Surfactant protein A binds to HIV and inhibits direct infection of CD4+ cells, but enhances dendritic cell-mediated viral transfer

The identification of surfactant protein A (SP-A) as an important innate immune factor of the lungs, amniotic fluid, and the vaginal tract suggests that it could play an important role during various stages of HIV disease progression and transmission. Therefore, we examined whether SP-A could bind to HIV and also had any effect on viral infectivity. Our data demonstrate that SP-A binds to HIV in a calcium-dependent manner that is inhibitable by mannose and EDTA. Affinity capture of the HIV viral lysate reveals that SP-A targets the envelope glycoprotein of HIV (gp120), which was confirmed by ELISA using recombinant gp120. Digestion of gp120 with endoglycosidase H abrogates the binding of SP-A, indicating that the high mannose structures on gp120 are the target of the collectin. Infectivity studies reveal that SP-A inhibits the infection of CD4+ T cells by two strains of HIV (BaL, IIIB) by >80%. Competition assays with CD4 and mAbs F105 and b12 suggest that SP-A inhibits infectivity by occlusion of the CD4-binding site. Studies with dendritic cells (DCs) demonstrate that SP-A enhances the binding of gp120 to DCs, the uptake of viral particles, and the transfer of virus from DCs to CD4+ T cells by >5-fold at a pH representative of the vaginal tract. Collectively, these results suggest that SP-A acts as a dual modulator of HIV infection by protecting CD4+ T cells from direct infection but enhancing the transfer of infection to CD4+ T cells mediated by DCs.     

Functional Mimetics of the HIV-1 CCR5 Co-Receptor Displayed on the Surface of Magnetic Liposomes

Chemokine G protein coupled receptors, principally CCR5 or CXCR4, function as co-receptors for HIV-1 entry into CD4⁺ T cells. Initial binding of the viral envelope glycoprotein (Env) gp120 subunit to the host CD4 receptor induces a cascade of structural conformational changes that lead to the formation of a high-affinity co-receptor-binding site on gp120. Interaction between gp120 and the co-receptor leads to the exposure of epitopes on the viral gp41 that mediates fusion between viral and cell membranes. Soluble CD4 (sCD4) mimetics can act as an activation-based inhibitor of HIV-1 entry in vitro, as it induces similar structural changes in gp120, leading to increased virus infectivity in the short term but to virus Env inactivation in the long term. Despite promising clinical implications, sCD4 displays low efficiency in vivo, and in multiple HIV strains, it does not inhibit viral infection. This has been attributed to the slow kinetics of the sCD4-induced HIV Env inactivation and to the failure to obtain sufficient sCD4 mimetic levels in the serum. Here we present uniquely structured CCR5 co-receptor mimetics. We hypothesized that such mimetics will enhance sCD4-induced HIV Env inactivation and inhibition of HIV entry. Co-receptor mimetics were derived from CCR5 gp120-binding epitopes and functionalized with a palmitoyl group, which mediated their display on the surface of lipid-coated magnetic beads. CCR5-peptidoliposome mimetics bound to soluble gp120 and inhibited HIV-1 infectivity in a sCD4-dependent manner. We concluded that CCR5-peptidoliposomes increase the efficiency of sCD4 to inhibit HIV infection by acting as bait for sCD4-primed virus, catalyzing the premature discharge of its fusion potential.     

Surfactant Protein D Inhibits HIV-1 Infection of Target Cells via Interference with gp120-CD4 Interaction and Modulates Pro-Inflammatory Cytokine Production

Surfactant Protein SP-D, a member of the collectin family, is a pattern recognition protein, secreted by mucosal epithelial cells and has an important role in innate immunity against various pathogens. In this study, we confirm that native human SP-D and a recombinant fragment of human SP-D (rhSP-D) bind to gp120 of HIV-1 and significantly inhibit viral replication in vitro in a calcium and dose-dependent manner. We show, for the first time, that SP-D and rhSP-D act as potent inhibitors of HIV-1 entry in to target cells and block the interaction between CD4 and gp120 in a dose-dependent manner. The rhSP-D-mediated inhibition of viral replication was examined using three clinical isolates of HIV-1 and three target cells: Jurkat T cells, U937 monocytic cells and PBMCs. HIV-1 induced cytokine storm in the three target cells was significantly suppressed by rhSP-D. Phosphorylation of key kinases p38, Erk1/2 and AKT, which contribute to HIV-1 induced immune activation, was significantly reduced in vitro in the presence of rhSP-D. Notably, anti-HIV-1 activity of rhSP-D was retained in the presence of biological fluids such as cervico-vaginal lavage and seminal plasma. Our study illustrates the multi-faceted role of human SP-D against HIV-1 and potential of rhSP-D for immunotherapy to inhibit viral entry and immune activation in acute HIV infection.     

Concanavalin A binding to HIV envelope protein is less sensitive to mutations in glycosylation sites than monoclonal antibody 2G12

Many mannose-binding proteins inhibit divergent strains of human immunodeficiency virus type 1 (HIV-1) in in vitro models of viral infectivity, suggesting that targeting mannose residues in vaccine applications might offset the strain restriction typically observed in antibody responses to HIV vaccine preparations. Concanavalin A (ConA) behaves like neutralizing antibodies that do not interfere with CD4 binding of gp120 but rather with later events in virus entry. The design of mannose-based vaccines, therefore, depends on understanding the mode of binding of ConA to the envelope protein in comparison with other mannose-binding proteins. Here, we further compare the binding affinity and fine specificity of ConA for the envelope protein to that of the human antibody 2G12. The 2G12 antibody is of unusual structure recognizing a cluster of 12 linked mannose residues associated with Man9GlcNAc2. Molecular structure comparison for Man9GlcNAc2 recognition by ConA and 2G12 indicates that 2G12 has a more restricted specificity to high mannose glycans of gp120 which correlates with kinetic analysis assessed by surface plasmon resonance (SPR) and ConA inhibits 2G12 binding to gp120 but 2G12 does not inhibit ConA binding to gp120. ConA binding to Env proteins from four different HIV strains proves significantly less sensitive to mutations in the glycosylation sites than 2G12 binding to the proteins. Thus, antibodies directed toward mannose epitopes reactive with ConA may prove to be more effective in the long run to thwart HIV infection and transmission.     

Differences in the mannose oligomer specificities of the closely related lectins from Galanthus nivalis and Zea mays strongly determine their eventual anti-HIV activity

Background

In a recent report, the carbohydrate-binding specificities of the plant lectins Galanthus nivalis (GNA) and the closely related lectin from Zea mays (GNA_maize) were determined by glycan array analysis and indicated that GNA_maize recognizes complex-type N-glycans whereas GNA has specificity towards high-mannose-type glycans. Both lectins are tetrameric proteins sharing 64% sequence similarity.

Results

GNA_maize appeared to be ~20- to 100-fold less inhibitory than GNA against HIV infection, syncytia formation between persistently HIV-1-infected HuT-78 cells and uninfected CD4⁺ T-lymphocyte SupT1 cells, HIV-1 capture by DC-SIGN and subsequent transmission of DC-SIGN-captured virions to uninfected CD4⁺ T-lymphocyte cells. In contrast to GNA, which preferentially selects for virus strains with deleted high-mannose-type glycans on gp120, prolonged exposure of HIV-1 to dose-escalating concentrations of GNA_maize selected for mutant virus strains in which one complex-type glycan of gp120 was deleted. Surface Plasmon Resonance (SPR) analysis revealed that GNA and GNA_maize interact with HIV III_B gp120 with affinity constants (K_D) of 0.33 nM and 34 nM, respectively. Whereas immobilized GNA specifically binds mannose oligomers, GNA_maize selectively binds complex-type GlcNAcβ1,2Man oligomers. Also, epitope mapping experiments revealed that GNA and the mannose-specific mAb 2G12 can independently bind from GNA_maize to gp120, whereas GNA_maize cannot efficiently bind to gp120 that contained prebound PHA-E (GlcNAcβ1,2man specific) or SNA (NeuAcα2,6X specific).

Conclusion

The markedly reduced anti-HIV activity of GNA_maize compared to GNA can be explained by the profound shift in glycan recognition and the disappearance of carbohydrate-binding sites in GNA_maize that have high affinity for mannose oligomers. These findings underscore the need for mannose oligomer recognition of therapeutics to be endowed with anti-HIV activity and that mannose, but not complex-type glycan binding of chemotherapeutics to gp120, may result in a pronounced neutralizing activity against the virus.     

Selection, characterization and application of new RNA HIV gp 120 aptamers for facile delivery of Dicer substrate siRNAs into HIV infected cells

The envelope glycoprotein of human immunodeficiency virus (HIV) consists of an exterior glycoprotein (gp120) and a trans-membrane domain (gp41) and has an important role in viral entry into cells. HIV-1 entry has been validated as a clinically relevant anti-viral strategy for drug discovery. In the present work, several 2′-F substituted RNA aptamers that bind to the HIV-1_BaL gp120 protein with nanomole affinity were isolated from a RNA library by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure. From two of these aptamers we created a series of new dual inhibitory function anti-gp120 aptamer–siRNA chimeras. The aptamers and aptamer–siRNA chimeras specifically bind to and are internalized into cells expressing HIV gp160. The Dicer-substrate siRNA delivered by the aptamers is functionally processed by Dicer, resulting in specific inhibition of HIV-1 replication and infectivity in cultured CEM T-cells and primary blood mononuclear cells (PBMCs). Moreover, we have introduced a ‘sticky’ sequence onto a chemically synthesized aptamer which facilitates attachment of the Dicer substrate siRNAs for potential multiplexing. Our results provide a set of novel inhibitory agents for blocking HIV replication and further validate the use of aptamers for delivery of Dicer substrate siRNAs.     

Cooperative anti-influenza activities of respiratory innate immune proteins and neuraminidase inhibitor

The surfactant collectins, surfactant proteins A and D (SP-A and D), and scavenger receptor-rich glycoprotein 340 (gp340) inhibit influenza A virus (IAV) in the following order of potency: SP-D>gp340>SP-A. SP-D binds in a calcium-dependent manner to carbohydrate attachments on the viral hemagglutinin (HA) and neuraminidase (NA). By contrast, gp340 and SP-A act like mucins in that they provide sialic acid ligands that bind to the viral HA. In this study, SP-D, SP-A, and gp340 showed cooperative antiviral interactions. These cooperative effects were most evident in viral aggregation but were also observed in at least some hemagglutination inhibition and viral neutralization assays. The mechanism of binding between gp340 and SP-D was further characterized using monoclonal antibodies. Although gp340 can bind to SP-D at a site distinct from the mannan-binding site, binding of gp340 to SP-D did not contribute to cooperative antiviral interactions. SP-D and mucin showed cooperative interactions, apparently dependent on NA inhibition by SP-D. The commercial NA inhibitor oseltamivir had a similar effect and also enhanced the neutralizing activity of SP-A and bronchoalveolar lavage fluid. Hence, oseltamivir collaborates with innate immune proteins in inhibiting the initial infection of epithelial cells.     

Human glutaredoxin-1 catalyzes the reduction of HIV-1 gp120 and CD4 disulfides and its inhibition reduces HIV-1 replication

Reduction of intramolecular disulfides in the HIV-1 envelope protein gp120 occurs after its binding to the CD4 receptor. Protein disulfide isomerase (PDI) catalyzes the disulfide reduction in vitro and inhibition of this enzyme blocks viral entry. PDI belongs to the thioredoxin protein superfamily that also includes human glutaredoxin-1 (Grx1). Grx1 is secreted from cells and the protein has also been found within the HIV-1 virion. We show that Grx1 efficiently catalyzes gp120, and CD4 disulfide reduction in vitro, even at low plasma levels of glutathione. Grx1 catalyzes the reduction of two disulfide bridges in gp120 in a similar manner as PDI. Purified anti-Grx1 antibodies were shown to inhibit the Grx1 activity in vitro and block HIV-1 replication in cultured peripheral blood mononuclear cells. Also, the polyanion PRO2000, that was previously shown to prevent HIV entry, inhibits the Grx1- and PDI-dependent reduction of gp120 disulfides. Our findings suggest that Grx1 activity is important for HIV-1 entry and that Grx1 and the gp120 intramolecular disulfides are novel pharmacological targets for rational drug development.     

Trypsin-resistant gp120 receptors are upregulated on short-term cultured human epidermal Langerhans cells

The CD4 molecule is known to be the preferential receptor for the HIV1 envelope glycoprotein. Epidermal Langerhans cells (LC) are dendritic cells which express several surface antigens, among them the CD4 antigens. LC infection was suggested when these cells were seen to present buddings coincident with membrane thickening of roughly 100 nm in size. These buddings were similar in ultrastructural aspect to HIV buddings on in vitro infected promonocytic cells (U937). To clarify the exact role of CD4 molecules in LC infection induced by HIV1, we investigated the possible involvement of the interactions between native and recombinant HIV1 gp 120 and the LC surface. We also assessed the expression of CD4 molecules on LC membranes dissociated by means of trypsin from their neighbouring keratinocytes. The cellular phenotype was monitored using flow cytometry. We show that human LC can bind the viral envelope protein and that this binding does not depend on CD4 protein expression. The amount of surface bound gp120 was not consistent with the amount of CD4 antigens present on LC membranes. The gp 120-binding sites on LC in suspension appear to be trypsin-resistant while the CD4 antigens (at least the epitopes known to bind HIV1) are trypsin-sensitive. A burst of gp 120 receptor expression was detected on 1-day cultured LC while the CD4 antigens disappeared. These findings lead to the logical conclusion that the binding of gp 120 is due to the presence of a LC surface molecule which is different from CD4 antigens.     

Peptide Ligands to Human Immunodeficiency Virus Type 1 gp120 Identified from Phage Display Libraries

We have used phage-displayed peptide libraries to identify novel ligands to the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein gp120. Screening of libraries of random 12-mers, 7-mers, and cyclic 9-mers produced two families of gp120 binding peptides. Members of a family with the prototype sequence RINNIPWSEAMM (peptide 12p1) inhibit the interaction between gp120 and both four-domain soluble CD4 (4dCD4) and monoclonal antibody (MAb) 17b, a neutralizing antibody that covers the chemokine receptor binding surface on gp120. Peptide 12p1 inhibits the interaction of 4dCD4 with gp120 from three different HIV strains, implying that it binds to a conserved site on gp120. Members of a second family of peptides, with the prototype sequence TSPYEDWQTYLM (peptide 12p2), bind more weakly to gp120. They do not detectably affect its interaction with 4dCD4, but they enhance its binding to MAb 17b. A common sequence motif in the two peptide families and cross-competition for gp120 binding suggest that they have overlapping contacts. Their divergent effects on the affinity of gp120 for MAb 17b may indicate that their binding stabilizes distinct conformational states of gp120. The functional properties of 12p1 suggest that it might be a useful lead for the development of inhibitors of HIV entry.     

The dendritic cell-specific C-type lectin DC-SIGN is a receptor for Schistosoma mansoni egg antigens and recognizes the glycan antigen Lewis x

Schistosoma mansoni soluble egg antigens (SEAs) are crucially involved in modulating the host immune response to infection by S. mansoni. We report that human dendritic cells bind SEAs through the C-type lectin dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN). Monoclonal antibodies against the carbohydrate antigens Lewisx (Lex) and GalNAcbeta1-4(Fucalpha1-3)GlcNAc (LDNF) inhibit binding of DC-SIGN to SEAs, suggesting that these glycan antigens may be critically involved in binding. In a solid-phase adhesion assay, DC-SIGN-Fc binds polyvalent neoglycoconjugates that contain the Lex antigen, whereas no binding was observed to Galbeta1-4GlcNAc, and binding to neoglycoconjugates containing only alpha-fucose or oligosaccharides with a terminal alpha1-2-linked fucose is low. These data indicate that binding of DC-SIGN to Lex antigen is fucose-dependent and that adjacent monosaccharides and/or the anomeric linkage of the fucose are important for binding activity. Previous studies have shown that DC-SIGN binds HIV gp120 that contains high-mannose-type N-glycans. Site-directed mutagenesis within the carbohydrate recognition domain (CRD) of DC-SIGN demonstrates that amino acids E324 and E347 are involved in binding to HIV gp120, Lex, and SEAs. By contrast, mutation of amino acid Val351 abrogates binding to SEAs and Lex but not HIV gp120. These data suggest that DC-SIGN recognizes these ligands through different (but overlapping) regions within its CRD. Our data imply that DC-SIGN not only is a pathogen receptor for HIV gp120 but may also function in pathogen recognition by interaction with the carbohydrate antigens Lex and possibly LDNF, which are found on important human pathogens, such as schistosomes and the bacterium Helicobacter pylori.     

Human immunodeficiency virus envelope (gp120) binding to DC-SIGN and primary dendritic cells is carbohydrate dependent but does not involve 2G12 or cyanovirin binding sites: implications for structural analyses of gp120-DC-SIGN binding

The calcium-dependent lectin, DC-SIGN, binds to human immunodeficiency virus (HIV) (and simian immunodeficiency virus) gp120 and mediates the binding and transfer of HIV from monocyte-derived dendritic cells (MDDCs) to permissive T cells. However, it has been recently reported that DC-SIGN binding to HIV gp120 may be carbohydrate independent. Here, we formally demonstrate that gp120 binding to DC-SIGN and MDDCs is largely if not wholly carbohydrate dependent. Endo-beta-N-glucosaminidase H (EndoH) treatment of gp120-Fc under conditions that maintained wild-type CD4 binding-and the full complement of complex glycans-significantly decreased (>90%) binding to DC-SIGN expressing cell lines, as well as to MDDCs. Any residual binding of EndoH-treated gp120-Fc to DC-SIGN was completely competed off with mannan. Mutational analysis indicated that no single glycosylation site affected the ability of gp120-Fc to bind DC-SIGN. To further guide our efforts in mapping the DC-SIGN binding sites on gp120, we used two well-characterized HIV inhibitory agents (2G12 monoclonal antibody and cyanovirin) that bind to high-mannose sugars on gp120. We showed that 2G12 and DC-SIGN bound to nonoverlapping sites in gp120 because (i) 2G12 did not block soluble gp120 or virion binding to DC-SIGN, (ii) 2G12 bound to gp120-Fc that was prebound to cell surface DC-SIGN, and (iii) gp120-Fc mutants that lack glycosylation sites involved in 2G12's epitope were also fully capable of binding DC-SIGN. These data were substantiated by the inability of cyanovirin to block gp120-Fc binding to DC-SIGN. Cyanovirin has been shown to effectively compete for 2G12 binding to gp120. Indeed, high concentrations of cyanovirin dramatically enhanced gp120-Fc binding to cell surfaces in the presence or absence of DC-SIGN. We provide evidence that this enhancement may be due to cyanovirin's ability to bridge gp120 to mannosylated cell surface proteins. These results have implications for antiviral therapeutics and for ongoing efforts to finely map the glycan structures on gp120 responsible for DC-SIGN binding.     

A Strategy for Eliciting Antibodies against Cryptic,Conserved, Conformationally Dependent Epitopes of HIV Envelope Glycoprotein

Background

Novel strategies are needed for the elicitation of broadly neutralizing antibodies to the HIV envelope glycoprotein, gp120. Experimental evidence suggests that combinations of antibodies that are broadly neutralizing in vitro may protect against challenge with HIV in nonhuman primates, and a small number of these antibodies have been selected by repertoire sampling of B cells and by the fractionation of antiserum from some patients with prolonged disease. Yet no additional strategies for identifying conserved epitopes, eliciting antibodies to these epitopes, and determining whether these epitopes are accessible to antibodies have been successful to date. The defining of additional conserved, accessible epitopes against which one can elicit antibodies will increase the probability that some may be the targets of broadly neutralizing antibodies.

Methodology/Principal Findings

We postulate that additional cryptic epitopes of gp120 are present, against which neutralizing antibodies might be elicited even though these antibodies are not elicited by gp120, and that many of these epitopes may be accessible to antibodies should they be formed. We demonstrate a strategy for eliciting antibodies in mice against selected cryptic, conformationally dependent conserved epitopes of gp120 by immunizing with multiple identical copies of covalently linked peptides (MCPs). This has been achieved with MCPs representing 3 different domains of gp120. We show that some cryptic epitopes on gp120 are accessible to the elicited antibodies, and some epitopes in the CD4 binding region are not accessible. The antibodies bind to gp120 with relatively high affinity, and bind to oligomeric gp120 on the surface of infected cells.

Conclusions/Significance

Immunization with MCPs comprised of selected peptides of HIV gp120 is able to elicit antibodies against conserved, conformationally dependent epitopes of gp120 that are not immunogenic when presented as gp120. Some of these cryptic epitopes are accessible to the elicited antibodies.     

Identification of the optimal DC-SIGN binding site on human immunodeficiency virus type 1 gp120

Human immunodeficiency virus type 1 (HIV-1) envelope (gp120) binding to DC-SIGN, a C-type lectin that can facilitate HIV infection in cis and in trans, is largely dependent on high-mannose-content moieties. Here, we delineate the N-linked glycosylation (N-glycan) sites in gp120 that contribute to optimal DC-SIGN binding. Soluble DC-SIGN was able to block 2G12 binding to gp120, but not vice versa, suggesting that DC-SIGN binds to a more flexible combination of N-glycans than 2G12. Consistent with this observation, HIV strain JRCSF gp120 prebound to 2G12 was 10-fold more sensitive to mannan competition than gp120 that was not prebound in a DC-SIGN cell surface binding assay. The analysis of multiple mutant forms of the 2G12 epitope revealed one triple glycosylation mutant form, termed 134mut (carrying N293Q, N382Q, and N388Q mutations), that exhibited a significant increase in sensitivity to both mannan competition and endoglycosidase H digestion compared to that of the 124mut form (carrying N293Q, N328Q, and N388Q mutations) and wild-type gp120 in a DC-SIGN binding assay. Importantly, no such differences were observed when binding to Galanthus nivalis was assessed. The 134mut form of gp120 also exhibited decreased binding to DC-SIGN in the context of native envelope spikes on a virion, and virus bearing 134mut exhibited less efficient DC-SIGN-mediated infection in trans. Significantly, 124mut and 134mut differed by only one glycosylation site mutation in each construct, and both 124mut and 134mut viruses exhibited wild-type levels of infectivity when used in a direct infection assay. In summary, while DC-SIGN can bind to a flexible combination of N-glycans on gp120, its optimal binding site overlaps with specific N-glycans within the 2G12 epitope. Conformationally intact envelopes that are DC-SIGN binding deficient can be used to probe the in vivo biological functions of DC-SIGN.     

A polyanion binding site on the CD4 molecule. Proximity to the HIV-gp120 binding region

Recent studies have demonstrated that sulfated polyanions (SP) are potent inhibitors of HIV infection in vitro, appearing to inhibit virus attachment. To understand the mode of action of these compounds a large panel of SP were examined for their ability to inhibit HIV infection, block anti-CD4 mAb binding and, when immobilized, bind soluble CD4 and virion gp120. Based on anti-CD4 mAb binding-inhibition studies a SP binding site was identified on the CD4 molecule. Dextran sulfate (DXS)-500 kDa, polyvinylsulfate (PVS), and polyanethole sulfonate were particularly potent SP inhibitors, blocking the binding of 11 of the 12 anti-CD4 mAb tested. These 11 mAb are known to interact with the two amino-terminal Ig-like domains of CD4. In fact, DXS-500 kDa exhibited an hierarchy of inhibition of anti-CD4 mAb which suggests that SP bind to a conformational site incorporating the first two Ig-like domains of CD4. This SP binding site is clearly distinct but closely associated with the gp120 binding region of CD4. In terms of anti-HIV activity there was no evidence that SP act at the virion level as rgp120 did not bind to immobilized SP and preincubation of virions with SP did not affect infectivity. In contrast, many of the SP tested showed some affinity for CD4 based on anti-CD4 mAb blocking studies and binding of soluble CD4 to immobilized SP. The most active in this regard were DXS-500 kDa and PVS, whose anti-HIV activity could be entirely due to disruption of the CD4-gp120 interaction. However, with SP such as heparin, fucoidan, the carrageenans, and polyanethole sulfonate, although CD4 blocking may contribute to anti-HIV activity, some other anti-viral mechanism is also operating. Finally, pentosan sulfate, a SP with anti-HIV activity comparable to DXS-500 kDa and PVS, showed little or no reactivity with CD4 and must inhibit HIV infection by a totally CD4-independent mechanism.     

An aptamer that neutralizes R5 strains of human immunodeficiency virus type 1 blocks gp120-CCR5 interaction

We recently described the isolation and structural characterization of 2'-fluoropyrimidine-substituted RNA aptamers that bind to gp120 of R5 strains of human immunodeficiency virus type 1 and thereby potently neutralize the infectivity of phylogenetically diverse R5 strains. Here we investigate the physical basis of their antiviral action. We show that both N-linked oligosaccharides and the variable loops V1/V2 and V3 are not required for binding of one aptamer, B40, to gp120. Using surface plasmon resonance binding analyses, we show that the aptamer binds to the CCR5-binding site on gp120 in a relatively CD4-independent manner, providing a mechanistic explanation for its neutralizing potency.     

A pH-sensitive heparin-binding sequence from Baculovirus gp64 protein is important for binding to mammalian cells but not to Sf9 insect cells

Binding to heparan sulfate is essential for baculovirus transduction of mammalian cells. Our previous study shows that gp64, the major glycoprotein on the virus surface, binds to heparin in a pH-dependent way, with a stronger binding at pH 6.2 than at 7.4. Using fluorescently labeled peptides, we mapped the pH-dependent heparin-binding sequence of gp64 to a 22-amino-acid region between residues 271 and 292. Binding of this region to the cell surface was also pH dependent, and peptides containing this sequence could efficiently inhibit baculovirus transduction of mammalian cells at pH 6.2. When the heparin-binding peptide was immobilized onto the bead surface to mimic the high local concentration of gp64 on the virus surface, the peptide-coated magnetic beads could efficiently pull down cells expressing heparan sulfate but not cells pretreated with heparinase or cells not expressing heparan sulfate. Interestingly, although this heparin-binding function is essential for baculovirus transduction of mammalian cells, it is dispensable for infection of Sf9 insect cells. Virus infectivity on Sf9 cells was not reduced by the presence of heparin or the identified heparin-binding peptide, even though the peptide could bind to Sf9 cell surface and be efficiently internalized. Thus, our data suggest that, depending on the availability of the target molecules on the cell surface, baculoviruses can use two different methods, electrostatic interaction with heparan sulfate and more specific receptor binding, for cell attachment.     

Fusion of a Short Peptide that Binds Immunoglobulin G to a Recombinant Protein Substantially Increases Its Plasma Half-Life in Mice

We explore a strategy to substantially increase the half-life of recombinant proteins by genetic fusion to FcIII, a 13-mer IgG-Fc domain binding peptide (IgGBP) originally identified by DeLano and co-workers at Genentech [DeLano WL, et al. (2000) Science 287∶1279–1283]. IgGBP fusion increases the in vivo half-life of proteins by enabling the fusion protein to bind serum IgG, a concept originally introduced by DeLano and co-workers in a patent but that to the best of our knowledge has never been pursued in the scientific literature. To further investigate the in vitro and in vivo properties of IgGBP fusion proteins, we fused FcIII to the C-terminus of a model fluorescent protein, monomeric Katushka (mKate). mKate-IgGBP fusions are easily expressed in Escherichia coli and bind specifically to human IgG with an affinity of ∼40 nM and ∼20 nM at pH 7.4 and pH 6, respectively, but not to mouse or rat IgG isotypes. mKate-IgGBP binds the Fc-domain of hIgG1 at a site overlapping the human neonatal Fc receptor (hFcRn) and as a consequence inhibits the binding of hIgG1 to hFcRn in vitro. High affinity binding to human IgG also endows mKate-IgGBP with a long circulation half-life of ∼8 hr in mice, a 75-fold increase compared to unmodified mKate. Thus, IgGBP fusion significantly reduces protein clearance by piggybacking on serum IgG without substantially increasing protein molecular weight due to the small size of the IgGBP. These attractive features could result in protein therapies with reduced dose frequency and improved patient compliance.     

Homology models of the HIV‐1 attachment inhibitor BMS‐626529 bound to gp120 suggest a unique mechanism of action

HIV‐1 gp120 undergoes multiple conformational changes both before and after binding to the host CD4 receptor. BMS‐626529 is an attachment inhibitor (AI) in clinical development (administered as prodrug BMS‐663068) that binds to HIV‐1 gp120. To investigate the mechanism of action of this new class of antiretroviral compounds, we constructed homology models of unliganded HIV‐1 gp120 (UNLIG), a pre‐CD4 binding‐intermediate conformation (pCD4), a CD4 bound‐intermediate conformation (bCD4), and a CD4/co‐receptor‐bound gp120 (LIG) from a series of partial structures. We also describe a simple pathway illustrating the transition between these four states. Guided by the positions of BMS‐626529 resistance substitutions and structure–activity relationship data for the AI series, putative binding sites for BMS‐626529 were identified, supported by biochemical and biophysical data. BMS‐626529 was docked into the UNLIG model and molecular dynamics simulations were used to demonstrate the thermodynamic stability of the different gp120 UNLIG/BMS‐626529 models. We propose that BMS‐626529 binds to the UNLIG conformation of gp120 within the structurally conserved outer domain, under the antiparallel β20–β21 sheet, and adjacent to the CD4 binding loop. Through this binding mode, BMS‐626529 can inhibit both CD4‐induced and CD4‐independent formation of the “open state” four‐stranded gp120 bridging sheet, and the subsequent formation and exposure of the chemokine co‐receptor binding site. This unique mechanism of action prevents the initial interaction of HIV‐1 with the host CD4+ T cell, and subsequent HIV‐1 binding and entry. Our findings clarify the novel mechanism of BMS‐626529, supporting its ongoing clinical development. Proteins 2015; 83:331–350. © 2014 Wiley Periodicals, Inc.