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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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Competitive inhibition of 125I-CD4 binding to gp120. A  fixed concentration of 125I-labeled soluble recombinant CD4 (50 nM) was  added to immobilized rgp160 (solid bars, MN; hatched bars, LAV isolate-derived) in the absence or presence of 10-fold molar excess (0.5 μM) of  unlabeled TSP1, TSP1-derived peptides CSVTCG or GRGDS, scrambled control peptide VGSCCT, fusion proteins containing the TSP1-binding domain of CD36 (CFP, aa 67–157) or LIMPII (aa 75–155), downstream fusion protein LFP156–243, or an equal volume of saliva (final  twofold dilution). Samples were incubated and bound TSP1 was measured as in Fig. 2. Shown is a single data set of triplicate samples. (n = 3,  error as SD).
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Figure 5: Competitive inhibition of 125I-CD4 binding to gp120. A fixed concentration of 125I-labeled soluble recombinant CD4 (50 nM) was added to immobilized rgp160 (solid bars, MN; hatched bars, LAV isolate-derived) in the absence or presence of 10-fold molar excess (0.5 μM) of unlabeled TSP1, TSP1-derived peptides CSVTCG or GRGDS, scrambled control peptide VGSCCT, fusion proteins containing the TSP1-binding domain of CD36 (CFP, aa 67–157) or LIMPII (aa 75–155), downstream fusion protein LFP156–243, or an equal volume of saliva (final twofold dilution). Samples were incubated and bound TSP1 was measured as in Fig. 2. Shown is a single data set of triplicate samples. (n = 3, error as SD).

Mentions: An obvious question is whether TSP1 could compromise the ability of gp120 to bind to CD4. Fig. 5 shows competitive inhibition of radiolabeled CD4-binding to gp120 derived from two different viral isolates (mn and lav). In the presence of 10-fold molar excess TSP1, complete inhibition of 125I-CD4–binding to immobilized gp120 was seen. CSVTCG peptide showed a partial but significant inhibition of CD4–gp120 complex formation (53 ± 9%), confirming a TSP-specific effect, while the RGD-containing peptide had little effect (4 ± 4%), and the scrambled control actually enhanced binding. Consistent with structural homology data, TSP1-binding CD36- and LIMPII-derived fusion proteins proved strong competitors (both ∼89% inhibition), whereas downstream LIMPII control protein had a minimal effect (32 ± 5%). For comparison, a 1:2 dilution of whole saliva was a potent inhibitor in this assay system. These observations support a role for salivary TSP1 as a direct inhibitor of HIV infectivity.


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)

Competitive inhibition of 125I-CD4 binding to gp120. A  fixed concentration of 125I-labeled soluble recombinant CD4 (50 nM) was  added to immobilized rgp160 (solid bars, MN; hatched bars, LAV isolate-derived) in the absence or presence of 10-fold molar excess (0.5 μM) of  unlabeled TSP1, TSP1-derived peptides CSVTCG or GRGDS, scrambled control peptide VGSCCT, fusion proteins containing the TSP1-binding domain of CD36 (CFP, aa 67–157) or LIMPII (aa 75–155), downstream fusion protein LFP156–243, or an equal volume of saliva (final  twofold dilution). Samples were incubated and bound TSP1 was measured as in Fig. 2. Shown is a single data set of triplicate samples. (n = 3,  error as SD).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199189&req=5

Figure 5: Competitive inhibition of 125I-CD4 binding to gp120. A fixed concentration of 125I-labeled soluble recombinant CD4 (50 nM) was added to immobilized rgp160 (solid bars, MN; hatched bars, LAV isolate-derived) in the absence or presence of 10-fold molar excess (0.5 μM) of unlabeled TSP1, TSP1-derived peptides CSVTCG or GRGDS, scrambled control peptide VGSCCT, fusion proteins containing the TSP1-binding domain of CD36 (CFP, aa 67–157) or LIMPII (aa 75–155), downstream fusion protein LFP156–243, or an equal volume of saliva (final twofold dilution). Samples were incubated and bound TSP1 was measured as in Fig. 2. Shown is a single data set of triplicate samples. (n = 3, error as SD).
Mentions: An obvious question is whether TSP1 could compromise the ability of gp120 to bind to CD4. Fig. 5 shows competitive inhibition of radiolabeled CD4-binding to gp120 derived from two different viral isolates (mn and lav). In the presence of 10-fold molar excess TSP1, complete inhibition of 125I-CD4–binding to immobilized gp120 was seen. CSVTCG peptide showed a partial but significant inhibition of CD4–gp120 complex formation (53 ± 9%), confirming a TSP-specific effect, while the RGD-containing peptide had little effect (4 ± 4%), and the scrambled control actually enhanced binding. Consistent with structural homology data, TSP1-binding CD36- and LIMPII-derived fusion proteins proved strong competitors (both ∼89% inhibition), whereas downstream LIMPII control protein had a minimal effect (32 ± 5%). For comparison, a 1:2 dilution of whole saliva was a potent inhibitor in this assay system. These observations support a role for salivary TSP1 as a direct inhibitor of HIV infectivity.

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