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Identification of a CD36-related thrombospondin 1-binding domain in HIV-1 envelope glycoprotein gp120: relationship to HIV-1-specific inhibitory factors in human saliva.

Crombie R, Silverstein RL, MacLow C, Pearce SF, Nachman RL, Laurence J - J. Exp. Med. (1998)

Bottom Line: In parallel, purified TSP1 inhibited HIV-1 infection of peripheral blood mononuclear cells and transformed T and promonocytic cell lines.Levels of TSP1 required for both viral aggregation and direct blockade of HIV-1 infection were physiologic, and affinity depletion of salivary TSP1 abrogated >70% of the inhibitory effect of whole saliva on HIV infectivity.Characterization of TSP1-gp120 binding specificity suggests a mechanism for direct blockade of HIV infectivity that might be exploited to retard HIV transmission that occurs via mucosal routes.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, The New York Hospital-Cornell Medical Center, New York 10021, USA.

ABSTRACT
Human and non-human primate salivas retard the infectivity of HIV-1 in vitro and in vivo. Because thrombospondin 1 (TSP1), a high molecular weight trimeric glycoprotein, is concentrated in saliva and can inhibit the infectivity of diverse pathogens in vitro, we sought to determine the role of TSP1 in suppression of HIV infectivity. Sequence analysis revealed a TSP1 recognition motif, previously defined for the CD36 gene family of cell adhesion receptors, in conserved regions flanking the disulfide-linked cysteine residues of the V3 loop of HIV envelope glycoprotein gp120, important for HIV binding to its high affinity cellular receptor CD4. Using solid-phase in vitro binding assays, we demonstrate direct binding of radiolabeled TSP1 to immobilized recombinant gp120. Based on peptide blocking experiments, the TSP1-gp120 interaction involves CSVTCG sequences in the type 1 properdin-like repeats of TSP1, the known binding site for CD36. TSP1 and fusion proteins derived from CD36-related TSP1-binding domains were able to compete with radiolabeled soluble CD4 binding to immobilized gp120. In parallel, purified TSP1 inhibited HIV-1 infection of peripheral blood mononuclear cells and transformed T and promonocytic cell lines. Levels of TSP1 required for both viral aggregation and direct blockade of HIV-1 infection were physiologic, and affinity depletion of salivary TSP1 abrogated >70% of the inhibitory effect of whole saliva on HIV infectivity. Characterization of TSP1-gp120 binding specificity suggests a mechanism for direct blockade of HIV infectivity that might be exploited to retard HIV transmission that occurs via mucosal routes.

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(A) Map showing the position of snythetic gp120 peptides  with respect to gp120 C2-V3-C3 domains and TSP-binding motifs. A set  of gp120mn peptides (∼20 aa) were immobilized either singly (numbered  1–7) or in pairs as indicated. Corresponding amino acid positions are: 1,  aa 271–290; 2, aa 281–300; 3, aa 291–310; 4, aa 311–330; 5, aa 331–351;  6, aa 351–370; 7, aa 3361–380; 1+2 and 6+7 overlap by 10 aa; gp120,  full length rgp120mn; V3 loop, aa 305–332; motif 1, aa 272–321; motif 2,  aa 331–384. (B) Binding of 125I-TSP1 to gp120 peptides. Increasing concentrations of soluble 125I-labeled TSP1 (25 nM–1 μM) were added to  immobilized gp120mn peptides. Samples were incubated and bound  TSP1 was measured as in Fig. 2. Shown is a single data set for 25 nM  TSP1 binding to ∼75–130 μmol peptide or ∼2 μmol gp120.
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Figure 4: (A) Map showing the position of snythetic gp120 peptides with respect to gp120 C2-V3-C3 domains and TSP-binding motifs. A set of gp120mn peptides (∼20 aa) were immobilized either singly (numbered 1–7) or in pairs as indicated. Corresponding amino acid positions are: 1, aa 271–290; 2, aa 281–300; 3, aa 291–310; 4, aa 311–330; 5, aa 331–351; 6, aa 351–370; 7, aa 3361–380; 1+2 and 6+7 overlap by 10 aa; gp120, full length rgp120mn; V3 loop, aa 305–332; motif 1, aa 272–321; motif 2, aa 331–384. (B) Binding of 125I-TSP1 to gp120 peptides. Increasing concentrations of soluble 125I-labeled TSP1 (25 nM–1 μM) were added to immobilized gp120mn peptides. Samples were incubated and bound TSP1 was measured as in Fig. 2. Shown is a single data set for 25 nM TSP1 binding to ∼75–130 μmol peptide or ∼2 μmol gp120.

Mentions: Fig. 4 A shows the location of a series of 20 aa synthetic peptides with respect to gp120 domain structure (mn isolate). Given the constraints of the solid phase assay, peptides were immobilized either singly or in pairs, and tested for an ability to bind radiolabeled TSP1. As shown in Fig. 4 B, active peptides corresponded to regions of gp120 containing homologous CLESH-1 motifs. Peptide pairs that extended represented motif sequences showed augmented binding compared to either peptide alone. In addition, single peptides representing sequences outside strongly homologous split motif half-sites also showed significant TSP1 binding. Interestingly, a V3-containing peptide (No. 4, aa311–330) with TSP1-binding activity also displayed augmented binding in combination with an inactive C2 peptide within the first CLESH-1 motif (No. 3, aa291–310). These data suggest the presence of two competent TSP1-binding elements in the predicted regions of gp120, including V3 sequences in the first motif, with the potential for interchangeable combinations of site usage, and possible TSP-mediated structural alterations that might disrupt conformation-dependent binding of gp120 to CD4 receptor.


Identification of a CD36-related thrombospondin 1-binding domain in HIV-1 envelope glycoprotein gp120: relationship to HIV-1-specific inhibitory factors in human saliva.

Crombie R, Silverstein RL, MacLow C, Pearce SF, Nachman RL, Laurence J - J. Exp. Med. (1998)

(A) Map showing the position of snythetic gp120 peptides  with respect to gp120 C2-V3-C3 domains and TSP-binding motifs. A set  of gp120mn peptides (∼20 aa) were immobilized either singly (numbered  1–7) or in pairs as indicated. Corresponding amino acid positions are: 1,  aa 271–290; 2, aa 281–300; 3, aa 291–310; 4, aa 311–330; 5, aa 331–351;  6, aa 351–370; 7, aa 3361–380; 1+2 and 6+7 overlap by 10 aa; gp120,  full length rgp120mn; V3 loop, aa 305–332; motif 1, aa 272–321; motif 2,  aa 331–384. (B) Binding of 125I-TSP1 to gp120 peptides. Increasing concentrations of soluble 125I-labeled TSP1 (25 nM–1 μM) were added to  immobilized gp120mn peptides. Samples were incubated and bound  TSP1 was measured as in Fig. 2. Shown is a single data set for 25 nM  TSP1 binding to ∼75–130 μmol peptide or ∼2 μmol gp120.
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Figure 4: (A) Map showing the position of snythetic gp120 peptides with respect to gp120 C2-V3-C3 domains and TSP-binding motifs. A set of gp120mn peptides (∼20 aa) were immobilized either singly (numbered 1–7) or in pairs as indicated. Corresponding amino acid positions are: 1, aa 271–290; 2, aa 281–300; 3, aa 291–310; 4, aa 311–330; 5, aa 331–351; 6, aa 351–370; 7, aa 3361–380; 1+2 and 6+7 overlap by 10 aa; gp120, full length rgp120mn; V3 loop, aa 305–332; motif 1, aa 272–321; motif 2, aa 331–384. (B) Binding of 125I-TSP1 to gp120 peptides. Increasing concentrations of soluble 125I-labeled TSP1 (25 nM–1 μM) were added to immobilized gp120mn peptides. Samples were incubated and bound TSP1 was measured as in Fig. 2. Shown is a single data set for 25 nM TSP1 binding to ∼75–130 μmol peptide or ∼2 μmol gp120.
Mentions: Fig. 4 A shows the location of a series of 20 aa synthetic peptides with respect to gp120 domain structure (mn isolate). Given the constraints of the solid phase assay, peptides were immobilized either singly or in pairs, and tested for an ability to bind radiolabeled TSP1. As shown in Fig. 4 B, active peptides corresponded to regions of gp120 containing homologous CLESH-1 motifs. Peptide pairs that extended represented motif sequences showed augmented binding compared to either peptide alone. In addition, single peptides representing sequences outside strongly homologous split motif half-sites also showed significant TSP1 binding. Interestingly, a V3-containing peptide (No. 4, aa311–330) with TSP1-binding activity also displayed augmented binding in combination with an inactive C2 peptide within the first CLESH-1 motif (No. 3, aa291–310). These data suggest the presence of two competent TSP1-binding elements in the predicted regions of gp120, including V3 sequences in the first motif, with the potential for interchangeable combinations of site usage, and possible TSP-mediated structural alterations that might disrupt conformation-dependent binding of gp120 to CD4 receptor.

Bottom Line: In parallel, purified TSP1 inhibited HIV-1 infection of peripheral blood mononuclear cells and transformed T and promonocytic cell lines.Levels of TSP1 required for both viral aggregation and direct blockade of HIV-1 infection were physiologic, and affinity depletion of salivary TSP1 abrogated >70% of the inhibitory effect of whole saliva on HIV infectivity.Characterization of TSP1-gp120 binding specificity suggests a mechanism for direct blockade of HIV infectivity that might be exploited to retard HIV transmission that occurs via mucosal routes.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, The New York Hospital-Cornell Medical Center, New York 10021, USA.

ABSTRACT
Human and non-human primate salivas retard the infectivity of HIV-1 in vitro and in vivo. Because thrombospondin 1 (TSP1), a high molecular weight trimeric glycoprotein, is concentrated in saliva and can inhibit the infectivity of diverse pathogens in vitro, we sought to determine the role of TSP1 in suppression of HIV infectivity. Sequence analysis revealed a TSP1 recognition motif, previously defined for the CD36 gene family of cell adhesion receptors, in conserved regions flanking the disulfide-linked cysteine residues of the V3 loop of HIV envelope glycoprotein gp120, important for HIV binding to its high affinity cellular receptor CD4. Using solid-phase in vitro binding assays, we demonstrate direct binding of radiolabeled TSP1 to immobilized recombinant gp120. Based on peptide blocking experiments, the TSP1-gp120 interaction involves CSVTCG sequences in the type 1 properdin-like repeats of TSP1, the known binding site for CD36. TSP1 and fusion proteins derived from CD36-related TSP1-binding domains were able to compete with radiolabeled soluble CD4 binding to immobilized gp120. In parallel, purified TSP1 inhibited HIV-1 infection of peripheral blood mononuclear cells and transformed T and promonocytic cell lines. Levels of TSP1 required for both viral aggregation and direct blockade of HIV-1 infection were physiologic, and affinity depletion of salivary TSP1 abrogated >70% of the inhibitory effect of whole saliva on HIV infectivity. Characterization of TSP1-gp120 binding specificity suggests a mechanism for direct blockade of HIV infectivity that might be exploited to retard HIV transmission that occurs via mucosal routes.

Show MeSH
Related in: MedlinePlus