR chemical shift perturbation (CSP) model of NCp7 bound to the DIS kissing complex. The observed CSP of amide resonances of NCp7 are mapped onto the previously determined NMR structure of NCp7 bound to HIV SL2 (PDB id. 1F6U). Residues whose amide resonances showed a significant CSP in both HN and N dimensions are colored red, and the residues with moderate or less CSP are colored in brown. Zinc atoms are shown in green. The N-terminal, C-terminal and Linker regions are denoted by N, C and L, respectively. The terminal residues on both the N and C terminus are not shown, as they were not observed in the HSQC spectrum. More residues in the proximal Zn finger show significant CSP and both the aromatic residues in the fingers F16 and W37 (highlighted with an asterisks) show a significant CSP on binding the RNA kissing complex. Mutating these residues to alanine results in a severe loss of chaperoning activity of the protein. Interestingly, alanine at position 30, presumed to be at a distant site from the RNA-binding interface, also shows significant CSP on binding the kissing complex, suggesting a conformational change in the linker structure that might reorient the two Zn fingers to accommodate proper stacking interactions between the kissing helix residues of the RNA and the aromatic acid residues of the protein. The hydrophobic patch that facilitates binding of RNA is also indicated.