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. 2006 Jan 24;34(2):472-84.
doi: 10.1093/nar/gkj442. Print 2006.

Complex interactions of HIV-1 nucleocapsid protein with oligonucleotides

Robert J Fisher  1 , Matthew J Fivash, Andrew G Stephen, Nathan A Hagan, Shilpa R Shenoy, Maxine V Medaglia, Lindsey R Smith, Karen M Worthy, John T Simpson, Robert Shoemaker, Karen L McNitt, Donald G Johnson, Catherine V Hixson, Robert J Gorelick, Daniele Fabris, Louis E Henderson, Alan Rein
Affiliations

Complex interactions of HIV-1 nucleocapsid protein with oligonucleotides

Robert J Fisher et al. Nucleic Acids Res. .

Erratum in

  • Nucleic Acids Res. 2006;34(3):1082

Abstract

The HIV-1 nucleocapsid (NC) protein is a small, basic protein containing two retroviral zinc fingers. It is a highly active nucleic acid chaperone; because of this activity, it plays a crucial role in virus replication as a cofactor during reverse transcription, and is probably important in other steps of the replication cycle as well. We previously reported that NC binds with high-affinity to the repeating sequence d(TG)n. We have now analyzed the interaction between NC and d(TG)4 in considerable detail, using surface plasmon resonance (SPR), tryptophan fluorescence quenching (TFQ), fluorescence anisotropy (FA), isothermal titration calorimetry (ITC) and electrospray ionization Fourier transform mass spectrometry (ESI-FTMS). Our results show that the interactions between these two molecules are surprisngly complex: while the K(d) for binding of a single d(TG)4 molecule to NC is only approximately 5 nM in 150 mM NaCl, a single NC molecule is capable of interacting with more than one d(TG)4 molecule, and conversely, more than one NC molecule can bind to a single d(TG)4 molecule. The strengths of these additional binding reactions are quantitated. The implications of this multivalency for the functions of NC in virus replication are discussed.

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Figures

Figure 1
Figure 1
Sequences of NC and NC mutants. Changes from wild-type (WT) are shown in bold.
Figure 2
Figure 2
SPR results: (A) Binding of NC to a 5.4 RU surface of (TG)4 in 150 mM NaCl. Solutions of 1.7, 3.13, 6.25, 12.5, 25, 50, 100 and 200 nM NC were passed over the surface at 100 µl/min. The horizontal line represents the response expected for a 1:1 complex of NC and (TG)4. The grey lines are the profiles expected from fitting the data to a simple 1:1 binding model as described in the Supplementary Data. (B) Binding of NC to (A)8. Solutions of NC as in (A) were passed over a 5.8 RU (A)8 surface. (C) Binding of NC to a 156.6 RU surface of (TG)4. Solutions of 1, 2.5, 5, 10, 25, 50, 100, 200, 400, 600, 800 and 1000 nM NC were passed over this surface. The two horizontal lines represent the signals expected if one (lower line) or two (upper line) NC molecules binds to each (TG)4 molecule. The grey lines are the profiles expected from fitting the data to the complex binding model shown in Figure 3, as described in the Supplementary Data. (D) Binding of NC to (TG)4 in buffer containing 250 mM rather than 150 mM NaCl. Solutions of 5, 10, 25, 50, 100, 200, 400, 800 and 1200 nM NC were passed over a surface containing 159.4 RU of (TG)4. The horizontal line represents the response expected for a 1:1 complex of NC and (TG)4. Grey lines are as in (C).
Figure 3
Figure 3
Proposed reaction-scheme for interaction of NC with (TG)4, showing three possible binding reactions, each with its respective Kd. N, NC; O, (TG)4.
Figure 4
Figure 4
TFQ results. (TG)4 was titrated into a 400 nM solution of NC and tryptophan fluorescence was monitored as described in Materials and Methods. The fluorescence readings were divided by the fluorescence of NC before addition of (TG)4 to obtain the normalized fluorescence shown here. Attempts were made to fit the data assuming a 1:1 binding system (dashed line) (giving a Kd of 3.4 ± 0.07 nM) or the more complex model shown in Figure 3 (solid grey line). The results shown here are normalized by dividing by the fluorescence value of NC alone. As indicated in Supplementary equation 5, the final fluorescence obtained after several equivalents of (TG)4 had been added [i.e. the residual fluorescence of NC bound to (TG)4] was subtracted from these values before attempting to fit the curves.
Figure 5
Figure 5
FA results. NC was titrated into solutions containing 10, 50, 100, 300, 600 or 1000 nM fluoresceinated (TG)4 and the anisotropy of the oligonucleotide was measured. Grey lines are fits to the data using the model shown in Figure 3. FA of two aliquots was measured independently for each point in the titrations. Inset: the FA results in the figure were plotted vs. the NC:(TG)4 ratio in the solution.
Figure 6
Figure 6
Analysis of competition between oligonucleotides in solution and immobilized (TG)4 for NC. (A) Binding of NC to (TG)4. One min injections of (from bottom to top) 0, 1, 2.5, 5, 10, 25, 50, 100 and 200 nM NC were passed over a surface of 344.2 RU's (TG)4 at 64 µl/min. (B) Rate of binding of NC to (TG)4 as a function of NC concentration. Initial slopes in (A) were measured using BIAevaluation 3.01, and the plot of initial slopes versus NC was fit using linear regression. (C) Effect of (TG)4 in solution on binding of NC to immobilized (TG)4. A total of 100 nM NC was incubated at 4°C with (from top to bottom) 0, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10.0, 25.0, 50.0 and 100.0 µM (TG)4 for at least 1 h before injecting a sample for 1 min at 64 µl/min.
Figure 7
Figure 7
Competition by oligonucleotides in solution with the binding of NC to immobilized (TG)4. Solutions of 100 nM NC were incubated with varying amounts of different oligonucleotides for at least 60 min. These solutions were then passed over an SPR surface containing 327 RU's of (TG)4 and the initial slope of the SPR profile, representing the initial rate of binding to the immobilized (TG)4, was measured. The open squares represent preincubation with 0.1, 0.25, 0.5, 1.0, 2.5, 5, 10, 25 and 50 µM (A)30; open triangles: preincubation with 2.5, 5.0, 10, 25, 50, 100, 250, 500, 1000 and 5000 nM (TG)10; open circles: preincubation with 20, 40, 60, 80, 100, 300, 500 and 1000 nM (A)10(TG)5(A)10; closed circles: preincubation with 20, 40, 60, 80, 100, 300, 500 and 1000 nM (TG)5(A)10(TG)5; closed triangles: preincubation with 0.1, 0.25, 0.5, 1.0, 2.5, 5, 10 and 25 µM (TG)4. The lines are fits to a 4-parameter logistic equation, as described in Supplementary Data, yielding the values shown in Table 3. Asterisks highlight the two curves in which ISmin is greater than zero.
Figure 8
Figure 8
ESI-FTMS spectra of (TG)4 and (TG)4-NC complexes. (A) Control 5 µM (TG)4, providing a 2470.46 Da experimental mass (2470.44 Da monoisotopic mass calculated from sequence, for a ∼8 p.p.m. mass accuracy). The signal labeled −G corresponds to a product in which a deoxyguanine is formally replaced by deoxyribose (observed decremental mass of ∼133 Da). D indicates (TG)4/(TG)4-G dimeric species. (B) Mixture containing 5 µM each of (TG)4 and NC. NC provided a mass of 6488.89 Da (6488.91 Da monoisotopic from sequence), while the 1:1 NO complex was 8959.33 Da (8959.35 Da monoisotopic from sequence). (C) Sample containing 5 µM (TG)4 and 15 µM NC. The 1:2 NON complex provided a mass of 15 447.93 Da (15 448.25 Da monoisotopic from sequence).
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