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.     

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.     

Conformational preferences of the HIV-1 principal neutralizing determinant

The model describing the conformational properties of the HIV-1 principal neutralizing determinant in the geometric space of dihedrals was generated in terms of NMR spectroscopy data published in literature. To gain an object in view, the following successive steps were put into effect: (i) the NMR-based local structures for the HIV(MN) V3 loop were determined in water and in a mixed water/trifluoroethanol (TFE) solvent (7:3), (ii) in either case, the conformations of its irregular segments were analyzed and the secondary structure elements identified, (iii) to appreciate the degree of conformational mobility of the stretch of interest, the simulated structures were compared with each other, (iv) to detect the amino acids retaining their conformations inside the diverse HIV-1 isolates, the structures computed were collated with the one derived previously for the V3 loop from Thailand isolate, and (v) as a matter of record, the structurally rigid residues, that may present the forward-looking targets for AIDS drug researches, were revealed. Summing up the principal results arising from these studies, the following conclusions were drawn: I. The HIV(MN) V3 loop offers the highly mobile fragment of gp120 sensitive to its environment whose changes trigger the large-scale structural reforms, bringing in substantial altering the secondary structure of this functionally important site of the virus envelope. II. In water, it exhibits extended site 1-14 separated by double beta-turn 15-20 with unordered region 21-35. III. Adding the TFE gives rise to destruction of the regular structure in the V3 loop N-terminal, stimulates the formation of 3(10)-helix in site 24-31, and affects also its central region 20-25 forming the HIV-1 immunogenic crown. IV. Regardless of statistically significant differences between local structures of the HIV(MN) V3 loop in water and in water/TFE solution, over one-third of residues keeps their conformational states; 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. V. There are no conserved structural motifs within the V3 loops from Minnesota and Thailand HIV-1 strains. However, perceptible portion of amino acids (more than 35%), including those appearing in the functionally important regions of gp120, holds the values of dihedral angles in which case. The implications are discussed in conjunction with the data on the experimental observations for the HIV-1 principal neutralizing determinant.     

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.     

Human immunodeficiency virus type 1 tropism for brain microglial cells is determined by a region of the env glycoprotein that also controls macrophage tropism.

Human immunodeficiency virus type 1 (HIV-1), the agent of AIDS, frequently infects the central nervous system. We inoculated adult human brain cultures with chimeric viruses containing parts of the env gene of a cloned primary isolate from brain tissue, HIV-1 JRFl, inserted into the cloned DNA of a T-cell-tropic strain. A chimeric virus containing the carboxy-terminal portion of HIV-1 JRFl env did not replicate in these brain tissue cultures, while a chimera expressing an env-encoded protein containing 158 amino acids of HIV-1 JRFl gp120, including the V3 loop, replicated well in brain microglial cells, as it does in blood macrophages. Infection of brain microglial cells with such a chimera was blocked by an antibody to the V3 loop of gp 120. Thus, env determinants in the region of gp120, outside the CD4-binding site and comprising the V3 loop, are critical for efficient viral binding to and/or entry into human brain microglia.     

Role of Naturally Occurring Basic Amino Acid Substitutions in the Human Immunodeficiency Virus Type 1 Subtype E Envelope V3 Loop on Viral Coreceptor Usage and Cell Tropism

To assess the role of naturally occurring basic amino acid substitutions in the V3 loop of human immunodeficiency virus type 1 (HIV-1) subtype E on viral coreceptor usage and cell tropism, we have constructed a panel of chimeric viruses with mutant V3 loops of HIV-1 subtype E in the genetic background of HIV-1LAI. The arginine substitutions naturally occurring at positions 8, 11, and 18 of the V3 loop in an HIV-1 subtype E X4 strain were systematically introduced into that of an R5 strain to generate a series of V3 loop mutant chimera. These chimeric viruses were employed in virus infectivity assays using HOS-CD4 cells expressing either CCR5 or CXCR4, peripheral blood mononuclear cells, T-cell lines, or macrophages. The arginine substitution at position 11 of the V3 loop uniformly caused the loss of infectivity in HOS-CD4-CCR5 cells, indicating that position 11 is critical for utilization of CCR5. CXCR4 usage was conferred by a minimum of two arginine substitutions, regardless of combination, whereas arginine substitutions at position 8 and 11 were required for T-cell line tropism. Nonetheless, macrophage tropism was not conferred by the V3 loop of subtype E R5 strain per se. We found that the specific combinations of amino acid changes in HIV-1 subtype E env V3 loop are critical for determining viral coreceptor usage and cell tropism. However, the ability to infect HOS-CD4 cells through either CXCR4 or CCR5 is not necessarily correlated with T-cell or macrophage tropism, suggesting that cellular tropism is not dictated solely by viral coreceptor utilization.     

Inhibition of human immunodeficiency virus type 1 infection and syncytium formation in human cells by V3 loop synthetic peptides from gp120.

Because V3 loop-specific antibodies have been shown to inhibit human immunodeficiency virus type 1 (HIV-1) infection of human cells and because specific mutations in the V3 loop render the virus ineffective for infection and syncytium formation, we tested the anti-HIV effects of V3 loop peptides from different HIV-1 strains. We obtained evidence that V3 loop synthetic peptides of 8 to 15 amino acids at nanogram concentrations efficiently blocked HIV-1 IIIB infection of several human T-cell lines and of freshly prepared normal human T cells. More importantly, syncytium formation by three different primary clinical HIV isolates was inhibited by the V3 loop peptide from HIV-1 IIIB at a concentration of 1 micrograms/ml. Concentrations of V3 peptides up to 50 micrograms/ml were not toxic to any of the human cells studied. Additionally, V3 peptides incubated in normal human serum or plasma exhibited biological and physical stability for up to 24 h. Taken together, these results suggest that the V3 loop peptides have medical utility as therapeutic reagents to either prevent HIV-1 infection in humans or reduce the spread of virus infection in HIV-infected individuals. These findings are especially significant because a number of reports in the literature indicate that the V3 loop region in gp120 plays an important role in the initial stages of HIV-1 infection of cells.     

Linkage of Amino Acid Variation and Evolution of Human Immunodeficiency Virus Type 1 gp120 Envelope Glycoprotein (Subtype B) with Usage of the Second Receptor

To clarify the relationship between the amino acid variations of the gp120 of human immunodeficiency virus type 1 (HIV-1) and the chemokine receptors that are used as the second receptor for HIV, we evaluated amino acid site variation of gp120 between the X4 strains (use CXCR4) and the R5 strains (use CCR5) from 21 sequences of subtype B. Our analysis showed that residues 306 and 322 in the V3 loop and residue 440 in the C4 region were associated with usage of the second receptor. The polymorphism at residue 440 is clearly associated with the usage of the second receptor: The amino acid at position 440 was a basic amino acid in the R5 strains, and a nonbasic and smaller amino acid in the X4 strains, while the V3 loop of the X4 strains was more basic than that of the R5 strains. This suggests that residue 440 in the C4 region, which is close to the V3 loop in the three-dimensional structure, is critical in determining which second receptor is used. Analysis of codon frequency suggests that, in almost all cases, the difference at residue 440 between basic amino acids in the R5 strains and nonbasic amino acids in the X4 strains could be due to a single nucleotide change. These findings predict that the evolutionary changes in amino acid residue 440 may be correlated with evolutionary changes in the V3 loop. One possibility is that a change in electric charge at residue 440 compensates for a change in electric charge in the V3 loop. The amino acid polymorphism at position 440 can be useful to predict the cell tropism of a strain of HIV-1 subtype B.     

Chimeras from a human rhinovirus 14-human immunodeficiency virus type 1 (HIV-1) V3 loop seroprevalence library induce neutralizing responses against HIV-1.

A chimeric virus library was designed whereby sequences corresponding to the V3 loop of human immunodeficiency virus type 1 (HIV-1) were presented on the surface of human rhinovirus 14. The V3 loop sequences consisted of a relatively conserved segment of seven amino acids and five adjacent residues that were allowed to vary in proportion to their seroprevalence among HIV-1 isolates of North America and Europe. A technique called random systematic mutagenesis was used to incorporate the composite V3 loop sequences flanked by zero to two randomized amino acids. This library could contain 2.7 x 10(8) members having diverse sequences and conformations. Immunoselection of a portion of this library by using two neutralizing V3 loop-directed monoclonal antibodies followed by selection for desirable growth and purification characteristics yielded a set of chimeric rhinoviruses, five of which are described. The inserted sequences in the five chimeras do not match those of any known isolate of HIV-1. Nonetheless, all five chimeras were neutralized by antibodies directed against different strains of HIV-1 and were able to elicit the production of antibodies that bind V3 loop peptides from diverse HIV-1 isolates. Moreover, antisera derived from four of the five chimeras were capable of neutralizing one or more strains of HIV-1 in cell culture. This study demonstrates that random systematic mutagenesis in conjunction with antibody screening is a powerful and efficient means to obtain antigenic chimeras with relevant immunogenic properties.     

Structural analysis of the envelope gp120 V3 loop for some HIV-1 variants circulating in the countries of Eastern Europe

The V3 loop on gp120 from human immunodeficiency virus type 1 (HIV-1) is a focus of many research groups involved in anti-AIDS drug development because this region of the protein is a principal target for neutralizing antibodies and a major determinant for cell tropism and syncytium formation. In this study, the nucleotide sequences of the env gene region coding the V3 loop were determined by DNA sequencing methods for four novel HIV-1 strains that circulate in the countries of Eastern Europe, such as Russia, Belarus, Ukraine, etc. Based on the empirical data obtained, the 3D structures of the V3 loops associated with these viral modifications were generated by computer modeling and then compared to discover similarities in the spatial arrangement of this functionally important site of gp120. Despite the HIV-1 genetic variety, several regions of the V3 loop that contain residues critical for cell tropism were shown to be structurally invariant, which may explain its exceptional role in a co-receptor usage. These data together with those on the biological activity of the V3 individual residues clearly show that these conserved structural motifs of gp120 represent potential HIV-1 weak points most suitable for therapeutic intervention.     

A disulfide-bound HIV-1 V3 loop sequence on the surface of human rhinovirus 14 induces neutralizing responses against HIV-1

An immunogenic sequence from the V3 loop of the MN isolate of human immunodeficiency virus type 1 (HIV-1), His-Ile-Gly-Pro-Gly-Arg-Ala-Phe, was transplanted onto a surface loop of the VP2 capsid protein of human rhinovirus 14. To optimize for virus viability and immunogenicity of the transplanted sequence, the HIV sequence was flanked by (1) a cysteine residue that could form a disulfide bond and (2) randomized amino acids (in either of two arrangements) to generate numerous presentations of the Cys-Cys loop. The location for engineering in VP2 was chosen by searching the geometries of disulfide-bound loops in known protein structures. A model for the structure of the transplanted V3 loop sequence was developed using molecular dynamics and energy minimization calculations. Proteolytic digestion with and without reducing agent demonstrated the presence of the disulfide bond in the chimeric virus examined. Monoclonal and polyclonal antibodies directed against the V3 region of the HIV-1MN strain potently neutralized two chimeric viruses. Guinea pig antisera against two chimeric viruses were able to neutralize HIV-1MN and HIV-1ALA-1 in cell culture. The ability of chimeric viruses to elicit antibodies capable of neutralizing the source of the transplanted sequence could be favorable for vaccine development.     

Molecular mimicry between the human immunodeficiency virus type 1 gp120 V3 loop and human brain proteins.

Immunologically cross-reactive proteins in the human brain that resemble the V3 loop of human immunodeficiency virus type 1 (HIV-1) gp120 have been identified. When several homogenized tissues from normal brains were used, a monoclonal antibody raised against amino acids 308 to 320 of the V3 loop reacted with three prominent human brain proteins (HBP) of 35, 55, and 110 kDa. Among the three, the 55-kDa HBP appears to be specific to the central nervous system. These results indicate that the V3 loop of HIV-1 gp120 shares an epitope with HBP. An immune response to the V3 loop that generates cross-reactive antibodies to cellular proteins may be an autoimmune mechanism by which HIV-1 can damage the central nervous system.     

Adaptive Mutations in the V3 Loop of gp120 Enhance Fusogenicity of Human Immunodeficiency Virus Type 1 and Enable Use of a CCR5 Coreceptor That Lacks the Amino-Terminal Sulfated Region

To identify sites in gp120 that interact with the CCR5 coreceptor and to analyze the mechanisms of infection, we selected variants of the CCR5-dependent JRCSF molecular clone of human immunodeficiency virus type 1 (HIV-1) that adapted to replicate in HeLa-CD4 cells that express the mutant coreceptor CCR5(Y14N) or CCR5(G163R), which were previously shown to bind purified gp120-CD4 complexes only weakly. Correspondingly, these mutant CCR5s mediate infections of wild-type virus only at relatively high cell surface concentrations, demonstrating a concentration-dependent assembly requirement for infection. The plots of viral infectivity versus concentration of coreceptors had sigmoidal shapes, implying involvement of multiple coreceptors, with an estimated stoichiometry of four to six CCR5s in the active complexes. All of the adapted viruses had mutations in the V3 loops of their gp120s. The titers of recombinant HIV-1 virions with these V3 mutations were determined in previously described panels of HeLa-CD4 cell clones that express discrete amounts of CCR5(Y14N) or CCR5(G163R). The V3 loop mutations did not alter viral utilization of wild-type CCR5, but they specifically enhanced utilization of the mutant CCR5s by two distinct mechanisms. Several mutant envelope glycoproteins were highly fusogenic in syncytium assays, and these all increased the efficiency of infection of the CCR5(Y14N) or CCR5(G163R) clonal panels without enhancing virus adsorption onto the cells or viral affinity for the coreceptor. In contrast, V3 loop mutation N300Y was selected during virus replication in cells that contained only a trace of CCR5(Y14N) and this mutation increased the apparent affinity of the virus for this coreceptor, as indicated by a shift in the sigmoid-shaped infectivity curve toward lower concentrations. Surprisingly, N300Y increased viral affinity for the second extracellular loop of CCR5(Y14N) rather than for the mutated amino terminus. Indeed, the resulting virus was able to use a mutant CCR5 that lacks 16 amino acids at its amino terminus, a region previously considered essential for CCR5 coreceptor function. Our results demonstrate that the role of CCR5 in infection involves at least two steps that can be strongly and differentially altered by mutations in either CCR5 or the V3 loop of gp120: a concentration-dependent binding step that assembles a critical multivalent virus-coreceptor complex and a postassembly step that likely involves a structural rearrangement of the complex. The postassembly step can severely limit HIV-1 infections and is not an automatic consequence of virus-coreceptor binding, as was previously assumed. These results have important implications for our understanding of the mechanism of HIV-1 infection and the factors that may select for fusogenic gp120 variants during AIDS progression.     

Isolation of TAK-779-resistant HIV-1 from an R5 HIV-1 GP120 V3 loop library

The human immunodeficiency virus (HIV-1) envelope glycoprotein (GP) 120 interacts with CD4 and the CCR5 coreceptor for viral entry. The V3 loop in GP120 is a crucial region for determining coreceptor usage during viral entry, and a variety of amino acid substitutions has been observed in clinical isolates. To construct an HIV-1 V3 loop library, we chose 10 amino acid positions in the V3 loop and incorporated random combinations (27,648 possibilities) of the amino acid substitutions derived from 31 R5 viruses into the V3 loop of HIV-1(JR-FL) proviral DNA. The constructed HIV-1 library contained 6.6 x 10(6) independent clones containing a set of 0-10 amino acid substitutions in the V3 loop. To address whether restricted steric alteration in the V3 loop could confer resistance to an entry inhibitor, TAK-779, we selected entry inhibitor-resistant HIV-1 by increasing the concentration of TAK-779 from 0.10 to 0.30 microM in PM1-CCR5 cells with high expression of CCR5. The selected viruses at passage 8 contained five amino acid substitutions in the V3 loop without any other mutations in GP120 and showed 15-fold resistance compared with the parental virus. These results indicated that a certain structure of the V3 loop containing amino acid substitutions derived from 31 R5 viruses can contribute to the acquisition of resistance to entry inhibitors binding to CCR5. Taken together, this type of HIV-1 V3 loop library is useful for isolating and analyzing the specific biological features of HIV-1 with respect to alterations of the V3 loop structure.     

Distinct human immunodeficiency virus strains in the bone marrow are associated with the development of thrombocytopenia

We analyzed bone marrow and blood from human immunodeficiency virus type 1 (HIV-1)-infected individuals and described the HIV-1 quasispecies in these cellular compartments. HIV-1 isolates from the bone marrow of thrombocytopenic patients contained distinct amino acids in the V3 loop and infected T-cell lines, implicating this virus in the development of thrombocytopenia.     

Determining the invariant structure elements of the HIV-1 variable V3 loops: insight into the HIV-MN and HIV-Haiti isolates

The computer approaches that combined the 3D protein structure modeling with the mathematical statistics methods were used to compute the NMR-based 3D structures of the HIV-1 gp120 V3 loop for the HIV-MN and HIV-Haiti isolates in water as well as to compare their conformational characteristics with the purpose of determining the structure elements common for the two virus modifications. As a result, the variability of the amino acid sequence was found to stimulate the considerable structural rearrangements of the V3 loop. However, despite this fact, one functionally crucial stretch of V3 and a greater portion of its residues were shown to preserve the conformations in the viral strains of interest. To reveal the structural motifs and individual amino acids giving rise to the close conformations in the HIV-MN and HIV-Haiti V3 loops regardless of the sequence and environment variability, the simulated structures were collated with those deciphered previously in terms of NMR data in a water/trifluoroethanol mixed solvent. The structure elements and single residues of V3 residing in its biologically significant sites and keeping the conformations in all of the cases at question are considered to be the promising targets for anti-AIDS drugs studies. In this context, the structurally inflexible motifs of V3 presenting the weak units in the virus protection system may be utilized as the most convenient landing-places for molecular docking of the V3 loop and ligand structures followed by selecting chemical compounds suitable as a basis for the design of safe and effective antiviral agents.     

Human immunodeficiency virus type 1 clones chimeric for the envelope V3 domain differ in syncytium formation and replication capacity.

Chimeric human immunodeficiency virus type 1 (HIV-1) molecular clones differing only in the envelope V3 region were constructed. The V3 regions were derived from two HIV-1 isolates with a non-syncytium-inducing, non-T-cell-tropic phenotype and from four HIV-1 isolates with a syncytium-inducing, T-cell-tropic phenotype. When assayed in SupT1 cells, the two chimeric viruses with a V3 region derived from the non-syncytium-inducing isolates did not induce syncytia and showed a low level of replication. The four chimeric viruses with a V3 region derived from the syncytium-inducing isolates did induce syncytia and replicated efficiently in SupT1 cells. In A3.01 cells, which do not support syncytium formation, the V3 loop affected replication similarly. Upon prolonged culture in SupT1 cells, the phenotype of a non-syncytium-inducing, low-replicating chimeric HIV-1 converted into a syncytium-inducing, high-replicating phenotype. Mutations within the usually conserved GPGR tip of the loop, which were shown to be responsible for the conversion into the syncytium-inducing, high-replicating phenotype, had occurred. In vitro mutagenesis showed that coupled changes of amino acids at both sides of the tip of the V3 loop were able to convert the viral phenotype from non-syncytium-inducing, low replicating into syncytium inducing, high replicating. Our data show that the V3 loop is involved in both syncytium forming and replicative capacity of HIV-1.     

Mapping of independent V3 envelope determinants of human immunodeficiency virus type 1 macrophage tropism and syncytium formation in lymphocytes.

The V3 region of the human immunodeficiency virus type 1 (HIV-1) envelope protein is known to have a major influence on macrophage tropism as well as the ability to cause syncytium formation or fusion in CD4-positive lymphocyte cultures. Using infectious molecular HIV-1 clones, a series of mutant clones was created which allowed detailed mapping of V3 amino acid positions involved in these properties. In these experiments the non-syncytium-inducing phenotype in T cells did not always correlate with macrophage tropism. Macrophage tropism appeared to depend on the presence of certain combinations of amino acids at five specific positions within and just outside of the V3 loop itself, whereas syncytium formation in lymphocytes was influenced by substitution of particular residues at two to four positions within V3. In most cases, different V3 amino acid positions were found to independently influence macrophage tropism and syncytium formation in T cells and position 13 was the only V3 location which appeared to simultaneously influence both macrophage tropism and syncytium formation in lymphocytes.     

Analysis of mutations in the V3 domain of gp160 that affect fusion and infectivity.

The third hypervariable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein has been proposed to play an important role in mediating viral entry. Antibodies to the V3 domain block HIV-1 infection but not virus binding to CD4. At the center of the V3 domain is a relatively conserved sequence of amino acids, GPGRA. It has previously been shown that mutation of some of these amino acids reduced the ability of gp160 expressed on the surface of cells to induce fusion with CD4-bearing cells. In order to analyze the role of V3 domain sequences in mediating HIV entry, we introduced several amino acid substitution mutations in the GPGRA sequence of gp160 derived from HIV-1 strain HXB2 and in the analogous sequence of strain SF33, GPGKV. Virus was generated by cotransfecting the env constructs and a selectable env-negative HIV vector, HIV-gpt. When complemented with a retrovirus env gene, infectious virus capable of a single round of replication was produced. The viral particles produced were analyzed biochemically for core and envelope proteins and for infectious titer. The transfected envs were also analyzed for ability to bind to CD4 and mediate cell fusion. Several of the amino acid substitutions resulted in moderate to severe decreases in virus infectivity and fusion activity. Envelope glycoprotein assembly onto particles and CD4 binding were not affected. These results provide evidence that V3 sequences are involved in mediating the fusion step of HIV-1 entry.     

Human Rhinovirus Type 14:Human Immunodeficiency Virus Type 1 (HIV-1) V3 Loop Chimeras from a Combinatorial Library Induce Potent Neutralizing Antibody Responses against HIV-1

In an effort to develop a useful AIDS vaccine or vaccine component, we have generated a combinatorial library of chimeric viruses in which the sequence IGPGRAFYTTKN from the V3 loop of the MN strain of human immunodeficiency virus type 1 (HIV-1) is displayed in many conformations on the surface of human rhinovirus 14 (HRV14). The V3 loop sequence was inserted into a naturally immunogenic site of the cold-causing HRV14, bridged by linkers consisting of zero to three randomized amino acids on each side. The library of chimeric viruses obtained was subjected to a variety of immunoselection schemes to isolate viruses that provided the most useful presentations of the V3 loop sequence for potential use in a vaccine against HIV. The utility of the presentations was assessed by measures of antigenicity and immunogenicity. Most of the immunoselected chimeras examined were potently neutralized by each of the four different monoclonal anti-V3 loop antibodies tested. Seven of eight chimeric viruses were able to elicit neutralizing antibody responses in guinea pigs against the MN and ALA-1 strains of HIV-1. Three of the chimeras elicited HIV neutralization titers that exceeded those of all but a small number of previously described HIV immunogens. These results indicate that HRV14:HIV-1 chimeras may serve as useful immunogens for stimulating immunity against HIV-1. This method can be used to flexibly reconstruct varied immunogens on the surface of a safe and immunogenic vaccine vehicle.