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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 TSP1 anti-HIV effect by LIMPII  TSP1-binding domain. PMA-stimulated primary PBMCs were infected  with 0.02 moi HIV/IIIB that had been preincubated with TSP1 alone, or  with LIMPII peptide LFP75–155 alone, or in the presence of TSP1, and  filtered before incubation as in Fig. 7. TSP-mediated inhibition is expressed as percentage of decrease in p24 relative to maximum p24 detected in the absence of TSP1. Data shown represent the average of two  independent experiments (error as SD).
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Figure 8: Competitive inhibition of TSP1 anti-HIV effect by LIMPII TSP1-binding domain. PMA-stimulated primary PBMCs were infected with 0.02 moi HIV/IIIB that had been preincubated with TSP1 alone, or with LIMPII peptide LFP75–155 alone, or in the presence of TSP1, and filtered before incubation as in Fig. 7. TSP-mediated inhibition is expressed as percentage of decrease in p24 relative to maximum p24 detected in the absence of TSP1. Data shown represent the average of two independent experiments (error as SD).

Mentions: To further delineate a TSP-specific effect, LIMPII TSP-binding peptide L75–155 (10-kD product purified after removal of GST moiety by proteolytic cleavage) was included in HIV–TSP1 preincubation mixtures as a competitor. Fig. 8 shows that 1 μM LIMPII peptide abrogated the inhibitory effect of even high concentrations of TSP1 (50– 100 μg/ml) by 83–90%. Incubation of virus in the presence of LIMPII peptide alone resulted in minimal decrease of HIV-1 infectivity (∼9%), suggesting that the amounts of peptide able to block the TSP1 antiviral effect were not sufficient to compete for HIV-1 envelope binding sites on PBMC target cells. The ability of the LIMPII peptide to restore infectivity supports a direct role for a CD36/ LIMPII-related TSP1-binding domain on HIV-1 gp120, and provides further evidence of a common binding site on TSP1.


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 TSP1 anti-HIV effect by LIMPII  TSP1-binding domain. PMA-stimulated primary PBMCs were infected  with 0.02 moi HIV/IIIB that had been preincubated with TSP1 alone, or  with LIMPII peptide LFP75–155 alone, or in the presence of TSP1, and  filtered before incubation as in Fig. 7. TSP-mediated inhibition is expressed as percentage of decrease in p24 relative to maximum p24 detected in the absence of TSP1. Data shown represent the average of two  independent experiments (error as SD).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Competitive inhibition of TSP1 anti-HIV effect by LIMPII TSP1-binding domain. PMA-stimulated primary PBMCs were infected with 0.02 moi HIV/IIIB that had been preincubated with TSP1 alone, or with LIMPII peptide LFP75–155 alone, or in the presence of TSP1, and filtered before incubation as in Fig. 7. TSP-mediated inhibition is expressed as percentage of decrease in p24 relative to maximum p24 detected in the absence of TSP1. Data shown represent the average of two independent experiments (error as SD).
Mentions: To further delineate a TSP-specific effect, LIMPII TSP-binding peptide L75–155 (10-kD product purified after removal of GST moiety by proteolytic cleavage) was included in HIV–TSP1 preincubation mixtures as a competitor. Fig. 8 shows that 1 μM LIMPII peptide abrogated the inhibitory effect of even high concentrations of TSP1 (50– 100 μg/ml) by 83–90%. Incubation of virus in the presence of LIMPII peptide alone resulted in minimal decrease of HIV-1 infectivity (∼9%), suggesting that the amounts of peptide able to block the TSP1 antiviral effect were not sufficient to compete for HIV-1 envelope binding sites on PBMC target cells. The ability of the LIMPII peptide to restore infectivity supports a direct role for a CD36/ LIMPII-related TSP1-binding domain on HIV-1 gp120, and provides further evidence of a common binding site on TSP1.

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