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.     

Induction of antibodies in guinea pigs and rhesus monkeys against the human immunodeficiency virus type 1 envelope: neutralization of nonpathogenic and pathogenic primary isolate simian/human immunodeficiency virus strains

We have compared the abilities of human immunodeficiency virus type 1 (HIV-1) envelope V3 peptides and recombinant gp120 to induce antibodies that neutralize simian/human immunodeficiency viruses (SHIVs). SHIV-89.6 is a nonpathogenic SHIV that expresses the envelope protein of primary HIV-1 isolate 89.6. SHIV-89.6P, clone KB9, is a pathogenic SHIV variant derived from SHIV-89.6. Infection of rhesus monkeys with these SHIVs rarely induces anti-V3 region antibodies. To determine the availability of the gp120 V3 loop for neutralizing antibody binding on SHIV-89.6 and KB9 virions, we have constructed immunogenic C4-V3 peptides from these SHIVs and induced anti-V3 antibodies in guinea pigs and rhesus monkeys. We found that both SHIV-89.6 and KB9 C4-V3 peptides induced antibodies that neutralized SHIV-89.6 but that only SHIV-KB9 C4-V3 peptide induced antibodies that neutralized SHIV-KB9. Immunoprecipitation assays demonstrated that SHIV-KB9 C4-V3 peptide-induced antibodies had a greater ability to bind SHIV-KB9 envelope proteins than did antibodies raised against SHIV-89.6 C4-V3 peptide. We have used a series of mutant HIV-1 envelope constructs to map the gp120 determinants that affect neutralization by anti-V3 antibodies. The residue change at position 305 of arginine (in SHIV-89.6) to glutamic acid (in SHIV-KB9) played a central role in determining the ability of peptide-induced anti-V3 antiserum to neutralize primary isolate SHIVs. Moreover, residue changes in the SHIV-89.6 V1/V2 loops also played roles in regulating the availability of the V3 neutralizing epitope on SHIV-89.6 and -KB9. Thus, SHIV-89.6 and -KB9 V3 region peptides are capable of inducing neutralizing antibodies against these primary isolate SHIVs, although the pathogenic SHIV-KB9 is less easily neutralized than its nonpathogenic variant SHIV-89.6. In contrast to natural infection with SHIV-89.6, in which few animals make anti-V3 antibodies, C4-V3 peptides frequently induced anti-V3 antibodies that neutralized primary isolate SHIV strains.     

Applications of biotinylated V3 loop peptides of human immunodeficiency virus type 1 to flow cytometric analyses and affinity chromatographic techniques

A principal neutralizing determinant of human immunodeficiency virus type 1 (HIV-1) lies within the V3 loop of gp120, the external major envelope glycoprotein. V3 loop peptides derived from two HIV-1 strains, HTLV-III BH-10 (V3-BH10) and LAV_ELI (V3-ELI), were synthesized and biotinylated. The binding of both biotinylated V3-BH10 and V3-ELI to the surfaces of MOLT-4 clone 8 cells was demonstrated by flow cytometric analyses. Both the peptides (more than 2 μM) bound to the cells (2 · 10⁵) in a dose-dependent manner. The binding of biotinylated V3-BH10 was specifically inhibited by a neutralizing monoclonal antibody (0.5β). The binding of both of the biotinylated V3 loop peptides was enhanced by the addition of unlabeled V3-BH10. In addition, the peptides were employed as ligands on affinity columns. A major V3 loop binding protein (V3BP) was purified from the membrane soluble fraction of MOLT-4 cells by successive application to two different V3 loop columns. V3BP consisted of two major polypeptides (32 and 33 kDa). The SDS-PAGE profile of V3BP did not change under non-reducing conditions, but only a single band was observed after analysis on native PAGE. The major peak of the eluate as determined by size exclusion chromatography was abroad and the estimated relative molecular mass was much larger than 33 kDa, suggesting that V3BP comprises several subunits. Taken together, we confirmed that the V3 loop peptides are useful in the characterization of V3BP(s) of which they are conformational ligands.     

The V1/V2 loop of HIV-1 gp120 is necessary for Tat binding and consequent modulation of virus entry

Preventing cell entry of human immunodeficiency virus 1 (HIV-1) is of interest for the development of innovative therapies. We previously reported a specific interaction between HIV-1 envelope glycoprotein 120 (gp120) and Tat at the cell surface, which enhances virus attachment and entry. We also identified a gp120-mimicking peptide, CT319, that competes with gp120 for Tat binding, thus inhibiting HIV-1 infection. Here we report a molecular dissection of gp120 regions involved in this mechanism. Our findings identify the V1/V2 loop of gp120 as involved in Tat binding, and define this interaction as functionally relevant for HIV-1 entry into host cells.     

Synthetic multimeric peptides derived from the principal neutralization domain (V3 loop) of human immunodeficiency virus type 1 (HIV-1) gp120 bind to galactosylceramide and block HIV-1 infection in a human CD4-negative mucosal epithelial cell line.

The glycosphingolipid galactosylceramide (GalCer), which binds gp120 with high affinity and specificity, is a potential alternative receptor for human immunodeficiency virus type 1 (HIV-1) in some CD4-negative neural and epithelial human cells, including the human colonic epithelial cell line HT-29. In the present study, we demonstrate that synthetic multibranched peptides derived from the consensus sequence of the HIV-1 V3 loop block HIV-1 infection in HT-29 cells. The most active peptide was an eight-branched multimer of the motif Gly-Pro-Gly-Arg-Ala-Phe which at a concentration of 1.8 microM induced a 50% inhibition of HIV-1 infection in competition experiments. This peptide was not toxic to HT-29 cells, and preincubation with HIV-1 did not affect viral infectivity, indicating that the antiviral activity was not due to a nonspecific virucidal effect. Using a high-performance thin-layer chromatography binding assay, we found that multibranched V3 peptides recognized GalCer and inhibited binding of recombinant gp120 to the glycosphingolipid. In addition, these peptides abolished the binding of an anti-GalCer monoclonal antibody to GalCer on the surface of live HT-29 cells. These data provide additional evidence that the V3 loop is involved in the binding of gp120 to the GalCer receptor and show that multibranched V3 peptides are potent inhibitors of the GalCer-dependent pathway of HIV-1 infection in CD4-negative mucosal epithelial cells.     

Vaccine-elicited V3 loop-specific antibodies in rhesus monkeys and control of a simian-human immunodeficiency virus expressing a primary patient human immunodeficiency virus type 1 isolate envelope

Vaccine-elicited antibodies specific for the third hypervariable domain of the surface gp120 of human immunodeficiency virus type 1 (HIV-1) (V3 loop) were assessed for their contribution to protection against infection in the simian-human immunodeficiency virus (SHIV)/rhesus monkey model. Peptide vaccine-elicited anti-V3 loop antibody responses were examined for their ability to contain replication of SHIV-89.6, a nonpathogenic SHIV expressing a primary patient isolate HIV-1 envelope, as well as SHIV-89.6P, a pathogenic variant of that virus. Low-titer neutralizing antibodies to SHIV-89.6 that provided partial protection against viremia following SHIV-89.6 infection were generated. A similarly low-titer neutralizing antibody response to SHIV-89.6P that did not contain viremia after infection with SHIV-89.6P was generated, but a trend toward protection against CD4+ T-lymphocyte loss was seen in these infected monkeys. These observations suggest that the V3 loop on some primary patient HIV-1 isolates may be a partially effective target for neutralizing antibodies induced by peptide immunogens.     

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.     

Multibranched V3 peptides inhibit human immunodeficiency virus infection in human lymphocytes and macrophages.

Synthetic polymeric constructions (SPCs) including the consensus sequence of the human immunodeficiency virus type 1 (HIV-1) surface envelope glycoprotein gp120 V3 loop (GPGRAF) blocked the fusion between HIV-1- and HIV-2-infected cells and CD4+ uninfected cells. A structure-activity relationship study using V3 SPC analogs showed that the most efficient inhibitor of cell fusion was an eight-branched SPC with the hexapeptide motif GPGRAF (i.e., [GPGRAF]8-SPC). N-terminal acetylation or incorporation of D-amino acids in the GPGRAF sequence of this SPC resulted in significant loss of activity. Analogs with fewer than six residues in the motif (i.e., GPGRA or GPGR), as well as SPCs with a nonrelevant sequence, did not inhibit cell fusion, demonstrating the high specificity of the antifusion activity. [GPGRAF]8-SPC, which was not toxic to CEM cells at concentrations of up to 50 microM, inhibited 50% of HIV-1(LAI) replication in these cells at a concentration of 0.07 microM. Moreover, [GPGRAF]8-SPC inhibited the infection of human peripheral blood mononuclear cells by several HIV-1 and HIV-2 isolates, including laboratory strains [HIV-1(LAI), HIV-1(NDK), and HIV-2(ROD)], and fresh primary isolates, including two zidovudine-resistant HIV-1 isolates and two HIV-2 isolates obtained from infected individuals. The multibranched peptide also inhibited infection of human primary macrophages by the highly cytopathic macrophage-tropic isolate HIV-1(89.6). The antiviral activity of [GPGRAF]8-SPC was not related to a virucidal effect, since preincubation of HIV-1 with the peptide did not affect its infectious titer. This result is in agreement with the concept that the multibranched peptide mimics a part of the V3 loop and thus interacts with the host cell. The therapeutic properties of synthetic multibranched peptides based on the V3 loop consensus motif should be evaluated in HIV-infected patients.     

A VSV-G pseudotyped HIV vector mediates efficient transduction of human pulmonary artery smooth muscle cells

Attempts were made to infect human vascular smooth muscle cells derived from the pulmonary artery (hPASMC) with two different human immunodeficiency virus (HIV) vector systems. ADA/Luc or HXB2/Luc were generated by cotransfection of luciferase reporter gene vector, pNL4-3-Luc-E- R-, and one of two envelope expressing vectors, pSMADA (R5) or pSMHXB2 (X4). The VSV-G/Luc or VSV-G/GFP were produced by a three-plasmid expression system which consisted of vesicular stomatitis virus G protein (VSV-G) expressing vector, packaging plasmid, and one of two reporter genes (pHR'-CMV-Luc or pHR'-CMV-GFP). We used hPASMC, U87.CD4.CCR5 and U87.CD4.CXCR4 for infection. Neither ADA/Luc nor HXB2/Luc could infect hPASMC, though they could infect U87.CD4 with corresponding coreceptors. On the other hand, the transduction of both VSV-G/Luc and VSV-G/GFP to hPASMC was remarkable. At day 3, the relative proportion of positive cells of hPASMC infected with VSV-G/GFP was 15%. The above finding indicates a direct role of HIV-1 infection in pulmonary hypertension 'a rare complication of HIV-1 infection' and HIV-based vectors could introduce foreign genes into hPASMC for gene therapy of pulmonary hypertension.     

Human immunodeficiency virus type 1 infection of SK-N-MC cells: domains of gp120 involved in entry into a CD4-negative, galactosyl ceramide/3' sulfo-galactosyl ceramide-positive cell line.

The primary receptor for human immunodeficiency virus (HIV) is the CD4 molecule; however, in vitro evidence suggests that a neutral glycolipid, galactosyl ceramide (GalCer) or a derivative molecule, 3' sulfogalactosyl ceramide (GalS), may serve as an alternative receptor for HIV type 1 (HIV-1) in cells of neural and colonic origin. Biochemical studies have demonstrated that recombinant gp120 envelope protein binds to GalCer/GalS in both solid-phase enzyme-linked immunosorbent assay and high-performance thin-layer chromatography overlays. We have used the SK-N-MC cell line, a CD4-negative, GalCer/GalS-positive cell line previously characterized as susceptible to HIV-1 infection, to identify virus isolates with either a positive infection phenotype, HIVHxB2, or a negative infection phenotype, HIV-1(89.6). Using a solid-phase virus binding assay, we determined the level of restriction in HIV-1(89.6) infection to be at the level of virus-glycolipid binding. Furthermore, using HIV-1HxB2-HIV-1(89.6) chimeras, we have identified a 193-amino-acid fragment from the envelope region of HIV-1HxB2 containing the V3, V4, and V5 regions which confers a positive infection phenotype on the HIV-1(89.6) background. Recombinant viruses which separate this 193-amino-acid fragment into two distinct chimeras are each able to confer a positive infection phenotype on the background of HIV89.6, suggesting that a stable GalCer/GalS-envelope interaction is dependent on the conformation of the envelope protein in the context of the viral membrane. Alternatively, the GalCer/GalS-gp120 bond may involve multiple sites on the oligomeric envelope protein.     

Regulation of epitope exposure in the gp41 membrane-proximal external region through interactions at the apex of HIV-1 Env

Glycoprotein Env of human immunodeficiency virus type 1 (HIV-1) mediates viral entry through membrane fusion. Composed of gp120 and gp41 subunits arranged as a trimer-of-heterodimers, Env adopts a metastable, highly dynamic conformation on the virion surface. This structural plasticity limits the temporospatial exposure of many highly conserved, neutralizing epitopes, contributing to the difficulty in developing effective HIV-1 vaccines. Here, we employed antibody neutralization of HIV-1 infectivity to investigate how inter- and intra-gp120 interactions mediated by variable loops V1/V2 and V3 at the Env apex regulate accessibility of the gp41 membrane-proximal external region (MPER) at the Env base. Swapping the V3 loop from Env_SF162 into the Env_HXB2 background shifted MPER exposure from the prefusogenic state to a functional intermediate conformation that was distinct from the prehairpin-intermediate state sensitive to gp41-targeted fusion inhibitors. The V3-loop swap had a profound impact on global protein dynamics, biasing the equilibrium to a closed conformation resistant to most anti-gp120 antibodies, stabilizing the protein to both cold- and soluble CD4-induced Env inactivation, and increasing the CD4 requirements for viral entry. Further dissection of the Env_HXB2 V3 loop revealed that residue 306 uniquely modulated epitope exposure and trimer stability. The R306S substitution substantially decreased sensitivity to antibodies targeting the gp41 MPER and, surprisingly, the gp120 V3-loop crown (residues 312–315), but had only modest effects on exposure of intervening gp120 epitopes. Furthermore, the point mutation reduced soluble CD4-induced inactivation, but had no impact on cold inactivation. The residue appeared to exert its effects by electrostatically modifying the strength of intra-subunit interactions between the V1/V2 and V3 loops. The distinct patterns of neutralization and stability pointed to a novel prefusogenic Env conformation along the receptor activation pathway and suggested that apical Env-regulation of gp41 MPER exposure can be decoupled from much of the dynamics of gp120 subunits.     

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.     

V3-independent determinants of macrophage tropism in a primary human immunodeficiency virus type 1 isolate.

Human immunodeficiency virus type 1 isolates differ in their ability to productively infect macrophages, and several groups have mapped the genetic basis for macrophage tropism to regions of env that include the third hypervariable region (V3 loop). We recently described a primary isolate (89.6) which is highly macrophage tropic and yet differs from other macrophage-tropic strains studied in that it is cytopathic in T cells. Genetic mapping of macrophage tropism determinants in this virus was done by using chimeras generated with the prototypic non-macrophage-tropic strain HXB2. Replacement of a 2.7-kb env-containing region of HXB with corresponding sequences from 89.6 conferred the macrophage-tropic phenotype, but insertion of the 89.6 V3 loop along with V4/V5 sequences did not. Conversely, placement of HXB sequences that included V3 into 89.6 did not impair this strain's ability to replicate in macrophages. Sequence analysis of V3 shows that 89.6 differs markedly from previously described macrophage-tropic consensus sequences and that it is more similar to highly charged non-macrophage-tropic strains. This suggests either that macrophage tropism is defined by structural determinants resulting from complex interactions among multiple env regions rather than V3 sequence-specific requirements or that there are multiple mechanisms by which different strains may establish productive macrophage infection. In addition, because the HXB V3 loop supports productive macrophage infection in the background of 89.6, phenotypic characterization of V3 sequences should be considered specific to the viral context in which they are placed.     

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.     

Presentation of native epitopes in the V1/V2 and V3 regions of human immunodeficiency virus type 1 gp120 by fusion glycoproteins containing isolated gp120 domains.

The immune response to viral glycoproteins is often directed against conformation- and/or glycosylation-dependent structures; synthetic peptides and bacterially expressed proteins are inadequate probes for the mapping of such epitopes. This report describes a retroviral vector system that presents such native epitopes on chimeric glycoproteins in which protein fragments of interest are fused to the C terminus of the N-terminal domain of the murine leukemia virus surface protein, gp70. The system was used to express two disulfide-bonded domains from gp120, the surface protein of human immunodeficiency virus type 1 (HIV-1), that include potent neutralization epitopes. The resulting fusion glycoproteins were synthesized at high levels and were efficiently transported and secreted. A fusion protein containing the HXB2 V1/V2 domain was recognized by an HIVIIIB-infected patient serum as well as by 17 of 36 HIV-1 seropositive hemophiliac, homosexual male and intravenous drug user patient sera. Many of these HIV+ human sera reacted with V1/V2 domains from several HIV-1 clones expressed in fusion glycoproteins, indicating the presence of cross-reactive antibodies against epitopes in the V1/V2 domain. Recognition of gp(1-263):V1/V2HXB2 by the HIVIIIB-infected human patient serum was largely blocked by synthetic peptides matching V1 but not V2 sequences, while recognition of this construct by a broadly cross-reactive hemophiliac patient serum was not blocked by individual V1 or V2 peptides or by mixtures of these peptides. A construct containing the V3 domain of the IIIB strain of HIV-1, gp(1-263):V3HXB2, was recognized by sera from a human and a chimpanzee that had been infected by HIVIIIB but not by sera from hemophiliac patients who had been infected with HIV-1 of MN-like V3 serotype. The reactive sera had significantly higher titers when assayed against gp(1-263):V3HXB2 than when assayed against matching V3 peptides. Immunoprecipitation of this fusion glycoprotein by the human serum was only partially blocked by V3 peptide, indicating that this infected individual produced antibodies against epitopes in V3 that were expressed on the fusion glycoprotein but not by synthetic peptides. These data demonstrated that the chimeric glycoproteins described here effectively present native epitopes present in the V1/V2 and V3 domains of gp120 and provide efficient methods for detection of antibodies directed against native epitopes in these regions and for characterization of such epitopes.     

Determinants of Neutralization Resistance in the Envelope Glycoproteins of a Simian-Human Immunodeficiency Virus Passaged In Vivo

In vivo passage of a simian-human immunodeficiency virus (SHIV-89.6) generated a virus, SHIV-89.6P, that exhibited increased resistance to some neutralizing antibodies (G. B. Karlsson et al., J. Exp. Med. 188:1159-1171, 1998). Here we examine the range of human immunodeficiency virus type 1 (HIV-1) neutralizing antibodies to which the passaged virus became resistant and identify envelope glycoprotein determinants of antibody resistance. Compared with the envelope glycoproteins derived from the parental SHIV-89.6, the envelope glycoproteins of the passaged virus were resistant to antibodies directed against the gp120 V3 variable loop and the CD4 binding site. By contrast, both viral envelope glycoproteins were equally sensitive to neutralization by two antibodies, 2G12 and 2F5, that recognize poorly immunogenic structures on gp120 and gp41, respectively. Changes in the V2 and V3 variable loops of gp120 were necessary and sufficient for full resistance to the IgG1b12 antibody, which is directed against the CD4 binding site. Changes in the V3 loop specified complete resistance to a V3 loop-directed antibody, while changes in the V1/V2 loops conferred partial resistance to this antibody. The epitopes of the neutralizing antibodies were not disrupted by the resistance-associated changes. These results indicate that in vivo selection occurs for HIV-1 envelope glycoproteins with variable loop conformations that restrict the access of antibodies to immunogenic neutralization epitopes.     

Antibodies to discontinuous or conformationally sensitive epitopes on the gp120 glycoprotein of human immunodeficiency virus type 1 are highly prevalent in sera of infected humans.

We have used an indirect-capture enzyme-linked immunosorbent assay to quantitate the reactivity of sera from human immunodeficiency virus type 1 (HIV-1)-infected humans with native recombinant gp120 (HIV-1 IIIB or SF-2) or with the gp120 molecule (IIIB or SF-2) denatured by being boiled in the presence of dithiothreitol with or without sodium dodecyl sulfate. Denaturation of IIIB gp120 reduced the titers of sera from randomly selected donors by at least 100-fold, suggesting that the majority of cross-reactive anti-gp120 antibodies present are directed against discontinuous or otherwise conformationally sensitive epitopes. When SF-2 gp120 was used, four of eight serum samples reacted significantly with the denatured protein, albeit with ca. 3- to 50-fold reductions in titer. Only those sera reacting with denatured SF-2 gp120 bound significantly to solid-phase-adsorbed SF-2 V3 loop peptide, and none bound to IIIB V3 loop peptide. Almost all antibody binding to reduced SF-2 gp120 was blocked by preincubation with the SF-2 V3 loop peptide, as was about 50% of the binding to native SF-2 gp120. When sera from a laboratory worker or a chimpanzee infected with IIIB were tested, the pattern of reactivity was reversed, i.e., there was significant binding to reduced IIIB gp120, but not to reduced SF-2 gp120. Binding of these sera to reduced IIIB gp120 was 1 to 10% that to native IIIB gp120 and was substantially decreased by preincubation with IIIB (but not SF-2) V3 loop peptide. To analyze which discontinuous or conformational epitopes were predominant in HIV-1-positive sera, we prebound monoclonal antibodies (MAbs) to IIIB gp120 and then added alkaline phosphatase-labelled HIV-1-positive sera. MAbs (such as 15e) that recognize discontinuous epitopes and compete directly with CD4 reduced HIV-1-positive sera binding by about 50%, whereas neutralizing MAbs to the C4, V2, and V3 domains of gp120 were either not inhibitory or only weakly so. Thus, antibodies to the discontinuous CD4-binding site on gp120 are prevalent in HIV-1-positive sera, antibodies to linear epitopes are less common, most of the antibodies to linear epitopes are directed against the V3 region, and most cross-reactive antibodies are directed against discontinuous epitopes, including regions involved in CD4 binding.     

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.     

Brucella abortus conjugated with a gp120 or V3 loop peptide derived from human immunodeficiency virus (HIV) type 1 induces neutralizing anti-HIV antibodies, and the V3-B. abortus conjugate is effective even after CD4+ T-cell depletion.

Human immunodeficiency virus type 1 (HIV-1) infection is associated with loss of function and numbers of CD4+ T-helper cells. In order to bypass the requirement for CD4+ cells in antibody responses, we have utilized heat-inactivated Brucella abortus as a carrier. In this study we coupled a 14-mer V3 loop peptide (V3), which is homologous to 9 of 11 amino acids from the V3 loop of HIV-1 MN, and gp120 from HIV-1 SF2 to B. abortus [gp120(SF2)-B. abortus]. Our results showed that specific antibody responses, dominated by immunoglobulin G2a in BALB/c mice, were induced by these conjugates. Sera from the immunized mice bound native gp120 expressed on the surfaces of cells infected with a recombinant vaccinia virus gp160 vector (VPE16). Sera from mice immunized with gp120(SF2)-B. abortus inhibited binding of soluble CD4 to gp120, whereas sera from mice immunized with V3-B. abortus were ineffective. Sera from mice immunized with either conjugate were capable of blocking syncytium formation between CD4+ CEM cells and H9 cells chronically infected with the homologous virus. Sera from mice immunized with gp120(SF2)-B. abortus were more potent than sera from mice immunized with V3-B. abortus in inhibiting syncytia from heterologous HIV-1 laboratory strains. Importantly, in primary and secondary responses, V3-B. abortus evoked anti-HIV MN antibodies in mice depleted of CD4+ cells, and sera from these mice were able to inhibit syncytia. These findings indicate that B. abortus can provide carrier function for peptides and proteins from HIV-1 and suggest that they could be used for immunization of individuals with compromised CD4+ T-cell function.