Characterization of the Interactions of vMIP-II, and a Dimeric Variant of vMIP-II, with Glycosaminoglycans

Chemokines are important immune proteins, carrying out their function by binding to glycosaminoglycans (GAGs) on the endothelial surface and to cell surface chemokine receptors. A unique viral chemokine analogue, viral macrophage inflammatory protein-II (vMIP-II), encoded by human herpesvirus-8, has garnered interest because of its ability to bind to multiple chemokine receptors, including both HIV coreceptors. In addition, vMIP-II binds to cell surface GAGs much more tightly than most human chemokines, which may be the key to its anti-inflammatory function in vivo. The goal of this work was to determine the mechanism of binding of GAG by vMIP-II. The interaction of vMIP-II with a heparin-derived disaccharide was characterized using NMR. Important binding sites were further analyzed by mutagenesis studies, in which corresponding vMIP-II mutants were tested for GAG binding ability using heparin chromatography and NMR. We found that despite having many more basic residues than some chemokines, vMIP-II shares a characteristic binding site similar to that of its human analogues, utilizing basic residues R18, R46, and R48. Interestingly, a particular mutation (Leu13Phe) caused vMIP-II to form a pH-dependent CC chemokine-type dimer as determined by analytical ultracentrifugation and NMR. To the best of our knowledge, this is the first example of engineering a naturally predominantly monomeric chemokine into a dissociable dimer by a single mutation. This dimeric vMIP-II mutant binds to heparin much more tightly than wild-type vMIP-II and provides a new model for studying the relationship between chemokine quaternary structure and various aspects of function. Structural differences between monomeric and dimeric vMIP-II upon GAG binding were characterized by NMR and molecular docking

SPR-based competition experiment between CBA and gp120 IIIB for binding to immobilized DC-SIGN (chip density 1050 RU ∼16 fmol).

<p>Effect of HHA exposure on the HIV-1(III_B) gp120 complex with DC-SIGN. 200 nM gp120 was injected (time point a), followed after 2 min by an additional injection (time point b) of varying concentrations of HHA (0.4 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Balzarini1" target="_blank">[1]</a>, 4 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Balzarini2" target="_blank">[2]</a>, 40 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Balzarini3" target="_blank">[3]</a>, 100 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Hladik1" target="_blank">[4]</a>, 400 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Balzarini4" target="_blank">[5]</a> and 1000 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Mori1" target="_blank">[6]</a>).</p

Inhibitory activity of GRFT and its mutant variants against DC-SIGN-mediated virus transmission of HIV-1-captured virions to CD4⁺ T-lymphocyte cells.

<p>The HIV-1-captured Raji/DC-SIGN cultures used for virus transmission in the presence or absence of the wild-type and mutant GRFT variants contained following amounts of captured HIV-1: 5.50±2.09 ng p24/10⁶ cells (for the WT GRFT experiments), 4.30±0.15 ng p24/10⁶ cells (for the mutant D30A GRFT experiments), 3.66±0.59 ng p24/10⁶ cells (for the mutant D70A GRFT experiments), 4.04±0.72 ng p24/10⁶ cells (for the mutant D112A GRFT experiments), and 3.78±0.77 ng p24/10⁶ cells (for the mutant Triple A GRFT experiments). Data represent the mean of three independent experiments.</p

SPR-based competition experiment between CBA and gp120 III_B for binding to immobilized DC-SIGN (chip density 715 RU ∼10.9 fmol).

<p>Panel A: Effect of GRFT exposure on the HIV-1(III_B) gp120 complex with DC-SIGN. 200 nM gp120 was injected (time point a), followed after 2 min by an additional injection (time point b) of varying concentrations of GRFT (0.1 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Balzarini1" target="_blank">[1]</a>, 1 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Balzarini2" target="_blank">[2]</a>, 10 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Balzarini3" target="_blank">[3]</a>, 25 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Hladik1" target="_blank">[4]</a>, 100 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Balzarini4" target="_blank">[5]</a> and 250 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Mori1" target="_blank">[6]</a>). Similar experiments are depicted in Panels B and D but for varying concentrations of mutant D30A GRFT variant and mutant D112A GRFT variant (0.2 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Balzarini1" target="_blank">[1]</a>, 2 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Balzarini2" target="_blank">[2]</a>, 20 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Balzarini3" target="_blank">[3]</a>, 50 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Hladik1" target="_blank">[4]</a>, 200 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Balzarini4" target="_blank">[5]</a> and 500 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Mori1" target="_blank">[6]</a>), respectively. In Panel C the concentrations of mutant D70A GRFT variant were 0.4 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Balzarini1" target="_blank">[1]</a>, 4 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Balzarini2" target="_blank">[2]</a>, 40 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Balzarini3" target="_blank">[3]</a>, 100 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Hladik1" target="_blank">[4]</a>, 400 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Balzarini4" target="_blank">[5]</a> and 1000 nM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132-Mori1" target="_blank">[6]</a>. The curves show a representative example out of two independent experiments.</p

Role of the Carbohydrate-Binding Sites of Griffithsin in the Prevention of DC-SIGN-Mediated Capture and Transmission of HIV-1

<div><p>Background</p><p>The glycan-targeting C-type DC-SIGN lectin receptor is implicated in the transmission of the human immunodeficiency virus (HIV) by binding the virus and transferring the captured HIV-1 to CD4⁺ T lymphocytes. Carbohydrate binding agents (CBAs) have been reported to block HIV-1 infection. We have now investigated the potent mannose-specific anti-HIV CBA griffithsin (GRFT) on its ability to inhibit the capture of HIV-1 to DC-SIGN, its DC-SIGN-directed transmission to CD4⁺ T-lymphocytes and the role of the three carbohydrate-binding sites (CBS) of GRFT in these processes.</p><p>Findings</p><p>GRFT inhibited HIV-1(III_B) infection of CEM and HIV-1(NL4.3) infection of C8166 CD4⁺ T-lymphocytes at an EC₅₀ of 0.059 and 0.444 nM, respectively. The single mutant CBS variants of GRFT (in which a key Asp in one of the CBS was mutated to Ala) were about ∼20 to 60-fold less potent to prevent HIV-1 infection and ∼20 to 90-fold less potent to inhibit syncytia formation in co-cultures of persistently HIV-1 infected HuT-78 and uninfected C8166 CD4⁺ T-lymphocytes. GRFT prevents DC-SIGN-mediated virus capture and HIV-1 transmission to CD4⁺ T-lymphocytes at an EC₅₀ of 1.5 nM and 0.012 nM, respectively. Surface plasmon resonance (SPR) studies revealed that wild-type GRFT efficiently blocked the binding between DC-SIGN and immobilized gp120, whereas the point mutant CBS variants of GRFT were ∼10- to 15-fold less efficient. SPR-analysis also demonstrated that wild-type GRFT and its single mutant CBS variants have the capacity to expel bound gp120 from the gp120-DC-SIGN complex in a dose dependent manner, a property that was not observed for HHA, another mannose-specific potent anti-HIV-1 CBA.</p><p>Conclusion</p><p>GRFT is inhibitory against HIV gp120 binding to DC-SIGN, efficiently prevents DC-SIGN-mediated transfer of HIV-1 to CD4⁺ T-lymphocytes and is able to expel gp120 from the gp120-DC-SIGN complex. Functionally intact CBS of GRFT are important for the optimal action of GRFT.</p></div

Inhibitory activity of GRFT and its mutant variants against DC-SIGN-mediated capture of HIV-1(NL4.3) by Raji/DC-SIGN.

<p>The control cultures contained DC-SIGN-captured virus at 3.77±0.37 ng p24/10⁶ cells (data represent the mean of 2 independent experiments).</p

Anti-HIV-1 activity of GRFT and its mutant variants in different cell systems.

a<p>EC₅₀ required to inhibit virus-induced cytopathicity in CEM (HIV-1 III_B) cell cultures by 50%.</p>b<p>EC₅₀ required to inhibit virus (HIV-1 NL4.3)-induced cytopathicity in C8166 cell cultures by 50%.</p>c<p>50%-Effective concentration or compound concentration required to inhibit syncytia formation between persistently HIV-1(III_B)-infected HuT-78/HIV-1 cells and uninfected CD4⁺ T-lymphocyte Sup T1 cells by 50%.</p>d<p>50%-Effective concentration or compound concentration required to inhibit syncytia formation between persistently HIV-1(III_B)-infected HuT-78/HIV-1 cells and uninfected CD4⁺ T-lymphocyte C8166 cells by 50%.</p><p>The data from which the EC₅₀'s were derived are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132.s001" target="_blank">Figures S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132.s002" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132.s003" target="_blank">S3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132.s004" target="_blank">S4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132.s005" target="_blank">S5</a> and are the mean of at least 2 to 3 independent experiments.</p

Structural Comparison of Monomeric Variants of the Chemokine MIP-1β Having Differing Ability To Bind the Receptor CCR5^†^,^‡

MIP-1β, a member of the chemokine family of proteins, tightly binds the receptor CCR5 as part of its natural function in the immune response, and in doing so also blocks the ability of many strains of HIV to enter the cell. The single most important MIP-1β residue known to contribute to its interaction with the receptor is Phe13, which when mutated reduces the ability of MIP-1β to bind to CCR5 by more than 1000-fold. To obtain a structural understanding of the dramatic effect of the absence of Phe13 in MIP-1β, we used multidimensional heteronuclear NMR to determine the three-dimensional structure of the MIP-1β F13A variant. We had previously shown that, unlike the wild-type protein which has been shown to be a tight dimer, the F13A mutant is monomeric even at high concentrations [Laurence, J. S., Blanpain, C., Burgner, J. W., Parmentier, M., and LiWang, P. J. (2000) Biochemistry 39, 3401−3409], leading to significant changes in the NMR spectra of F13A and the wild-type protein. We have obtained a total of 940 structural restraints for MIP-1β F13A, and have calculated a family of structures having a backbone rmsd from the average of 0.55 Å (residues 12−67). A structural comparison of the F13A mutant with a fully active monomeric variant, P8A, shows that despite some differences in the 1H−15N HSQC spectra the two are nearly identical in NOE distance restraints and in backbone conformation. A comparison of F13A with the wild-type protein shows largely the same fold, although differences exist in the N-terminal and loop regions for which the loss of the dimer in F13A can mainly account. A dynamics comparison confirms greater flexibility in F13A than in the wild-type protein in regions of dimer contact in the wild-type protein. In an analysis to determine if the large functional effect resulting from the loss of Phe13 is due to the local side chain change or due to more global structural changes, we conclude that local effects predominate. This suggests that a strategy for designing tight binding anti-CCR5 therapeutics should include a Phe-like component

Binding efficiency of DC-SIGN to immobilized HIV-1 gp120 III_B and HIV-1 gp120 ADA that were pre-exposed to GRFT and its mutant variants.

a<p>Concentration used in the study represents a 60- to 70-fold K_D concentration of the particular CBA against HIV-1 gp120. Data are the mean of 2 independent experiments. The SPR data are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0064132#pone.0064132.s006" target="_blank">Figure S6</a>.</p

Increased (positive sign) or decreased (negative sign) amplitude (RU) upon GRFT exposure to the DC-SIGN-gp120 complex.

<p>Increased (positive sign) or decreased (negative sign) amplitude (RU) upon GRFT exposure to the DC-SIGN-gp120 complex.</p