Recognition properties of V3-specific antibodies to V3 loop peptides derived from HIV-1 gp120 presented in multiple conformations

To identify structural constraints and amino acid sequences important for antibody recognition of the third variable domain (V3) of HIV-1 gp120, we have studied the solution conformation of three 35-mer circular V3 loop peptides derived from HIV-1 strains which differ in syncytium- (SI) and non-syncytium-inducing (NSI) capacity. In addition to 2D NMR and CD analyses, fluid- and solid-phase immunoassays were performed using V3-specific antibodies to V3 peptides and gp120 derived from different strains of HIV-1. NMR and CD spectroscopy indicated that circular and linear V3 loops exist in water as a dynamic ensemble of multiple conformations. Amino acid substitutions and biochemical modifications of the V3 loop were found to affect antibody binding depending on the presentation of the antigens. From NMR observations and immunological experiments, we provide evidence for a V3 loop specific monoclonal antibody interaction which is directed predominantly against linear epitopes rather than against discontinuous epitopes. The absence of a single defined solution conformation of 35-mer circular V3 peptides should be taken into account when using V3-related peptides to investigate structural elements in the V3 domain of the gp120 envelope protein of HIV-1 involved in biological processes of the virus.     

Conformation of the HIV-1 gp120 V3 loop. Structure functional analysis of the HIV-Haiti viral strain

The structural model describing the conformational preferences of the HIV-Haiti gp120 V3 loop in geometric space of dihedral angles was generated in terms of NMR spectroscopy data using the methods of computer modeling. The elements of secondary structure anti conformations of irregular stretches were deciphered for the fragment making the virus principal neutralizing determinant as well as the determinants of cell tropism and syncytium formation. The structurally conserved amino acids of the HIV-1 V3 loop, that may present the forward-looking targets for AIDS drug design, were identified based on the combined analysis of the results obtained with those derived previously. In particular, it was demonstrated that the register of these amino acids comprises Asn-25 critical for virus binding with primary cell receptor CD4 as well as Arg-3 critical for utilization of CCR5 coreceptor and heparan sulfate proteoglycan syndecans. The results obtained are discussed in conjunction with the literature data on the biological activity of individual amino acid residues of the HIV-1 gpl20 V3 loop.     

Structural analysis of the HIV-1 gp120 V3 loop: application to the HIV-Haiti isolates

The model describing the structure and conformational preferences of the HIV-Haiti V3 loop in the geometric spaces of Cartesian coordinates and dihedral angles was generated in terms of NMR spectroscopy data published in literature. To this end, the following successive steps were put into effect: (i) the NMR-based 3D structure for the HIV-Haiti V3 loop in water was built by computer modeling methods; (ii) the conformations of its irregular segments were analyzed and the secondary structure elements identified; and (iii) to reveal a common structural motifs in the HIV-Haiti V3 loop regardless of its environment variability, the simulated structure was collated with the one deciphered previously for the HIV-Haiti V3 loop in a water/trifluoroethanol (TFE) mixed solvent. As a result, the HIV-Haiti V3 loop was found to offer the highly variable fragment of gp120 sensitive to its environment whose changes trigger the large-scale structural rearrangements, bringing in substantial altering the secondary and tertiary structures of this functionally important site of the virus envelope. In spite of this fact, over half of amino acid residues that reside, for the most part, in the functionally important regions of the gp120 protein and may present promising targets for AIDS drug researches, were shown to preserve their conformational states in the structures under review. In particular, the register of these amino acids holds Asn-25 that is critical for the virus binding with primary cell receptor CD4 as well as Arg-3 that is critical for utilization of CCR5 co-receptor and heparan sulfate proteoglycans. The conservative structural motif embracing one of the potential sites of the gp120 N-linked glycosylation was detected, which seems to be a promising target for the HIV-1 drug design. The implications are discussed in conjunction with the literature data on the biological activity of the individual amino acids for the HIV-1 gp120 V3 loop.     

Restraining the conformation of HIV-1 gp120 by removing a flexible loop

The trimeric HIV/SIV envelope glycoprotein, gp160, is cleaved to noncovalently associated fragments, gp120 and gp41. Binding of gp120 to viral receptors leads to large structural rearrangements in both fragments. The unliganded gp120 core has a disordered beta3-beta5 loop, which reconfigures upon CD4 binding into an ordered, extended strand. Molecular modeling suggests that residues in this loop may contact gp41. We show here that deletions in the beta3-beta5 loop of HIV-1 gp120 weaken the binding of CD4 and prevent formation of the epitope for monoclonal antibody (mAb) 17b (which recognizes the coreceptor site). Formation of an encounter complex with CD4 binding and interactions of gp120 with mAbs b12 and 2G12 are not affected by these deletions. Thus, deleting the beta3-beta5 loop blocks the gp120 conformational change and may offer a strategy for design of restrained immunogens. Moreover, mutations in the SIV beta3-beta5 loop lead to greater spontaneous dissociation of gp120 from cell-associated trimers. We suggest that the CD4-induced rearrangement of this loop releases structural constraints on gp41 and thus potentiates its fusion activity.     

The binding of a glycoprotein 120 V3 loop peptide to HIV-1 neutralizing antibodies. Structural implications

The structural and antigenic properties of a peptide (CRK) derived from the V3 loop of HIV-1 gp120 protein were studied using NMR and SPR techniques. The sequence of CRK corresponds to the central portion of the V3 loop containing the highly conserved GPGR residue sequence. Although the biological significance of this conserved sequence is unknown, the adoption of conserved secondary structure (type II beta-turn) in this region has been proposed. The tendency of CRK (while free or conjugated to protein), to adopt such structure and the influence of such structure upon CRK antigenicity were investigated by NMR and SPR, respectively. Regardless of conjugation, CRK is conformationally averaged in solution but a weak tendency of the CRK GPGR residues to adopt a beta-turn conformation was observed after conjugation. The influence of GPGR structure upon CRK antigenicity was investigated by measuring the affinities of two cognate antibodies: 5023A and 5025A, for CRK, protein-conjugated CRK and gp120 protein. Each antibody bound to all the antigens with nearly the same affinity. From these data, it appears that: (a) antibody binding most likely involves an induced fit of the peptide and (b) the gp120 V3 loop is probably conformationally heterogeneous. Since 5023A and 5025A are HIV-1 neutralizing antibodies, neutralization in these cases appears to be independent of adopted GPGR beta-turn structure.     

Probing hydrogen bonds in the antibody-bound HIV-1 gp120 V3 loop by solid state NMR REDOR measurements

We describe solid state NMR measurements on frozen solutions of the complex of the 24-residue HIV-1 gp120 V3 loop peptide RP135 with the Fab fragment of the anti-gp120 antibody 0.5, using rotational echo double resonance (REDOR). In order to probe possible hydrogen bonding between arginine side chains and glycine backbone carbonyls in the region of the conserved Gly-Pro-Gly-Arg (GPGR) motif of the V3 loop, RP135 samples were prepared with ¹⁵N labels at the nitrogen positions of arginine side chains and ¹³C labels at glycine carbonyl positions and ¹³C-detected ¹³C-¹⁵N REDOR measurements were performed on peptide/antibody complexes of these labeled samples. Such hydrogen bonding was previously observed in a crystal structure of the V3 loop peptide/antibody complex RP142/59.1 [Ghiara et al. (1994) Science, 264, 82–85], but is shown by the REDOR measurements to be absent in the RP135/0.5 complex. These results confirm the antibody-dependent conformational differences in the GPGR motif suggested by previously reported solid state NMR measurements of and backbone dihedral angles in the RP135/0.5 complex. In addition, we describe REDOR measurements on the helical synthetic peptide MB(i+4)EK in frozen solution that establish our ability to detect ¹³C-¹⁵N dipole–dipole couplings in the distance range appropriate to these hydrogen bonding studies. We also report the results of molecular modeling calculations on the central portion RP135, using a combination of the solid state NMR restraints of Weliky et al. [Nat. Struct. Biol., 6, 141–145, 1999] and the liquid state NMR restraints of Tugarinov et al. (Nat. Struct. Biol., 6, 331–335, 1999]. The dynamics calculations demonstrate the mutual compatibility of the two sets of experimental structural restraints and reduce ambiguities in the solid state NMR restraints that result from symmetry and signal-to-noise considerations.     

Identification of the potential regions of Epap-1 that interacts with V3 loop of HIV-1 gp120

Early pregnancy associated protein-1 (Epap-1), a 90 kDa glycoprotein present in first trimester placental tissue, inhibits HIV-1 entry through interaction with HIV-1 gp120 at V3 and C5 regions. In the present study, we have identified the specific 32 mer region of Epap-1 that can interact with V3 loop. This was achieved by docking between Epap-1 molecular model and gp120 and studying the interaction of peptides with gp120 in vitro. Out of four peptides analyzed, two peptides (P-2 and P-3) showed significant interaction with V3 domain (N = 8; N = 7) of gp120. In the studies conducted using soluble gp120 and virus, peptide P-2 has shown conserved interaction at V3 loop regions recognized by 257D and F425 antibodies and higher anti-viral activity. Also, P-2 inhibited cell fusion mediated dye transfer between gp120 expressing HL2/3 and CD4 expressing Sup T1 cells suggesting its inhibition of viral entry, which is further confirmed by its action on HIV infection mediated by Tat activated beta gal expression in TZM-bl cells. Further optimization of P-2 peptide showed that the anti-viral activity and gp120 interaction residues lie in the N-terminal region of the peptide. These results together suggest that P-2 inhibits viral entry through specific interaction at V3 loop region.     

A tyrosine-sulfated peptide derived from the heavy-chain CDR3 region of an HIV-1-neutralizing antibody binds gp120 and inhibits HIV-1 infection

Sulfated tyrosines at the amino terminus of the principal HIV-1 coreceptor CCR5 play a critical role in its ability to bind the HIV-1 envelope glycoprotein gp120 and mediate HIV-1 entry. Human antibodies that recognize the CCR5-binding region of gp120 are also modified by tyrosine sulfation, which is necessary for their ability to neutralize HIV-1. Here we demonstrate that a sulfated peptide derived from the CDR3 region of one of these antibodies, E51, can efficiently bind gp120. Association of this peptide, pE51, with gp120 requires tyrosine sulfation and is enhanced by, but not dependent on, CD4. Alteration of any of four pE51 tyrosines, or alteration of gp120 residues 420, 421, or 422, critical for association with CCR5, prevents gp120 association with pE51. pE51 neutralizes HIV-1 more effectively than peptides based on the CCR5 amino terminus and may be useful as a fusion partner with other protein inhibitors of HIV-1 entry. Our data provide further insight into the association of the CCR5 amino terminus with gp120, show that a conserved, sulfate-binding region of gp120 is accessible to inhibitors in the absence of CD4, and suggest that soluble mimetics of CCR5 can be more effective than previously appreciated.     

Interaction of the HIV-1 gp120 viral protein V3 loop with bacterial lipopolysaccharide: a pattern recognition inhibition

HIV-1 represents an elusive target for therapeutic compounds due to its high rate of mutation. Targeting structural patterns instead of a constantly changing specific three-dimensional structure may represent an approach that is less sensitive to viral mutations. The V3 loop of gp120 of HIV-1, which is responsible for binding of viral gp120 to CCR5 or CXCR4 coreceptors, has already been identified as an effective target for the inhibition of viral entry. The peptide derived from the V3 loop of gp120 specifically interacts with the lipid A moiety of LPS, as does the full gp120 protein. NMR analysis of V3 in complex with LPS shows formation of an amphipathic turn. The interaction between LPS and V3 relies on the structural pattern, comprising a combination of hydrophobic and charge interactions, similar to the interaction between antimicrobial peptides and LPS. LPS inhibited binding of gp120 to the surface of target T cells. Nonendotoxic LPS antagonists inhibited viral infection, demonstrating the possibility for the development of an inhibitor of HIV-1 attachment to T cells based on the recognition of a conserved structural pattern.     

Cross-reactive potential of rabbit antibodies raised against a cyclic peptide representing a chimeric V3 loop of HIV-1 gp120 studied by biosensor technique and ELISA

Abstract Rabbit antibodies were induced against a free cyclic peptide representing the chimeric sequence of a consensus V3 loop of HIV-1 gp120. The reactivity of these antibodies was tested in a biosensor system (BIAcore^™, Pharmacia AB, Uppsala, Sweden) and in ELISA with the peptide immunogen in its cyclic and linear forms, as well as with peptides corresponding to the V3 region of different HIV-1 variants. The antibodies reacted with all the peptides tested both in ELISA and in biosensor assays and recognized the cyclic form of the chimeric peptide better than the linear form. Although antibodies raised against the V3 region of particular HIV-1 variants cross-react with other HIV-1 strains, it seems that the use of a chimeric peptide as immunogen improved the cross-reactivity spectrum of recognition of the antibodies. The anti-V3 antibodies were also tested for their ability to neutralize in vitro four HIV-1 laboratory strains. Only the HIV_MN variant was found to be neutralized. Compared to conventional solid phase immunoassays, the BIAcore presents several advantages for measuring the differential reactivity of peptide analogues. In view of their broadly cross-reactive potential, antibodies raised against a consensus sequence should be useful in immunodiagnosis of viral antigenic variants.     

Molecular Recognition of CCR5 by an HIV-1 gp120 V3 Loop

The binding of protein HIV-1 gp120 to coreceptors CCR5 or CXCR4 is a key step of the HIV-1 entry to the host cell, and is predominantly mediated through the V3 loop fragment of HIV-1 gp120. In the present work, we delineate the molecular recognition of chemokine receptor CCR5 by a dual tropic HIV-1 gp120 V3 loop, using a comprehensive set of computational tools predominantly based on molecular dynamics simulations and free energy calculations. We report, what is to our knowledge, the first complete HIV-1 gp120 V3 loop : CCR5 complex structure, which includes the whole V3 loop and the N-terminus of CCR5, and exhibits exceptional agreement with previous experimental findings. The computationally derived structure sheds light into the functional role of HIV-1 gp120 V3 loop and CCR5 residues associated with the HIV-1 coreceptor activity, and provides insights into the HIV-1 coreceptor selectivity and the blocking mechanism of HIV-1 gp120 by maraviroc. By comparing the binding of the specific dual tropic HIV-1 gp120 V3 loop with CCR5 and CXCR4, we observe that the HIV-1 gp120 V3 loop residues 13–21, which include the tip, share nearly identical structural and energetic properties in complex with both coreceptors. This result paves the way for the design of dual CCR5/CXCR4 targeted peptides as novel potential anti-AIDS therapeutics.     

Accurate and efficient gp120 V3 loop structure based models for the determination of HIV-1 co-receptor usage

Background  

HIV-1 targets human cells expressing both the CD4 receptor, which binds the viral envelope glycoprotein gp120, as well as either the CCR5 (R5) or CXCR4 (X4) co-receptors, which interact primarily with the third hypervariable loop (V3 loop) of gp120. Determination of HIV-1 affinity for either the R5 or X4 co-receptor on host cells facilitates the inclusion of co-receptor antagonists as a part of patient treatment strategies. A dataset of 1193 distinct gp120 V3 loop peptide sequences (989 R5-utilizing, 204 X4-capable) is utilized to train predictive classifiers based on implementations of random forest, support vector machine, boosted decision tree, and neural network machine learning algorithms. An in silico mutagenesis procedure employing multibody statistical potentials, computational geometry, and threading of variant V3 sequences onto an experimental structure, is used to generate a feature vector representation for each variant whose components measure environmental perturbations at corresponding structural positions.     

Molecular Recognition of CXCR4 by a Dual Tropic HIV-1 gp120 V3 Loop

HIV-1 cell entry is initiated by the interaction of the viral envelope glycoprotein gp120 with CD4, and chemokine coreceptors CXCR4 and CCR5. The molecular recognition of CXCR4 or CCR5 by the HIV-1 gp120 is mediated through the V3 loop, a fragment of gp120. The binding of the V3 loop to CXCR4 or CCR5 determines the cell tropism of HIV-1 and constitutes a key step before HIV-1 cell entry. Thus, elucidating the molecular recognition of CXCR4 by the V3 loop is important for understanding HIV-1 viral infectivity and tropism, and for the design of HIV-1 inhibitors. We employed a comprehensive set of computational tools, predominantly based on free energy calculations and molecular-dynamics simulations, to investigate the molecular recognition of CXCR4 by a dual tropic V3 loop. We report what is, to our knowledge, the first HIV-1 gp120 V3 loop:CXCR4 complex structure. The computationally derived structure reveals an abundance of polar and nonpolar intermolecular interactions contributing to the HIV-1 gp120:CXCR4 binding. Our results are in remarkable agreement with previous experimental findings. Therefore, this work sheds light on the functional role of HIV-1 gp120 V3 loop and CXCR4 residues associated with HIV-1 coreceptor activity.     

Studies on in vivo induction of HIV-1 envelope-specific cytotoxic T lymphocytes by synthetic peptides from the V3 loop region of HIV-1 IIIB gp120

We have previously reported the induction of MHC class I-restricted, CD8⁺ cytotoxic T lymphocytes (CTLs) specific to human immunodeficiency virus type 1 (HIV-1) in mice by a 15-amino acid peptide (R15K) from the V3 loop in gp120. We now present evidence showing that CTL activity induced by R15K was stable for 8–10 weeks after a single injection and that as little as 20 μg peptide was sufficient for efficient CTL induction in vivo. While induction of CTLs was efficient with R15K emulsified in either complete or incomplete Freund's adjuvant, only a low-level CTL response was observed in mice immunized with R15K in either alum or saline. We analyzed a series of carrier-free synthetic peptides ranging in length from 8 to 24 amino acids from the V3 loop region and observed that peptide R10I consisting of 10 amino acids from the middle portion of R15K was more efficient for CTL induction. Additionally, lymph node cells from mice immunized with 24 and 15 amino acid peptides (N24G and R15K, respectively) when restimulated in vitro with R10I exhibited greater HIV-1 env-specific CTL activity than when either of the longer peptides was used for restimulation. A peptide consisting of only 8 amino acids (R8K) was sufficient neither for inducing primary CTLs nor for in vitro restimulation of lymph node CTL precursors. These results establish that a carrier-free 10-amino acid synthetic peptide from the V3 loop region in HIV-1 gp120 has the optimal sequence for efficient induction of HIV env-specific CTLs in mice.     

Disulfide bond that constrains the HIV-1 gp120 V3 domain is cleaved by thioredoxin

A functional disulfide bond in both the HIV envelope glycoprotein, gp120, and its immune cell receptor, CD4, is involved in viral entry, and compounds that block cleavage of the disulfide bond in these proteins inhibit HIV entry and infection. The disulfide bonds in both proteins are cleaved at the cell surface by the small redox protein, thioredoxin. The target gp120 disulfide and its mechanism of cleavage were determined using a thioredoxin kinetic trapping mutant and mass spectrometry. A single disulfide bond was cleaved in isolated and cell surface gp120, but not the gp160 precursor, and the extent of the reaction was enhanced when gp120 was bound to CD4. The Cys(32) sulfur ion of thioredoxin attacks the Cys(296) sulfur ion of the gp120 V3 domain Cys(296)-Cys(331) disulfide bond, cleaving the bond. Considering that V3 sequences largely determine the chemokine receptor preference of HIV, we propose that cleavage of the V3 domain disulfide, which is facilitated by CD4 binding, regulates chemokine receptor binding. There are 20 possible disulfide bond configurations, and, notably, the V3 domain disulfide has the same unusual -RHStaple configuration as the functional disulfide bond cleaved in CD4.     

Expression, purification, and isotope labeling of a gp120 V3 peptide and production of a Fab from a HIV-1 neutralizing antibody for NMR studies

Most human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies in infected individuals and in immunized animals are directed against the third variable loop (V3) of the envelope glycoprotein (gp120) of the virus. This loop plays a crucial role in phenotypic determination, cytopathicity (syncytium induction), and coreceptor usage of HIV-1. The human monoclonal antibody 447-52D was found to neutralize a broad spectrum of HIV-1 strains. In order to solve the solution structure of the V3MN peptide bound to the 447-52D Fab fragment by NMR, large quantities of labeled peptide and a protocol for the purification of the Fab fragment were needed. An expression plasmid coding for the 23-residue V3 peptide of the HIV-1MN strain (V3MN peptide, YNKRKRIHIGPGRAFYTTKNIIG) linked to a derivative of the RNA-binding domain of hnRNCP1 was constructed. The fusion protein attached to the V3 peptide prevents its degradation. Using this system, U-15N, U-13C,15N, and U-13C,15N, 50% 2H labeled fusion protein molecules were expressed in Escherichia coli grown on rich Celtone medium with yields of about 240 mg/liter. The V3MN peptide was released by CNBr cleavage and purified by RP-HPLC, giving final yields of 6-13 mg/liter. This expression system is generally applicable for biosynthesis of V3-related peptides and was also used to prepare the V3JR-FL. The 447-52D Fab fragment was obtained by a short enzymatic papain cleavage of the whole antibody. Preliminary NMR spectra demonstrate that full structural analysis of the V3MN complexed to the 447-52D Fab is feasible. This system enables studies of the same epitope bound to different HIV-1 neutralizing antibodies.     

The HF-SCF energy of HIV-1 MNgp120 V3 hairpin loop conformers

The purpose of this study is to analyze the structure of the V3 loop of the HIV-1 gp120 molecule at the atomic level. The total energy of each member of the antibody-complexed 16-mer V3 conformer data set of Sharon et al. (PDB 1NJ0) was determined by the Hartree–Fock-self-consistent field (HF-SCF) method and with the GROMOS96 force field. There was no correlation between the results of the classical GROMOS96 force field analysis and the ab initio HF-SCF quantum mechanical analysis of the energy of the V3-loop-peptide conformers. HF-SCF optimization (AM1) of conformer geometries yielded structures in which HIS315 is displaced from its original position in the combining site of human antibody fragment 447-52D, but with the hairpin turn intact. The hairpin shape of the V3 loop remained detectable, albeit distorted, even with perturbation by a lithium dicationic electrostatic force field and by substitution of the PRO320 at the crown of the V3 hairpin by a GLY. These data suggest that the hairpin conformation is at least partially stable to long-range electrostatic perturbations, either with or without PRO in the tip of the crown of the V3-hairpin loop. Figure Molecular geometry of HIV-1 V3 conformer model 5 and a GLY320 substituted model 5. Space-filling models were obtained with ViewMol3D [Sharon et al. (2002) PDB 1NJ0]). Red=oxygen, blue=nitrogen, black=carbon, white=hydrogen and purple=lithium. End-to-end distance (D) was obtained with ViewMol3D and is in Ångströms. Geometry optimized GLY320 Model 5, D=4.74 ÅThis revised version was published online in October 2004 with corrections to the Graphical Abstract.     

Study on conformational homology of the HIV-1 gp120 protein V3 loop. Structural analysis of the HIV-RF and HIV-thailand viral strains

Based on NMR spectroscopy data, conformation of the HIV-RF gp120 protein V3 loop giving rise to the virus principal neutralizing determinant and also determinants of cell tropism and syncytium formation was calculated by computer modeling approaches. Elements of the HIV-RF V3 loop secondary structure and conformational states of its irregular stretches were determined. The calculated structure was compared with the conformation of the homologous stretch of the HIV-Thailand protein gp120 V3 loop, and structural elements preserved in the two viral strains were identified. Conservative elements of the HIV-1 V3 loop structure are considered to be promising targets for deriving chemically modified forms of this loop with the enhanced immunogenicity and cross-reactivity of neutralizing antibodies and also for creation of effective antiviral drugs on this base.     

Conformational model for the consensus V3 loop of the envelope protein gp120 of HIV-1 in a 20% trifluoroethanol/water solution.

Based on experimental NMR data, a model was generated for the conformation of the disulfide-bond-closed cyclic peptide corresponding to the whole V3 loop of the consensus HIV-1 strain in a 20% trifluoroethanol/water solution. The obtained family of structures shows a prominent and well-defined amphipathic alpha helix at the C-terminal end of the peptide from Thr23 to Gln32. A series of turns characterizes the central Gly15-Tyr21 region, while the N-terminal region is poorly defined. Independent experimental data confirms the features of this model, and suggests that this type of conformation can be readily adopted when the V3 loop is in contact with a membrane. The examined V3 loop belongs to a macrophage tropic strain, and using the model, a structural explanation is proposed for the different requirements of V3 loops belonging to macrophage and T-cell line tropic HIV-1 strains.