Affinity of the HIV-1 nucleocapsid protein for SL3 RNA: Effects due to salt and variation due to measurement technique

The mature nucleocapsid protein of HIV-1 (NCp7) and the NC-domains in gag-precursors are attractive targets for anti-AIDS drug discovery. NCp7 binds tightly to a 20mer mimic of stem-loop 3 RNA (SL3, also called psi-RNA, in the packaging domain of genomic RNA). The structure and stoichiometry of the NCp7-SL3 complex depend on the balance between sequence-specific binding of NCp7 to the G-rich loop bases of SL3 and non-sequence-specific interactions that are mainly electrostatic. This balance is primarily affected by the nature and concentration of the added salt, the concentration of wild-type NCp7 and SL3, and effects due to non-ideality of solutions. All these factors affect the measured or apparent dissociation constant (K d ). NC-domains recognize and specifically package genomic HIV-1 RNA, while electrostatic attractions and high concentrations of protein and RNA drive NCp7 to completely coat the RNA in the mature virion. The affinity of NCp7 for SL3 was studied using fluorescence (monitoring the fluorescence of Trp-37, the only tryptophan in the NCp7 sequence, which is quenched in a sequence-specific complex with SL3), surface plasmon resonance (SPR), isothermal titration calorimetry (ITC) and electrophoretic mobility shift assay (EMSA). High salt concentrations screen the ionic interactions between NCp7 and SL3 (net charges of +9 and -19, respectively, at neutral pH). It was found that 0.018 M MgCl 2 and 0.200 M added NaCl produce the same affinity for a 1:1 sequence-specific complex between NCp7 and SL3. Acetate ions stabilize the complex by more than a factor of 2 over the chloride ion and sulfate ions stabilize the complex by another factor of 2-3 over acetate. No change in the affinity of the complex occurs when substituting K + for Na + . The order of addition of NCp7 and SL3 in titrations monitored by tryptophan fluorescence and ITC, and hence the concentration of NCp7 and SL3, does not affect the balance between sequence-specific and non-sequence-specific binding in the physiological range of salt concentration. The presence of only one species of a 1:1 complex between NCp7 and SL3 at 0.200 M NaCl was also confirmed by EMSA

Mapping the chemical probing data from Schroeder <i>et al.</i>[7] onto the SHAPE-restrained secondary structure of <i>in vitro</i> transcribed STMV RNA.

<p>Red circles indicate nucleotides modified by DMS, kethoxal, or CMCT. The data do not appear to clearly rule out the proposed secondary structure of residues 1–730. A substantial number of the modifications occur in predicted loops, bulges, and single-stranded regions (67 out of 119 hits). Many of the reactive base-paired nucleotides are in A-U or G-U base pairs immediately adjacent to a predicted bulge loop (<i>e.g.</i>, 128, 185, 187, 192, 213, 413–414, 556, 561, 652–653, 663, 675), while others (382–390 and 503–515) are in a predicted stem that has two bulges and has no run of more than three consecutive base pairs, so it should be prone to fraying.</p

Distribution of double-helical RNA segments in the STMV virion.

<p>The crystal structure of STMV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054384#pone.0054384-Larson1" target="_blank">[4]</a> reveals 30 segments of double-helical RNA (blue). Each helix contains 9 base pairs, centered on a crystallographic two-fold axis. An icosahedral cage (pink) is shown for reference. Adopted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054384#pone.0054384-Zeng1" target="_blank">[8]</a>.</p

Predicted secondary structure at the 3′ end of STMV RNA.

<p>Secondary structure for the 406 3′-terminal nucleotides of STMV RNA proposed by Gultyaev <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054384#pone.0054384-Gultyaev1" target="_blank">[10]</a>. Nucleotides are colored according to their SHAPE reactivity (see scale). The SHAPE data supports the tRNA-like structure and the five stem-loops (nucleotides 728–1058), but does not support the second pseudoknot domain (nucleotides 653–727).</p

SHAPE-restrained secondary structure model for free STMV RNA.

<p>Nucleotides are colored according to their SHAPE reactivity (see scale). Inset shows a box plot comparing the distribution of SHAPE reactivity values between base paired and single-stranded nucleotides. Each grey box represents the interquartile range (IQR) of the data; the bottom and top edges of the box are the 25th and 75th percentiles, respectively. The band near the middle of each box is the median value. The whiskers above and below each box extend to the most extreme data points not considered outliers. Outliers are plotted individually as crosses. Points are outliers if they are greater than 1.5 IQR from the 75th percentile or less than 1.5 IQR from the 25th percentile. In this secondary structure model, the distribution for base paired nucleotides is narrower and has a much lower median value than the distribution for single-stranded nucleotides.</p

Effect of Mg²⁺ on the SHAPE reactivity profile of free STMV RNA.

<p>SHAPE reactivities for STMV RNA in the presence (top) and absence (middle) of Mg²⁺. The difference plot (bottom) shows that 10 mM Mg²⁺ has little effect on the SHAPE reactivity profile.</p

SHAPE-restrained secondary structure of free STMV RNA with a tRNA-like fold at the 3′ end.

<p>This alternate model of the STMV RNA was obtained by combining the SHAPE-restrained secondary structure predicted separately for nucleotides 1–727 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054384#pone-0054384-g002" target="_blank">Figure 2</a>) with the Gultyaev <i>et al.</i> prediction <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054384#pone.0054384-Gultyaev1" target="_blank">[10]</a> for nucleotides 728–1058 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054384#pone-0054384-g005" target="_blank">Figure 5</a>). Nucleotides are colored according to their SHAPE reactivity (see scale). The extended central domain (nucleotides 64–720) is identical to that of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054384#pone-0054384-g002" target="_blank">Figure 2</a>.</p

Minimum free energy (MFE) structure obtained for STMV RNA without the SHAPE data.

<p>The structure was predicted using RNAstructure with default parameters. Nucleotides are colored according to their SHAPE reactivity (see scale). The SHAPE data are not consistent with this model, since several base paired regions have high reactivity values.</p

Identification of possible double-helical stems corresponding to those seen in the crystal structure.

<p>There are 30 stems in the crystal structure, each containing nine base pairs with an additional base stacked at each 3′ end, <i>i.e.</i>, 20 nucleotides (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054384#pone-0054384-g001" target="_blank">Figure 1</a>). A model that connects successive stems would require something on the order of 5–10 nucleotides per connection. This figure shows how our secondary structure model might be organized to fit into the STMV capsid, with a sufficient number of stems to cover the 30 edges of the icosahedral frame, as required by <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054384#pone-0054384-g001" target="_blank">Figure 1</a>.</p