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.     

Structural Analysis of the HIV-1 gp120 V3 Loop: Application to the HIV-Haiti Isolates

Abstract

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.     

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.     

Solution structure of the mature HIV-1 protease monomer: insight into the tertiary fold and stability of a precursor

We present the first solution structure of the HIV-1 protease monomer spanning the region Phe1-Ala95 (PR1-95). Except for the terminal regions (residues 1-10 and 91-95) that are disordered, the tertiary fold of the remainder of the protease is essentially identical to that of the individual subunit of the dimer. In the monomer, the side chains of buried residues stabilizing the active site interface in the dimer, such as Asp25, Asp29, and Arg87, are now exposed to solvent. The flap dynamics in the monomer are similar to that of the free protease dimer. We also show that the protease domain of an optimized precursor flanked by 56 amino acids of the N-terminal transframe region is predominantly monomeric, exhibiting a tertiary fold that is quite similar to that of PR1-95 structure. This explains the very low catalytic activity observed for the protease prior to its maturation at its N terminus as compared with the mature protease, which is an active stable dimer under identical conditions. Adding as few as 2 amino acids to the N terminus of the mature protease significantly increases its dissociation into monomers. Knowledge of the protease monomer structure and critical features of its dimerization may aid in the screening and design of compounds that target the protease prior to its maturation from the Gag-Pol precursor.     

Modeling of the 3D structure of the HIV-Haiti immunodominant epitope

The 3D structure for the HIV-Haiti immunodominant epitope was computed in terms of NMR spectroscopy data using the theoretical procedure including a probabilistic approach in conjunction with molecular mechanics algorithms and quantum chemical methods. The immunogenic crown of the virus protein gp120 was shown to form in solution a prevalent conformation whose geometric parameters match the double beta-turn IV-IV. Two structures observed in crystal were found in the ensemble of the best-energy conformations of the HIV-Haiti principal neutralizing epitope. From a comparison of simulated structures with those computed previously for the HIV-Thailand and HIV-MN isolates, it was concluded that the immunogenic tip of gp120 gives rise to similar spatial backbone forms in different HIV-1 strains but has some inherent conformational flexibility of its individual amino acid residues. The differences in local fragment structures revealed in three isolates of HIV-1 are supposed to be important for the specificity of its binding with neutralizing antibodies.     

Cooperative effects of the human immunodeficiency virus type 1 envelope variable loops V1 and V3 in mediating infectivity for T cells.

Insertion of T-cell line-tropic V3 and V4 loops from the HXB2 strain into the macrophage-tropic YU-2 envelope resulted in a virus with delayed infectivity for HUT78 and Jurkat cells compared with HXB2. Sequence analysis of viral DNA derived from long-term cultures of Jurkat cells revealed a specific mutation that changed a highly conserved Asn residue in the V1 loop of Env to an Asp residue (N-136-->D). Introduction of this mutation into clones containing a T-cell line-tropic V3 loop, either with or without a T-cell line-tropic V4 loop, resulted in viruses that replicated to high levels in Jurkat cells and peripheral blood lymphocytes. The Env proteins from these constructs were expressed with the vaccinia virus/T7 hybrid system and were found to be translated, processed, and cleaved and to bind to soluble CD4 similar to the wild-type HXB2 and YU-2 Env proteins. Env-mediated fusion with HeLa T4+ cells, however, was regulated by both the altered V1 loop and T-cell line-tropic V3 loop. These results suggest that subsequent to the initial gp120-CD4 binding event, a functional interaction can occur between the altered V1 loop and T-cell line-tropic V3 loop that results in infection of Jurkat cells and peripheral blood lymphocytes.     

SAMHD1: a new insight into HIV-1 restriction in myeloid cells

Human myeloid-lineage cells are refractory to HIV-1 infection. The Vpx proteins from HIV-2 and sooty mangabey SIV render these cells permissive to HIV-1 infection through proteasomal degradation of a putative restriction factor. Two recent studies discovered the cellular protein SAMHD1 to be this restriction factor, demonstrating that Vpx induces proteasomal degradation of SAMHD1 and enhances HIV-1 infection in myeloid-lineage cells. SAMHD1 functions as a myeloid-cell-specific HIV-1 restriction factor by inhibiting viral DNA synthesis. Here we discuss the implications of these findings in delineating the mechanisms of HIV-1 restriction in myeloid-lineage cells and the potential role of Vpx in lentiviral pathogenesis.     

Relationship between the V3 loop and the phenotypes of human immunodeficiency virus type 1 (HIV-1) isolates from children perinatally infected with HIV-1.

The third variable region (V3) of the envelope protein of human immunodeficiency virus type 1 (HIV-1) contains group- and type-specific epitopes for neutralizing antibodies and contains determinants involved in viral tropism and syncytium-inducing (SI) activity. We studied the in vivo relationship between V3 sequences and viral phenotypes in 24 perinatally HIV-1-infected children. To avoid in vitro selection of intrapatient minor variants, genetic studies were performed directly on uncultured peripheral blood mononuclear cells (PBMC), and the tropisms of HIV-1 isolates were evaluated by culturing patients' PBMC directly with monocyte-derived macrophages, lymphocytes, and MT-2 cells. According to their phenotypes, we could define five types of primary isolates: (i) non-syncytium-inducing (NSI) macrophagetropic, (ii) NSI macrophage-lymphotropic, (iii) NSI lymphotropic, (iv) SI lympho-T-cell line-tropic, and (v) SI pleiotropic. The SI viral phenotype was correlated with a more advanced status of disease. Genetic analysis of intrapatient molecular variants revealed that no relationship between the degree of intrapatient V3 variability and the pattern of viral tropism existed; moreover, within a single patient, the values for V3 variability between CD4+ lymphocytes and CD14+ monocytes were similar, thus suggesting that in vivo variability of the monocytotropic variants is more extensive than previously appreciated. A comparison between the intrapatient major variants and the phenotype of primary isolates disclosed that a negatively charged amino acid at residue site 25 was associated with an NSI macrophage- and macrophage-lymphotropic viral phenotype. Finally, by comparing the V3 sequences derived from our study population with those of several prototypes, we observed that the majority of isolates circulating in Italy are related to the North American subtype B macrophagetropic isolates.     

Crystal structure of a human rhinovirus that displays part of the HIV-1 V3 loop and induces neutralizing antibodies against HIV-1

We report the 2.7 A resolution structure of a chimeric rhinovirus, MN-III-2, that displays part of the HIV-1 gp120 V3 loop and elicits HIV-neutralizing antibodies. The V3 loop insert is dominated by two type I beta turns. The structures of two adjacent tripeptides resemble those of analogous segments in three Fab/V3 loop peptide complexes. Although two of the three corresponding antibodies bind and neutralize MN-III-2 well, only one of the three can bind without significant rearrangement. These results suggest that the V3 loop insert: (1) can share some local conformational similarity to V3 loop sequences presented on different structural frameworks; (2) must be able to adopt multiple conformations, even in a relatively constrained environment; and (3) may mimic the conformational variability of the epitope on HIV-1, increasing the likelihood of eliciting appropriate neutralizing immune responses.     

Replication and neutralization of human immunodeficiency virus type 1 lacking the V1 and V2 variable loops of the gp120 envelope glycoprotein.

A human immunodeficiency virus type 1 (HIV-1) mutant lacking the V1 and V2 variable loops in the gp120 exterior envelope glycoprotein replicated in Jurkat lymphocytes with only modest delays compared with the wild-type virus. Revertants that replicated with wild-type efficiency rapidly emerged and contained only a few amino acid changes in the envelope glycoproteins compared with the parent virus. Both the parent and revertant viruses exhibited increased sensitivity to neutralization by antibodies directed against the V3 loop or a CD4-induced epitope on gp120 but not by soluble CD4 or an antibody against the CD4 binding site. This result demonstrates the role of the gp120 V1 and V2 loops in protecting HIV-1 from some subsets of neutralizing antibodies.     

Alternative conformations of HIV-1 V3 loops mimic beta hairpins in chemokines,suggesting a mechanism for coreceptor selectivity

The V3 loop of the HIV-1 envelope glycoprotein gp120 is involved in binding to the CCR5 and CXCR4 coreceptors. The structure of an HIV-1(MN) V3 peptide bound to the Fv of the broadly neutralizing human monoclonal antibody 447-52D was solved by NMR and found to be a beta hairpin. This structure of V3(MN) was found to have conformation and sequence similarities to beta hairpins in CD8 and CCR5 ligands MIP-1alpha, MIP-1beta, and RANTES and differed from the beta hairpin of a V3(IIIB) peptide bound to the strain-specific murine anti-gp120(IIIB) antibody 0.5beta. In contrast to the structure of the bound V3(MN) peptide, the V3(IIIB) peptide resembles a beta hairpin in SDF-1, a CXCR4 ligand. These data suggest that the 447-52D-bound V3(MN) and the 0.5beta-bound V3(IIIB) structures represent alternative V3 conformations responsible for selective interactions with CCR5 and CXCR4, respectively.     

Atomic insight into the CD4 binding-induced conformational changes in HIV-1 gp120

The entry of HIV-1 into a target cell requires gp120 and receptor CD4 as well as coreceptor CCR5/CXCR4 recognition events associated with conformational changes of the involved proteins. The binding of CD4 to gp120 is the initiation step of the whole process involving structural rearrangements that are crucial for subsequent pathways. Despite the wealth of knowledge about the gp120/CD4 interactions, details of the conformational changes occurring at this stage remain elusive. We have performed molecular dynamics simulations in explicit solvent based on the gp120/CD4/CD4i crystal structure in conjunction with modeled V3 and V4 loops to gain insight into the dynamics of the binding process. Three differentiated interaction modes between CD4 and gp120 were found, which involve electrostatics, hydrogen bond and van der Waals networks. A "binding funnel" model is proposed based on the dynamical nature of the binding interface together with a CD4-attraction gradient centered in gp120 at the CD4-Phe43-binding cavity. Distinct dynamical behaviors of free and CD4-bound gp120 were monitored, which likely represent the ground and pre-fusogenic states, respectively. The transition between these states revealed concerted motions in gp120 leading to: i) loop contractions around the CD4-Phe43-insertion cavity; ii) stabilization of the four-stranded "bridging sheet" structure; and iii) translocation and clustering of the V3 loop and the bridging sheet leading to the formation of the coreceptor binding site. Our results provide new insight into the dynamic of the underlying molecular recognition mechanism that complements the biochemical and structural studies.     

Insights into the oligomeric structure of the HIV-1 Vpu protein

The HIV-1-encoded protein Vpu forms an oligomeric ion channel/pore in membranes and interacts with host proteins to support the virus lifecycle. However, Vpu molecular mechanisms are currently not well understood. Here, we report on the Vpu oligomeric organization under membrane and aqueous conditions and provide insights into how the Vpu environment affects the oligomer formation. For these studies, we designed a maltose-binding protein (MBP)-Vpu chimera protein and produced it in E. coli in soluble form. We analyzed this protein using analytical size-exclusion chromatography (SEC), negative staining electron microscopy (nsEM), and electron paramagnetic resonance (EPR) spectroscopy. Surprisingly, we found that MBP-Vpu formed stable oligomers in solution, seemingly driven by Vpu transmembrane domain self-association. A coarse modeling of nsEM data as well as SEC and EPR data suggests that these oligomers most likely are pentamers, similar to what was reported regarding membrane-bound Vpu. We also noticed reduced MBP-Vpu oligomer stability upon reconstitution of the protein in β-DDM detergent and mixtures of lyso-PC/PG or DHPC/DHPG. In these cases, we observed greater oligomer heterogeneity, with MBP-Vpu oligomeric order generally lower than in solution; however, larger oligomers were also present. Notably, we found that in lyso-PC/PG, above a certain protein concentration, MBP-Vpu assembles into extended structures, which had not been reported for Vpu. Therefore, we captured various Vpu oligomeric forms, which can shed light on Vpu quaternary organization. Our findings could be useful in understanding Vpu organization and function in cellular membranes and could provide information regarding the biophysical properties of single-pass transmembrane proteins.     

Restricted variable residues in the C-terminal segment of HIV-1 V3 loop regulate the molecular anatomy of CCR5 utilization

The V3 loop of the HIV-1 envelope glycoprotein (Env) is the major determinant for coreceptor utilization, but the structural basis for this specificity remains to be defined. By characterizing a set of naturally occurring R5 Env variants, we demonstrate that Asp324 in the conserved IIGDIR motif of the V3 loop (CTRPN(300)NNTRKSIHIGP(311)GRAFYTTGEIIGD(324)IRQAHC) C-terminal segment regulates the molecular anatomy of CCR5 utilization. Whereas gp120 subunits with Asp or Asn at position 324 were fusogenic with coreceptor chimeras containing either the N-terminal domain or the body of CCR5, substitution of charged (Glu, Lys) or small hydrophobic (Gly, Ala) residues resulted in complete loss of fusogenic activity with the N terminus and markedly reduced utilization of the body of CCR5, although their ability to use wild-type CCR5 was unchanged. This phenotypic conversion was confirmed in both gain and loss of function experiments using Env from multiple subtypes. Alignment of sequences of R5 V3 loops (n=599) from the HIV database revealed that the mutation of Asp324 in the conserved IIGDIR motif is restricted to Asn324, with proportions of 71.5% and 28%, respectively. Infection of primary CD4(+)T cells demonstrated that Env bearing Asp324 was less sensitive to RANTES, suggesting that Asp or Asn in this position may be crucial for viral fitness. The CD4-dependent gp120 binding to CCR5 was decreased when Asp324 was replaced with a charged or hydrophobic residue, but unchanged when replaced with Asn. Molecular modeling analyses predicted that Asp/Asn324 forms a critical H-bond with Asn300. These findings indicate that Asp or Asn at position 324 of the V3 stem stabilizes the conformation of V3 loop and hence influences the intensities of interaction between CD4-activated gp120 and CCR5 which results in viral entry.     

HIV-1 GP120 V3 conformational and informational entropies

In an attempt to analyze structure, function and evolution of HIV-1 GP120 V3, interactions among the Hartree–Fock energy, the conformational entropy and the Shannon entropy were determined for the 1NJ0 set of antibody-bound V3 loop conformers. The Hartree–Fock energy of each conformer was determined at the MINI level with GAMESS. The conformational entropy was determined per conformer and per residue from the mass-weighted covariance matrices. The Shannon entropy per residue was determined from sequence-substitution frequencies. Correlations were determined by linear regression analysis. There was a negative correlation between the Hartree–Fock energy and the conformational entropy (R=−0.4840, p=0.0078, df =28) that enhanced the negative Helmholtz-free-energy change for the binding of the GP120 ligand to target CD4. The Shannon entropy of V3 was a function of the conformational entropy variance (R=0.7225, p=0.00157, df=15) and of the V3 Hartree–Fock energy. Biological implications of this work are that (1) conformational entropy interacts with V3 Hartree–Fock energy to enhance GP120 binding to CD4 cell receptors and that (2) the Hartree–Fock energy of V3 interacts with the evolutionary system to participate in the regulation of V3 diversity.     

CD81 extracellular domain 3D structure: insight into the tetraspanin superfamily structural motifs

Human CD81, a known receptor for hepatitis C virus envelope E2 glycoprotein, is a transmembrane protein belonging to the tetraspanin family. The crystal structure of human CD81 large extracellular domain is reported here at 1.6 A resolution. Each subunit within the homodimeric protein displays a mushroom-like structure, composed of five alpha-helices arranged in 'stalk' and 'head' subdomains. Residues known to be involved in virus binding can be mapped onto the head subdomain, providing a basis for the design of antiviral drugs and vaccines. Sequence analysis of 160 tetraspanins indicates that key structural features and the new protein fold observed in the CD81 large extracellular domain are conserved within the family. On these bases, it is proposed that tetraspanins may assemble at the cell surface into homo- and/or hetero-dimers through a conserved hydrophobic interface located in the stalk subdomain, while interacting with other liganding proteins, including hepatitis C virus E2, through the head subdomain. The topology of such interactions provides a rationale for the assembly of the so-called tetraspan-web.     

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.     

Evolution of the HIV-1 V3 region in the Italian epidemic

The epidemic of Human Immunodeficiency Virus type 1 infection in Italy is mostly ascribed to the B subtype, which represents the prevalent subtype in Western Countries. The virus isolates of the B subtype, moreover, show an increasing nucleotide heterogeneity over time, indicating a continuous intra-subtype dynamic evolution, typical of long-lasting epidemics. In recent years, however, the progressive decrease in the transmission rate among the historically defined risk groups (i.e. homosexuals and IDUs) and the parallel increase in heterosexual transmission are slowly introducing variants of non-B subtype into the Italian HIV-1 epidemic. This appears to be strictly linked to the growing number of immigrants from non-Western Countries, where non-B clades and CRFs are prevalent, and consequent inter-racial blending. The distribution of these novel genetic forms needs to be evaluated by continuous molecular monitoring nationwide to verify whether they will overcome the pre-existing B-clade epidemic, which could have significant implications for diagnosis, treatment and vaccine development. Here we review the genetic evolution of HIV-1 spreading within the Italian epidemic.