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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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Related in: MedlinePlus

125I-gp120 interacts with the CD36 binding peptide of TSP1.  Increasing concentrations of soluble 125I-labeled recombinant gp120lai (1  nM–1 μM) were incubated with immobilized ligand for 2 h at 37°C, and  bound radioactivity was measured as in Fig. 2. Shown are 125I-gp120  binding to CSVTCG peptide derived from TSP1 properdin-like type 1  repeat, scrambled control peptide VGSCCT, and GRGDS derived from  TSP1 calcium binding type 3 repeat. This plot represents a single data set  of triplicate samples (n = 2, errors as SD).
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Figure 3: 125I-gp120 interacts with the CD36 binding peptide of TSP1. Increasing concentrations of soluble 125I-labeled recombinant gp120lai (1 nM–1 μM) were incubated with immobilized ligand for 2 h at 37°C, and bound radioactivity was measured as in Fig. 2. Shown are 125I-gp120 binding to CSVTCG peptide derived from TSP1 properdin-like type 1 repeat, scrambled control peptide VGSCCT, and GRGDS derived from TSP1 calcium binding type 3 repeat. This plot represents a single data set of triplicate samples (n = 2, errors as SD).

Mentions: CSVTCG peptides found in the type 1 repeats of TSP1 are binding sites for CD36 and LIMPII (35, 52–54). Therefore, we tested whether gp120 shared this same specificity. Saturation isotherms showed significant binding of radiolabeled gp120 (lav isolate) to immobilized CSVTCG peptide (Fig. 3), with an apparent affinity of 300 nM. The activity was sequence-specific, as demonstrated by inefficient binding to scrambled control peptide VGSCCT, or to an RGD-containing peptide similar to the GRGDA cell adhesion sequence of the last TSP1 type 3 calcium-binding repeat. This is further evidence that the TSP1-gp120 interaction is mediated through a CD36/LIMPII-related structural domain.


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)

125I-gp120 interacts with the CD36 binding peptide of TSP1.  Increasing concentrations of soluble 125I-labeled recombinant gp120lai (1  nM–1 μM) were incubated with immobilized ligand for 2 h at 37°C, and  bound radioactivity was measured as in Fig. 2. Shown are 125I-gp120  binding to CSVTCG peptide derived from TSP1 properdin-like type 1  repeat, scrambled control peptide VGSCCT, and GRGDS derived from  TSP1 calcium binding type 3 repeat. This plot represents a single data set  of triplicate samples (n = 2, errors as SD).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199189&req=5

Figure 3: 125I-gp120 interacts with the CD36 binding peptide of TSP1. Increasing concentrations of soluble 125I-labeled recombinant gp120lai (1 nM–1 μM) were incubated with immobilized ligand for 2 h at 37°C, and bound radioactivity was measured as in Fig. 2. Shown are 125I-gp120 binding to CSVTCG peptide derived from TSP1 properdin-like type 1 repeat, scrambled control peptide VGSCCT, and GRGDS derived from TSP1 calcium binding type 3 repeat. This plot represents a single data set of triplicate samples (n = 2, errors as SD).
Mentions: CSVTCG peptides found in the type 1 repeats of TSP1 are binding sites for CD36 and LIMPII (35, 52–54). Therefore, we tested whether gp120 shared this same specificity. Saturation isotherms showed significant binding of radiolabeled gp120 (lav isolate) to immobilized CSVTCG peptide (Fig. 3), with an apparent affinity of 300 nM. The activity was sequence-specific, as demonstrated by inefficient binding to scrambled control peptide VGSCCT, or to an RGD-containing peptide similar to the GRGDA cell adhesion sequence of the last TSP1 type 3 calcium-binding repeat. This is further evidence that the TSP1-gp120 interaction is mediated through a CD36/LIMPII-related structural domain.

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