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.     

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.     

Determination of structurally conservative amino acids of the HIV-1 protein gp120 V3 loop as promising targets for drug design by protein engineering approaches

Based on the published NMR spectroscopy data, three-dimensional structures of the HIV-1 gp120 protein V3 loop were obtained by computer modeling in the viral strains HIV-Haiti and HIV-MN. In both cases, the secondary structure elements and conformations of irregular stretches were determined for the fragment representing the principal antigenic determinant of the virus, as well as determinants of the cellular tropism and syncytium formation. Notwithstanding the high variability of the amino acid sequence of gp120 protein, more than 50% of the V3 loop residues retained their conformations in the different HIV-1 virions. The combined analysis of the findings and the literature data on the biological activity of the individual residues of the HIV-1 V3 loop resulted in identification of its structurally conservative amino acids, which seem to be promising targets for antiviral drug design by protein engineering approaches.     

The region of the envelope gene of human immunodeficiency virus type 1 responsible for determination of cell tropism.

Different isolates of human immunodeficiency virus type 1 (HIV-1) vary in the cell tropisms they display, i.e., the range of cell types in which they are able to establish a productive infection. Here, we report on the phenotypes of recombinants between two molecularly cloned strains of HIV-1. Our results prove that the envelope glycoprotein gp120 is solely responsible for the difference in cell tropism between the two parental isolates and that no other genes or sequences are involved in determining the cell tropism of these strains. The region of the envelope involved in the determination of cell tropism includes sequences which encode the V3 loop of gp120. Control of cell tropism by this region of the virus env gene is a general phenomenon which applies to many different HIV-1 isolates.     

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.     

Structural requirements for and consequences of an antiviral porphyrin binding to the V3 loop of the human immunodeficiency virus (HIV-1) envelope glycoprotein gp120

Several porphyrin derivatives were reported to have anti-HIV-1 activity. Among them, meso-teta(4-carboxyphenyl)porphine (MYCPP) and other carboxyphenyl derivatives were the most potent inhibitors (EC₅₀ < 0.7 μM). MTCPP bound to the HIV-1 enveloope glycoprotein gp120 and to full-length V3 loop peptides corresponding to several HIV-1 isolates but not to other peptides from gp120+gp41. However, it remained possible that MTCPP bound to HIV-1 envelop glycoprotein gp120 and to full-length V3 loop peptides corresponding to several HIV-1 isolates but not to other peptides from gp120+gp41. However, it remained possible that MTCPP bound to regions on gp120 which cannot be mimicked by peptides. Further characterization of the binding domain for MTCPP is important for understanding the antiviral activity of porphyrins and for the design of anit-HIV-1 drugs interfering with functions of the virus envelope. Results presented here show that: (i) deletion of the V3 loop from the gp120 sequence resulted in drastically diminished MTCPP binding, suggesting that the V3 loop is the dominant if not the only target site on gp120; (ii) this site was only partially mimicked by full-length V3 loop peptides; (iii) MTCPP binding to the gp120 V3 loop elicited allosteric effects resulting in decreased accessibility of the CD4 receptor binding site; (iv) the binding site for MTCPP lies within the central portion of the V3 loop (KSIHIGPGRAFY for the HIV-1 subtype B consensus sequence) and does not involve directly the GPG apex of the loop. These results may help in designing antiviral compounds with improved activity.     

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.     

The Influence of N-Linked Glycans on the Molecular Dynamics of the HIV-1 gp120 V3 Loop

N-linked glycans attached to specific amino acids of the gp120 envelope trimer of a HIV virion can modulate the binding affinity of gp120 to CD4, influence coreceptor tropism, and play an important role in neutralising antibody responses. Because of the challenges associated with crystallising fully glycosylated proteins, most structural investigations have focused on describing the features of a non-glycosylated HIV-1 gp120 protein. Here, we use a computational approach to determine the influence of N-linked glycans on the dynamics of the HIV-1 gp120 protein and, in particular, the V3 loop. We compare the conformational dynamics of a non-glycosylated gp120 structure to that of two glycosylated gp120 structures, one with a single, and a second with five, covalently linked high-mannose glycans. Our findings provide a clear illustration of the significant effect that N-linked glycosylation has on the temporal and spatial properties of the underlying protein structure. We find that glycans surrounding the V3 loop modulate its dynamics, conferring to the loop a marked propensity towards a more narrow conformation relative to its non-glycosylated counterpart. The conformational effect on the V3 loop provides further support for the suggestion that N-linked glycosylation plays a role in determining HIV-1 coreceptor tropism.     

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.     

Replication-competent variants of human immunodeficiency virus type 2 lacking the V3 loop exhibit resistance to chemokine receptor antagonists

Entry of human immunodeficiency virus type 1 (HIV-1) and HIV-2 requires interactions between the envelope glycoprotein (Env) on the virus and CD4 and a chemokine receptor, either CCR5 or CXCR4, on the cell surface. The V3 loop of the HIV gp120 glycoprotein plays a critical role in this process, determining tropism for CCR5- or CXCR4-expressing cells, but details of how V3 interacts with these receptors have not been defined. Using an iterative process of deletion mutagenesis and in vitro adaptation of infectious viruses, variants of HIV-2 were derived that could replicate without V3, either with or without a deletion of the V1/V2 variable loops. The generation of these functional but markedly minimized Envs required adaptive changes on the gp120 core and gp41 transmembrane glycoprotein. V3-deleted Envs exhibited tropism for both CCR5- and CXCR4-expressing cells, suggesting that domains on the gp120 core were mediating interactions with determinants shared by both coreceptors. Remarkably, HIV-2 Envs with V3 deletions became resistant to small-molecule inhibitors of CCR5 and CXCR4, suggesting that these drugs inhibit wild-type viruses by disrupting a specific V3 interaction with the coreceptor. This study represents a proof of concept that HIV Envs lacking V3 alone or in combination with V1/V2 that retain functional domains required for viral entry can be derived. Such minimized Envs may be useful in understanding Env function, screening for new inhibitors of gp120 core interactions with chemokine receptors, and designing novel immunogens for vaccines.     

Correlated mutations at gp120 positions 322 and 440: implications for gp120 structure

Analysis of V3 and C4 sequences of HIV-1 reveals correlated mutations at gp120 positions 322 and 440, and a very strong preference for a positively charged residue at position 440 when position 322 is negatively charged. This observation suggests that these two residues are close to each other and interact electrostatically in R5 viruses. This interaction was used to model V3 in the context of gp120 using NMR data for the V3 loop and the crystal structure of the gp120-core. The interaction between residues 322 and 440 may serve as part of the molecular switch for HIV-1 phenotype conversion.     

Effects of partial deletions within the human immunodeficiency virus type 1 V3 loop on coreceptor tropism and sensitivity to entry inhibitors

The human immunodeficiency virus type 1 (HIV-1) V3 loop is critical for coreceptor binding and principally determines tropism for the CCR5 and CXCR4 coreceptors. The recent crystallographic resolution of V3 shows that its base is closely associated with the conserved coreceptor binding site on the gp120 core, whereas more distal regions protrude toward the cell surface, likely mediating interactions with coreceptor extracellular loops. However, these V3-coreceptor interactions and the structural basis for CCR5 or CXCR4 specificity are poorly understood. Using the dual-tropic virus HIV-1_R3A, which uses both CCR5 and CXCR4, we sought to identify subdomains within V3 that selectively mediate R5 or X4 tropism. An extensive panel of V3 mutants was evaluated for effects on tropism and sensitivity to coreceptor antagonists. Mutations on either side of the V3 base (residues 3 to 8 and 26 to 33) ablated R5 tropism and made the resulting X4-tropic Envs more sensitive to the CXCR4 inhibitor AMD3100. When mutations were introduced within the V3 stem, only a deletion of residues 9 to 12 on the N-terminal side ablated X4 tropism. Remarkably, this R5-tropic Δ9-12 mutant was completely resistant to several small-molecule inhibitors of CCR5. Envs with mutations in the V3 crown (residues 13 to 20) remained dual tropic. Similar observations were made for a second dual-tropic isolate, HIV-1_89.6. These findings suggest that V3 subdomains can be identified that differentially affect R5 and X4 tropism and modulate sensitivity to CCR5 and CXCR4 inhibitors. These studies provide a novel approach for probing V3-coreceptor interactions and mechanisms by which these interactions can be inhibited.     

Effect of amino acid substitution of the V3 and bridging sheet residues in human immunodeficiency virus type 1 subtype C gp120 on CCR5 utilization

The V3 loop and the bridging sheet domain of human immunodeficiency virus type 1 (HIV-1) subtype B envelope glycoprotein gp120 have been implicated in CCR5 coreceptor utilization. In this study, mutant envelope glycoproteins of a subtype C isolate containing substitutions in the V3 or C4 region were generated to determine which are required for efficient CCR5-dependent cell fusion and viral entry. We found that the V3 crown and C4 residues are relatively dispensable for cell-cell fusion, although some residues may be involved in the regulation of early postentry steps in viral replication. In contrast, seven highly conserved residues located in the V3 stem are critical for CCR5 utilization, which can explain the apparent paradox that the functional convergence in CCR5 usage by genetically divergent HIV-1 strains involves a variable region. The finding that C4 residues do not have a critical role may appear to contradict the current model that bridging sheet residues are involved in the gp120-CCR5 interaction. However, a plausible interpretation is that these C4 residues may have a distinct role in the binding and fusion steps of the gp120-CCR5 interaction.     

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.     

Functional role of the V1/V2 region of human immunodeficiency virus type 1 envelope glycoprotein gp120 in infection of primary macrophages and soluble CD4 neutralization.

We have examined the influence of the V1/V2 region of the human immunodeficiency virus type 1 (HIV-1) gp120 on certain biologic properties of the virus. We observed that on the genomic background of the T-cell-line-tropic strain, HIV-1SF2mc, both the V1 and V2 domains of the macrophage-tropic strain, HIV-1SF162mc, in addition to the required V3 domain, are necessary to attain full macrophage tropism. Furthermore, the V2 domain modulates the sensitivity of HIV-1 to soluble CD4 neutralization. Structural studies of recombinant and mutant envelope glycoproteins suggest that the function of the V1/V2 region is to interact with the V3 domain and confer on the envelope gp120 of HIV-1SF2mc a conformation more similar to that of the macrophage-tropic strain HIV-1SF162mc. The conformation of the envelope gp120 appears to be strain specific and plays an important role in determining HIV-1 tissue tropism and sensitivity to soluble CD4 neutralization.     

Analysis of the Critical Domain in the V3 Loop of Human Immunodeficiency Virus Type 1 gp120 Involved in CCR5 Utilization

Human immunodeficiency virus type 1 (HIV-1) infection of CD4(+) lymphocytes and macrophages involves interaction of the surface subunit of the envelope protein (gp120) with coreceptors. Isolates have been found with specific tropism for macrophages and/or T-cell lines, through the utilization of chemokine receptor CCR5 (R5) or CXCR4 (X4). The third hypervariable loop (V3 loop) of gp120 is the major determinant of tropism. Using chimeric envelopes between HXB2 (X4) and ADA (R5), we found that the C-terminal half of the V3 loop was sufficient to confer on HXB2 the ability to infect CCR5-expressing cells. A sequence motif was identified at positions 289 to 292 allowing 30% of wild-type levels of infection, whereas full activity was achieved with the conversion of Lys to Glu at position 287 in addition to the above motif. Moreover, V3 loops from either SF2 (X4R5) or SF162 (R5) also allowed infection of CCR5-expressing cells, supporting the importance of V3 loops in influencing CCR5 utilization. The effects of amino acid changes at position 287 on the level of infection via CCR5 showed that negatively charged residues (Glu and Asp) were optimal for efficient interaction whereas only bulky hydrophobic residues drastically reduced infection. In addition, sequences at the N terminus of the V3 loop independently modulated the level of infection via CCR5. This study also examined the susceptibility of chimeric envelopes to neutralization by anticoreceptor antibodies and suggested the presence of differential interaction between the chimeric envelopes and CCR5. These findings highlight the critical residues in the V3 loop that mediate HIV-1 infection.     

Prediction of the secondary structure of HIV-1 gp120

The secondary structure of HIV-1 gp120 was predicted using multiple alignment and a combination of two independent methods based on neural network and nearest-neighbor algorithms. The methods agreed on the secondary structure for 80% of the residues in BH10 gp120. Six helices were predicted in HIV strain BH10 gp120, as well as in 27 other HIV-1 strains examined. Two helical segments were predicted in regions displaying profound sequence variation, one in a region suggested to be critical for CD4 binding. The predicted content of helix, β-strand, and coil was consistent with estimates from Fourier transform infrared spectroscopy. The predicted secondary structure of gp120 compared well with data from NMR analysis of synthetic peptides from the V3 loop and the C4 region. As a first step towards modeling the tertiary structure of gp120, the predicted secondary structure may guide the design of future HIV sub-unit vaccine candidates. © 1996 Wiley-Liss, Inc.     

Mapping the determinants of the CCR5 amino-terminal sulfopeptide interaction with soluble human immunodeficiency virus type 1 gp120-CD4 complexes

CD4 and CCR5 mediate fusion and entry of R5 human immunodeficiency virus type 1 (HIV-1) strains. Sulfotyrosine and other negatively charged residues in the CCR5 amino-terminal domain (Nt) are crucial for gp120 binding and viral entry. We previously showed that a soluble gp120-CD4 complex specifically binds to a peptide corresponding to CCR5 Nt residues 2 to 18, with sulfotyrosines in positions 10 and 14. This sulfopeptide also inhibits soluble gp120-CD4 binding to cell surface CCR5 as well as infection by an R5 virus. Here we show that residues 10 to 18 constitute the minimal domain of the CCR5 Nt that is able to specifically interact with soluble gp120-CD4 complexes. In addition to sulfotyrosines in positions 10 and 14, negatively charged residues in positions 11 and 18 participate in this interaction. Furthermore, the CCR5 Nt binds to a CD4-induced surface on gp120 that is composed of conserved residues in the V3 loop stem and the C4 domain. Binding of gp120 to cell surface CCR5 is further influenced by residues in the crown of the V3 loop, C1, C2, and C3. Our data suggest that gp120 docking to CCR5 is a multistep process involving several independent regions of the envelope glycoprotein and the coreceptor.     

Structure and Dynamics of the gp120 V3 Loop That Confers Noncompetitive Resistance in R5 HIV-1JR-FL to Maraviroc

Maraviroc, an (HIV-1) entry inhibitor, binds to CCR5 and efficiently prevents R5 human immunodeficiency virus type 1 (HIV-1) from using CCR5 as a coreceptor for entry into CD4⁺ cells. However, HIV-1 can elude maraviroc by using the drug-bound form of CCR5 as a coreceptor. This property is known as noncompetitive resistance. HIV-1_V3-M5 derived from HIV-1_JR-FLan is a noncompetitive-resistant virus that contains five mutations (I304V/F312W/T314A/E317D/I318V) in the gp120 V3 loop alone. To obtain genetic and structural insights into maraviroc resistance in HIV-1, we performed here mutagenesis and computer-assisted structural study. A series of site-directed mutagenesis experiments demonstrated that combinations of V3 mutations are required for HIV-1_JR-FLan to replicate in the presence of 1 µM maraviroc, and that a T199K mutation in the C2 region increases viral fitness in combination with V3 mutations. Molecular dynamic (MD) simulations of the gp120 outer domain V3 loop with or without the five mutations showed that the V3 mutations induced (i) changes in V3 configuration on the gp120 outer domain, (ii) reduction of an anti-parallel β-sheet in the V3 stem region, (iii) reduction in fluctuations of the V3 tip and stem regions, and (iv) a shift of the fluctuation site at the V3 base region. These results suggest that the HIV-1 gp120 V3 mutations that confer maraviroc resistance alter structure and dynamics of the V3 loop on the gp120 outer domain, and enable interactions between gp120 and the drug-bound form of CCR5.     

Identification of conserved and variable structures in the human immunodeficiency virus gp120 glycoprotein of importance for CXCR4 binding

CD4 and the chemokine receptors, CXCR4 and CCR5, serve as receptors for human immunodeficiency virus type 1 (HIV-1). Binding of the HIV-1 gp120 envelope glycoprotein to the chemokine receptors normally requires prior interaction with CD4. Mapping the determinants on gp120 for the low-affinity interaction with CXCR4 has been difficult due to the nonspecific binding of this viral glycoprotein to cell surfaces. Here we examine the binding of a panel of gp120 mutants to paramagnetic proteoliposomes displaying CXCR4 on their surfaces. We show that the gp120 beta19 strand and third variable (V3) loop contain residues important for CXCR4 interaction. Basic residues from both elements, as well as a conserved hydrophobic residue at the V3 tip, contribute to CXCR4 binding. Removal of the gp120 V1/V2 variable loops allows the envelope glycoprotein to bind CXCR4 in a CD4-independent manner. These results indicate that although some variable gp120 residues contribute to the specific binding to CCR5 or CXCR4, gp120 elements common to CXCR4- or CCR5-using strains are involved in the interaction with both coreceptors.