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. 2003 Sep 15;31(18):5425-32.
doi: 10.1093/nar/gkg738.

DNA condensation by the nucleocapsid protein of HIV-1: a mechanism ensuring DNA protection

G Krishnamoorthy  1 , Bernard Roques, Jean-Luc Darlix, Yves Mély
Affiliations

DNA condensation by the nucleocapsid protein of HIV-1: a mechanism ensuring DNA protection

G Krishnamoorthy et al. Nucleic Acids Res. .

Abstract

The nucleocapsid (NC) protein NCp7 of the immunodeficiency virus type 1 is a small basic protein with two zinc finger motifs. NCp7 has key roles in virus replication and structure, which rely on its interactions with nucleic acids. Although most interactions involve RNAs, binding to the viral DNA is thought to be of importance to achieve protection of the DNA against cellular nucleases and its integration into the host genome. We investigated the interaction of NCp7 with plasmid DNA as a model system. The fluorescence probe YOYO-1 was used as the reporter. Binding of NCp7 to DNA caused DNA condensation, as inferred from the dramatic decrease in YOYO-1 fluorescence. Efficient condensation of DNA required the full length NCp7 with the zinc fingers. The fingerless peptide was less efficient in condensing DNA. Binding of both these NC peptides led to freezing of the segmental dynamics of DNA as revealed by anisotropy decay kinetics of YOYO-1. The truncated peptide NC(12-55) which retains the zinc fingers did not lead to DNA condensation despite its ability to bind and partially freeze the segmental motion of DNA. We propose that the histone-like property of NCp7 leading to DNA condensation contributes to viral DNA stability, in vivo.

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Figures

Figure 1
Figure 1
(A) NC peptides used in the present study. (B) Fluorescence emission spectra of DNA-bound YOYO-1 in the presence of NCp7 [NC(1–72)]. Samples had 400 nM of DNA phosphate, 8 nM of YOYO-1 (dye:phosphate = 1:50) in 100 mM NaCl, 20 mM HEPES at pH 7.4. (a) control; (b) 0.1 µM NC(1–72) and (c) 0.4µM NC(1–72). The excitation was at 470 nm.
Figure 1
Figure 1
(A) NC peptides used in the present study. (B) Fluorescence emission spectra of DNA-bound YOYO-1 in the presence of NCp7 [NC(1–72)]. Samples had 400 nM of DNA phosphate, 8 nM of YOYO-1 (dye:phosphate = 1:50) in 100 mM NaCl, 20 mM HEPES at pH 7.4. (a) control; (b) 0.1 µM NC(1–72) and (c) 0.4µM NC(1–72). The excitation was at 470 nm.
Figure 2
Figure 2
Titration of fluorescence intensity of DNA-bound YOYO-1 with NCp7 and the related peptides. (a) NC(1–72); (b) NC(1–72)dd and (c) NC(12–55). The concentration of DNA phosphate was 400 nM in (A) and 5 µM in (B). Dye:phosphate ratio was kept at 1:50. Fluorescence intensities refer to equilibrium values. The horizontal lines at 0.07 correspond to PEI–DNA complex having the ratio, of primary N of PEI to DNA phosphate, of 10.
Figure 2
Figure 2
Titration of fluorescence intensity of DNA-bound YOYO-1 with NCp7 and the related peptides. (a) NC(1–72); (b) NC(1–72)dd and (c) NC(12–55). The concentration of DNA phosphate was 400 nM in (A) and 5 µM in (B). Dye:phosphate ratio was kept at 1:50. Fluorescence intensities refer to equilibrium values. The horizontal lines at 0.07 correspond to PEI–DNA complex having the ratio, of primary N of PEI to DNA phosphate, of 10.
Figure 3
Figure 3
Titration of fluorescence intensity of DNA-bound YOYO-1 with NC(1–72) at various DNA concentrations. DNA phosphate concentrations were 0.1 µM (squares); 0.2 µM (circles); 0.4 µM (triangles) and 0.8µM (inverted triangles). Dye:phosphate ratio was 1:50.
Figure 4
Figure 4
Time dependence of DNA condensation process monitored through YOYO-1 fluorescence intensity. At zero time DNA–YOYO-1 complex [either 5 µM (A) or 1 µM (B) of DNA phosphate] was mixed with 1 µM of either NC(1–72) (a) or NC(1–72)dd (b). Dye:phosphate ratio was kept at 1:50. Other conditions were same as in Figure 1B. Fast and slow phases of the condensation process could be seen.
Figure 4
Figure 4
Time dependence of DNA condensation process monitored through YOYO-1 fluorescence intensity. At zero time DNA–YOYO-1 complex [either 5 µM (A) or 1 µM (B) of DNA phosphate] was mixed with 1 µM of either NC(1–72) (a) or NC(1–72)dd (b). Dye:phosphate ratio was kept at 1:50. Other conditions were same as in Figure 1B. Fast and slow phases of the condensation process could be seen.
Figure 5
Figure 5
Stern-Volmer plots associated with the quenching of DNA-bound YOYO-1 (10 µM DNA phosphate and 5 nM YOYO-1) by acrylamide. (Dye:phosphate ratio = 1:2000.) I0 and I are the fluorescence intensities in the absence and in the presence of acrylamide. (a) Control, slope, KSV ∼0; (b) 6 µM NCp7, KSV = 0.15 M–1; and (c) PEI at N:P = 1:5, KSV = 0.6 M–1.
Figure 6
Figure 6
Decay of fluorescence anisotropy of DNA-bound YOYO-1. The concentrations of DNA phosphate and YOYO-1 were 10 µM and 5 nM, respectively (dye:phosphate = 1:2000). (A) Control; (B) with 4 µM of NC(1–72); (C) with 4 µM of NC(1–72)dd; and (D) with 4 µM of NC(12–55). Parameters obtained by analysis of these traces by equation 1 are given in Table 11. Smooth lines correspond to fits according to the listed parameters.
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