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.     

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.     

Synthesis, gp120 binding and anti-HIV activity of fatty acid esters of 1,1-linked disaccharides

Inspired by the anti-human immunodeficiency virus (HIV) activity of analogues of β-galactosylceramide (GalCer), a set of mono- and di-saccharide fatty acid esters were designed as GalCer mimetics and their binding to the V3 loop peptide of HIV-1 and anti-HIV activity evaluated. 1,1-linked Gal-Man and Glu-Man disaccharides with an ester on the Man subunit bound the V3 loop peptide and inhibited HIV infectivity in single round infection assays with the TZM-bl cell line. IC(50)'s were in the 50 μM range with no toxicity to the cells at concentrations up to 200 μM. These compounds appear to inhibit virus entry at early steps in viral infection since they were inactive if added post viral entry. Although these compounds were found to bind to the V3 loop peptide of gp120, it is not clear that this interaction is responsible for their anti-HIV activity because the relative binding affinity of closely related analogues did not correlate with their antiviral behavior. The low cytotoxicity of these 1,1-linked disaccharide fatty acid esters, combined with the easy accessibility to structurally diverse analogues make these molecules attractive leads for new topical anti-viral agents.     

Evaluation of multibranched peptides as inhibitors of HIV infection

Summary Synthetic polymeric constructions (SPCs) containing the consensus sequence of the HIV-1 surface envelope gycoprotein gp120 V3 loop (GPGRAF) block the fusion between HIV-1- and HIV-2-infected cells and CD4⁺-uninfected lymphocytes. By testing the activity of a series of SPC analogs in a cell-to-cell fusion assay, we found that the most active construction is an eight-branched SPC with the hexapeptide motif GPGRAF. This compound is also able to inhibit the infection of human lymphocytes and macrophages by unrelated isolates of HIV-1 and HIV-2. This antiviral activity is specific, since no toxicity was observed at the concentrations that inhibit HIV replication and syncytia formation. These data suggest that V3 SPCs may represent a new class of therapeutic anti-HIV agents, able to neutralize a wide range of viral isolates in infected individuals.     

Neutralizing antibodies against the V3 loop of human immunodeficiency virus type 1 gp120 block the CD4-dependent and -independent binding of virus to cells.

The CD4 molecule is an essential receptor for human immunodeficiency virus type 1 (HIV-1) through high-affinity interactions with the viral external envelope glycoprotein gp120. Previously, neutralizing monoclonal antibodies (MAbs) specific to the third hypervariable domain of gp120 (the V3 loop) have been thought to block HIV infection without affecting the binding of HIV particles to CD4-expressing human cells. However, here we demonstrate that this conclusion was not correct and was due to the use of soluble gp120 instead of HIV particles. Indeed, neutralizing anti-V3 loop MAbs inhibited completely the binding and entry of HIV particles into CD4+ human cells. In contrast, the binding of virus was only partially inhibited by neutralizing anti-CD4 MAbs against the gp120 binding site in CD4, which, like the anti-V3 loop MAbs, completely inhibited HIV entry and infection. Nonneutralizing control MAbs against either the V3 loop or the N or C terminus of gp120 had no significant effect on HIV binding and entry. HIV-1 particles were also found to bind human and murine cells expressing or not expressing the human CD4 molecule. Interestingly, the binding of HIV to CD4+ murine cells was inhibited by both anti-V3 and anti-CD4 MAbs, whereas the binding to human and murine CD4- cells was affected only by anti-V3 loop MAbs. The effect of anti-V3 loop neutralizing MAbs on the HIV binding to cells appears not to be the direct consequence of gp120 shedding from HIV particles or of a decreased affinity of CD4 or gp120 for binding to its surface counterpart. Taken together, our results suggest the existence of CD4-dependent and -independent binding events involved in the attachment of HIV particles to cells; in both of these events, the V3 loop plays a critical role. As murine cells lack the specific cofactor CXCR4 for HIV-1 entry, other cell surface molecules besides CD4 might be implicated in stable binding of HIV particles to cells.     

V3 loop truncations in HIV-1 envelope impart resistance to coreceptor inhibitors and enhanced sensitivity to neutralizing antibodies

The V1/V2 region and the V3 loop of the human immunodeficiency virus type I (HIV-1) envelope (Env) protein are targets for neutralizing antibodies and also play an important functional role, with the V3 loop largely determining whether a virus uses CCR5 (R5), CXCR4 (X4), or either coreceptor (R5X4) to infect cells. While the sequence of V3 is variable, its length is highly conserved. Structural studies indicate that V3 length may be important for interactions with the extracellular loops of the coreceptor. Consistent with this view, genetic truncation of the V3 loop is typically associated with loss of Env function. We removed approximately one-half of the V3 loop from three different HIV-1 strains, and found that only the Env protein from the R5X4 strain R3A retained some fusion activity. Loss of V1/V2 (DeltaV1/V2) was well tolerated by this virus. Passaging of virus with the truncated V3 loop resulted in the derivation of a virus strain that replicated with wild-type kinetics. This virus, termed TA1, retained the V3 loop truncation and acquired several adaptive changes in gp120 and gp41. TA1 could use CCR5 but not CXCR4 to infect cells, and was extremely sensitive to neutralization by HIV-1 positive human sera, and by antibodies to the CD4 binding site and to CD4-induced epitopes in the bridging sheet region of gp120. In addition, TA1 was completely resistant to CCR5 inhibitors, and was more dependent upon the N-terminal domain of CCR5, a region of the receptor that is thought to contact the bridging sheet of gp120 and the base of the V3 loop, and whose conformation may not be greatly affected by CCR5 inhibitors. These studies suggest that the V3 loop protects HIV from neutralization by antibodies prevalent in infected humans, that CCR5 inhibitors likely act by disrupting interactions between the V3 loop and the coreceptor, and that altered use of CCR5 by HIV-1 associated with increased sensitivity to changes in the N-terminal domain can be linked to high levels of resistance to these antiviral compounds.     

Identification of the principal neutralizing determinant of human immunodeficiency virus type 1 as a fusion domain.

The V3 loop, located near the middle of the surface envelope glycoprotein gp120, is the major neutralizing domain of human immunodeficiency virus type 1 (HIV-1). Although the majority of the V3 loop is highly variable between different strains of HIV-1, a Gly-Pro-Gly-Arg motif at the tip of the loop is highly conserved. To determine whether this region plays a role in fusion mediated by the HIV-1 envelope glycoproteins, we introduced seven single-amino-acid changes in the V3 loop. The mutant envelope glycoproteins were expressed from an HIV-1 envelope expression vector and analyzed for their ability to induce cell fusion in the absence of virus replication. Our results indicated that single-amino-acid changes in the V3 loop were capable of completely abolishing or greatly reducing the ability of the HIV-1 envelope glycoproteins to induce cell fusion, thereby identifying the V3 loop as a fusion domain of HIV-1. Mutations in the highly conserved tip of the loop or in a nonconserved region flanking the highly conserved tip had no effect on envelope glycoprotein synthesis, processing, transport, or binding to the CD4 receptor molecule. Mutation of the putative disulfide bridge-forming Cys at residue 336 blocked gp160 cleavage and CD4 binding.     

Evidence for similar recognition of the conserved neutralization epitopes of human immunodeficiency virus type 1 envelope gp120 in humans and macaques

We compared the immune responses to the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins in humans and macaques with the use of clade A and clade B isogenic V3 loop glycan-possessing and -deficient viruses. We found that the presence or absence of the V3 loop glycan affects to similar extents immune recognition by a panel of anti-HIV human and anti-simian/human immunodeficiency virus (anti-SHIV) macaque sera. All sera tested neutralized the glycan-deficient viruses, in which the conserved CD4BS and CD4i epitopes are more exposed, better than the glycan-containing viruses. The titer of broadly neutralizing antibodies appears to be higher in the sera of macaques infected with glycan-deficient viruses. Collectively, our data add legitimacy to the use of SHIV-macaque models for testing the efficacy of HIV-1 Env-based immunogens. Furthermore, they suggest that antibodies to the CD4BS and CD4i sites of gp120 are prevalent in human and macaque sera and that the use of immunogens in which these conserved neutralizing epitopes are more exposed is likely to increase their immunogenicity.     

Design, synthesis, and biological evaluation of chicoric acid analogs as inhibitors of HIV-1 integrase

A series of analogs of the potent HIV-1 integrase (HIV IN) inhibitor chicoric acid (CA) was designed with the intention of ameliorating some of the parent natural product's undesirable properties, in particular its toxicity, instability, and poor membrane permeability. More than 70 analogs were synthesized and assayed for three types of activity: (1) the ability to inhibit 3'-end processing and strand transfer reactions using recombinant HIV IN in vitro, (2) toxicity against the CD4+ lymphoblastoid cell line, MT2, and (3) anti-HIV activity against HIV(LAI). CA analogs lacking one of the carboxyl groups of CA and with 3,4,5-trihydroxycinnamoyl sidechains in place of the caffeoyl group of CA exhibited the most potent inhibition of HIV replication and end-processing activity. Galloyl-substituted derivatives also displayed very potent in vitro and in vivo activities, in most cases exceeding the inhibitory effects of CA itself. Conversely, analogous monocarboxy caffeoyl analogs exhibited only modest inhibition, while the corresponding 3,4-dihydroxybenzoyl-substituted compounds were devoid of activity.     

Interplay between HIV-1 infection and host microRNAs

Using microRNA array analyses of in vitro HIV-1-infected CD4(+) cells, we find that several host microRNAs are significantly up- or downregulated around the time HIV-1 infection peaks in vitro. While microRNA-223 levels were significantly enriched in HIV-1-infected CD4(+)CD8(-) PBMCs, microRNA-29a/b, microRNA-155 and microRNA-21 levels were significantly reduced. Based on the potential for microRNA binding sites in a conserved sequence of the Nef-3'-LTR, several host microRNAs potentially could affect HIV-1 gene expression. Among those microRNAs, the microRNA-29 family has seed complementarity in the HIV-1 3'-UTR, but the potential suppressive effect of microRNA-29 on HIV-1 is severely blocked by the secondary structure of the target region. Our data support a possible regulatory circuit at the peak of HIV-1 replication which involves downregulation of microRNA-29, expression of Nef, the apoptosis of host CD4 cells and upregulation of microRNA-223.     

Determinants of CD4 independence for a human immunodeficiency virus type 1 variant map outside regions required for coreceptor specificity

Although infection by human immunodeficiency virus (HIV) typically requires an interaction between the viral envelope glycoprotein (Env), CD4, and a chemokine receptor, CD4-independent isolates of HIV and simian immunodeficiency virus have been described. The structural basis and underlying mechanisms for this phenotype are unknown. We have derived a variant of HIV-1/IIIB, termed IIIBx, that acquired the ability to utilize CXCR4 without CD4. This virus infected CD4-negative T and B cells and fused with murine 3T3 cells that expressed human CXCR4 alone. A functional IIIBx env clone exhibited several mutations compared to the CD4-dependent HXBc2 env, including the striking loss of five glycosylation sites. By constructing env chimeras with HXBc2, the determinants for CD4 independence were shown to map outside the V1/V2 and V3 hypervariable loops, which determine chemokine receptor specificity, and at least partly within an area on the gp120 core that has been implicated in forming a conserved chemokine receptor binding site. We also identified a point mutation in the C4 domain that could render the IIIBx env clone completely CD4 dependent. Mutations in the transmembrane protein (TM) were also required for CD4 independence. Remarkably, when the V3 loop of a CCR5-tropic Env was substituted for the IIIBx Env, the resulting chimera was found to utilize CCR5 but remained CD4 independent. These findings show that Env determinants for chemokine receptor specificity are distinct from those that mediate CD4-independent use of that receptor for cell fusion and provide functional evidence for multiple steps in the interaction of Env with chemokine receptors. Combined with our observation that the conserved chemokine receptor binding site on gp120 is more exposed on the IIIBx gp120 (T. L. Hoffman, C. C. LaBranche, W. Zhang, G. Canziani, J. Robinson, I. Chaiken, J. A. Hoxie, and R. W. Doms, Proc. Natl. Acad. Sci. USA 96:6359-6364, 1999), the findings from this study suggest novel approaches to derive and design Envs with exposed chemokine receptor binding sites for vaccine purposes.     

Polyvalent interactions of HIV-gp120 protein and nanostructures of carbohydrate ligands

This paper presents the initial effort in anti-HIV infection using glycosphingolipid-based nanostructures. HIV infection of CD4 negative cells is initiated by the binding of the viral envelope glycoprotein gp120 to galactosylceramide (GalCer), a glycosphingolipid that serves as the cellular receptor for viral recognition. A series of nanostructures of GalCer are designed and produced using an AFM-based lithography method known as nanografting. The geometry dependence of recombinant gp120 binding to these nanostructures is monitored using high-resolution AFM imaging. Gp120 molecules are found to favor binding sites that allow for polyvalent interactions. Increased adsorption at the intersection of two lines, or between two parallel lines with matching separation for trimeric binding, strongly suggests that trivalent interactions are dominant in gp120-GalCer nanostructure interactions. Systematic distance-dependence studies, using parallel nanolines with various separations, reveal a separation of 4.8 nm, matching the separation of V3 loops in gp120 trimers. This investigation demonstrates that nanotechnology provides a powerful tool for investigating and guiding polyvalent interactions among biological systems.     

The N-terminal V3 loop glycan modulates the interaction of clade A and B human immunodeficiency virus type 1 envelopes with CD4 and chemokine receptors

We investigated the underlying mechanism by which the highly conserved N-terminal V3 loop glycan of gp120 conferred resistance to neutralization of human immunodeficiency virus type 1 (HIV-1). We find that the presence or absence of this V3 glycan on clade A and B viruses accorded various degrees of susceptibility to neutralization by antibodies to the CD4 binding site, CD4-induced epitopes, and chemokine receptors. Our data suggest that this carbohydrate moiety on gp120 blocks access to the binding site for CD4 and modulates the chemokine receptor binding site of phenotypically diverse clade A and clade B isolates. Its presence also contributes to the masking of CD4-induced epitopes on clade B envelopes. These findings reveal a common mechanism by which diverse HIV-1 isolates escape immune recognition. Furthermore, the observation that conserved functional epitopes of HIV-1 are more exposed on V3 glycan-deficient envelope glycoproteins provides a basis for exploring the use of these envelopes as vaccine components.     

Enhanced in vitro human immunodeficiency virus type 1 replication in B cells expressing surface antibody to the TM Env protein.

The human immunodeficiency virus type 1 (HIV-1) external envelope glycoprotein gp120 tightly binds CD4 as its principal cellular receptor, explaining the tropism of HIV-1 for CD4+ cells. Nevertheless, reports documenting HIV infection or HIV binding in cells lacking CD4 surface expression have raised the possibility that cellular receptors in addition to CD4 may interact with HIV envelope. Moreover, the lymphocyte adhesion molecule LFA-1 appears to play an important role in augmenting HIV-1 viral spread and cytopathicity in vitro, although the mechanism of this function is still not completely defined. In the course of characterizing a human anti-HIV gp41 monoclonal antibody, we transfected a CD4-negative, LFA-1-negative B-cell line to express an anti-gp41 immunoglobulin receptor (surface immunoglobulin [sIg]/gp41). Despite acquiring the ability to bind HIV envelope, such transfected B cells could not be infected by HIV-1. These cells were not intrinsically defective for supporting HIV-1 infection, because when directed to produce surface CD4 by using retroviral constructs, they acquired the ability to replicate HIV-1. Interestingly, transfected cells expressing both surface CD4 and sIg/gp41 receptors replicated HIV much better than cells expressing only CD4. The enhancement resided specifically in sIg/gp41, because isotype-specific, anti-IgG1 antibodies directed against sIg/gp41 blocked the enhancement. These data directly establish the ability of a cell surface anti-gp41 receptor to enhance HIV-1 replication.     

A single injection of recombinant measles virus vaccines expressing human immunodeficiency virus (HIV) type 1 clade B envelope glycoproteins induces neutralizing antibodies and cellular immune responses to HIV

The anchored and secreted forms of the human immunodeficiency virus type 1 (HIV-1) 89.6 envelope glycoprotein, either complete or after deletion of the V3 loop, were expressed in a cloned attenuated measles virus (MV) vector. The recombinant viruses grew as efficiently as the parental virus and expressed high levels of the HIV protein. Expression was stable during serial passages. The immunogenicity of these recombinant vectors was tested in mice susceptible to MV and in macaques. High titers of antibodies to both MV and HIV-Env were obtained after a single injection in susceptible mice. These antibodies neutralized homologous SHIV89.6p virus, as well as several heterologous HIV-1 primary isolates. A gp160 mutant in which the V3 loop was deleted induced antibodies that neutralized heterologous viruses more efficiently than antibodies induced by the native envelope protein. A high level of CD8+ and CD4+ cells specific for HIV gp120 was also detected in MV-susceptible mice. Furthermore, recombinant MV was able to raise immune responses against HIV in mice and macaques with a preexisting anti-MV immunity. Therefore, recombinant MV vaccines inducing anti-HIV neutralizing antibodies and specific T lymphocytes responses deserve to be tested as a candidate AIDS vaccine.     

193 Computer-aided design of novel HIV-1 entry inhibitors based on glycosphingolipids

Novel HIV-1 entry inhibitors targeting the envelope gp120 V3 loop were designed by computer modeling based on glycosphingolipid β-galactosylceramide (β-GalCer) forming on the surface of some susceptible host cells the primary receptor for HIV-1 alternative to CD4, which is used by the virus to enter macrophages and T-lymphocytes (e.g. Fantini et al., 1993). To achieve this goal, 3D structures of the twelve water-soluble analogs of β-GalCer containing different substitutes of its fatty acid residue were determined by quantum chemical calculations and evaluation of their potential anti-HIV-1 activity was carried out by molecular docking, molecular dynamics, and free energy simulations. Analysis of the structural complexes of these β-GalCer derivatives with the HIV-1 V3 loops from the five diverse viral strains makes it clear that, in all of the cases of interest, the third variable domain of gp120 forms two potential binding sites for glycolipids concerning the immunogenic tip and the base of V3. At the same time, nonconventional XH … π hydrogen bonds between XH sugar groups (X designates C or O) and overlapping π-orbitals of the conserved Phe-20, Tyr-21, and His-34 residues of the V3 loop were shown to play a key role in specific binding of the designed glycosphingolipids to the above conserved structural motifs of V3 that include residues critical for cell tropism (Andrianov et al., 2011; Andrianov, Kornoushenko, et al., 2012). These findings testifying to the ability of the simulated chemicals to specifically and effectively interact with the functionally important sites of V3 were confirmed by those on molecular dynamics and calculating the free energy of formation of the complexes for these β-GalCer analogs with the HIV-1 V3 loops from different viral modifications. Finally, the majority of the designed molecules were found to form much more stable complexes with V3, compared to that of the β-GalCer-based anti-HIV-1 agent developed previously (Andrianov, Anishchenko, et al., 2012) and used in the calculations as a control. In the light of the data obtained, these potential HIV-1 entry inhibitors present the promising basic compounds for the development of novel, potent, and broad antiviral drugs.     

Importance of V3 Loop Flexibility and Net Charge in the Context of Co-Receptor Recognition. A Molecular Dynamics Study on HIV gp120

The entry of HIV-1 into a host cell requires the interaction of envelope glycoprotein gp120 with CD4 receptor as well as a co-receptor, which can be either CCR5 or CXCR4. The third variable loop (V3) of HIV-1 gp120 plays an important role in co-receptor selection (CCR5 or CXCR4) and also acts as an epitope for neutralizing antibodies against gp120. Here we have performed long time molecular dynamics simulations of two gp120 structures that are representatives of a R5 and X4 strains in the CD4-free and CD4-bound states. The results indicate some conserved features in both systems, such as the rigidity of the gp120 core, the conservation of the CD4 Phe43-gp120 binding cavity contacts, a high flexibility of the V3 loop particularly in the CD4 bound form. Analysis of the distribution of V3 loop's net charge shows it to be more positive for the gp120 sequences selecting CXCR4 co-receptor, letting us to propose that V3 loop net charge and flexibility are the two main elements in the co-receptor selection.     

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.     

Matrix fibronectin increases HIV stability and infectivity

HIV particles are detected extracellularly in lymphoid tissues, a major reservoir of the virus. We previously reported that a polymerized form of fibronectin (FN), superfibronectin (sFN), as well as a fragment of FN, III1-C, enhanced infection of primary CD4(+) T cells by HIV-1IIIB. We now show that sFN enhances infection of primary CD4(+) T cells by both R5 and X4 strains of HIV-1. Using HIV pseudotyped with different envelope glycoproteins (gp120) and HOS cells transfected with various chemokine receptors alone or in combination with the CD4 molecule, we show that sFN-mediated enhancement requires the CD4 receptor and does not alter the specificity of gp120 for different chemokine receptors. Because the III1-C fragment also resulted in enhancement, we asked whether proteolysis of FN generated fragments capable of enhancing HIV infection. We found that progressive proteolysis of FN by chymotrypsin correlates with an enhancement of HIV infection in both primary CD4(+) T cells and the IG5 reporter cell line. Furthermore, incubation of HIV with sFN significantly prolonged infectivity at 37 degrees C compared with dimeric FN or BSA. In conclusion, these results indicate that polymerized (matrix) or degraded (inflammation-associated), but not dimeric (plasma), FN are capable of enhancing infection by HIV-1, independent of the coreceptor specificity of the strains. Moreover, virions bound to matrix FN maintain infectivity for longer periods of time than do virions in suspension. This study suggests that matrix proteins and their conformational status may play a role in the pathogenesis of HIV.