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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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Inhibitory effect of TSP1 on HIV-1 infectivity. HIV-1 isolate IIIB was preincubated in the absence of TSP1, or with various concentrations of purified TSP1 for 1 h at 37°C. Preincubated virus–TSP1  mixtures were added to target cells either directly (−), or first passed  through 0.2 μm filters (+). PMA-activated primary PBMC, SK23 (T cell  line) or U937 (promonocytic line) were inoculated with the mois indicated. After an additional 1 h at 37°C, infected cells were washed, cultured for 7 d, and HIV-1 p24 antigen was measured by ELISA. Inhibition  is expressed for a single data set as percentage of maximum p24 detected  in the absence of TSP1.
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Figure 7: Inhibitory effect of TSP1 on HIV-1 infectivity. HIV-1 isolate IIIB was preincubated in the absence of TSP1, or with various concentrations of purified TSP1 for 1 h at 37°C. Preincubated virus–TSP1 mixtures were added to target cells either directly (−), or first passed through 0.2 μm filters (+). PMA-activated primary PBMC, SK23 (T cell line) or U937 (promonocytic line) were inoculated with the mois indicated. After an additional 1 h at 37°C, infected cells were washed, cultured for 7 d, and HIV-1 p24 antigen was measured by ELISA. Inhibition is expressed for a single data set as percentage of maximum p24 detected in the absence of TSP1.

Mentions: To investigate whether purified TSP1 could inhibit HIV-1 infection similarly to saliva, viral isolate IIIB was added to target cells after preincubation with TSP1. Many studies have shown that filtration of virus–saliva mixtures is required for maximum inhibition (10, 11, 18, 24). The physiologic equivalent of such filtration is thought to be the constant cleansing of the oral cavity by salivary flow. Therefore, in some experiments preincubated virus-TSP1 mixtures were passed through 0.2-μm filters. Different multiplicities of infectious virus per target cell were tested in both systems. At concentrations found in saliva (2–10 μg/ml), TSP1 reduced HIV-1 infection of PHA-activated donor PBMCs by >83% when prefiltered, as measured by ELISA detection of p24 viral antigen (Fig. 7). This was comparable to a 1:2 dilution of whole saliva. In contrast, fibrinogen, another high molecular weight adhesive glycoprotein in saliva, had no effect in this system. Specificity was documented by abrogation of the inhibitory effect in the presence of a specific anti-TSP1 polyclonal antibody (107% of control p24), but not control IgG (data not shown). Prolonged incubation of the TSP–virion mixture was unnecessary, as exposures as brief as 5 min appeared sufficient to reduce infectivity by ⩾50% at 1 μg/ml TSP1 (data not shown). The TSP1 effect also was apparent for HIV-1 IIIB infection of CD4+ T-lymphoblastoid and monocytoid cell lines (SK23 ⩾ 90.7% and U937 ⩾83.0% inhibition, respectively). In addition, two monocytotropic strains of HIV-1, p13 and HA593, representing patient isolates obtained from the NIH AIDS Retroviral Repository (Bethesda, MD), were susceptible to inhibition by purified TSP1. At viral mois of 0.8, 100 μg/ml TSP1 inhibited HIV infectivity by ⩾98 ± 1% (data not shown). In contrast to prefiltration experiments, direct addition of TSP1 was able to inhibit HIV-1 by 50–75% only when a high concentration of TSP1 and a low moi of inoculum was used (Fig. 7). In parallel assays, saliva could inhibit HIV-1 as a direct addition only when added in dilutions of <1:4. However, preincubation of virus with saliva permitted dilutions >1:10. The results again are consistent with levels of TSP1 found in these dilutions of saliva.


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)

Inhibitory effect of TSP1 on HIV-1 infectivity. HIV-1 isolate IIIB was preincubated in the absence of TSP1, or with various concentrations of purified TSP1 for 1 h at 37°C. Preincubated virus–TSP1  mixtures were added to target cells either directly (−), or first passed  through 0.2 μm filters (+). PMA-activated primary PBMC, SK23 (T cell  line) or U937 (promonocytic line) were inoculated with the mois indicated. After an additional 1 h at 37°C, infected cells were washed, cultured for 7 d, and HIV-1 p24 antigen was measured by ELISA. Inhibition  is expressed for a single data set as percentage of maximum p24 detected  in the absence of TSP1.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199189&req=5

Figure 7: Inhibitory effect of TSP1 on HIV-1 infectivity. HIV-1 isolate IIIB was preincubated in the absence of TSP1, or with various concentrations of purified TSP1 for 1 h at 37°C. Preincubated virus–TSP1 mixtures were added to target cells either directly (−), or first passed through 0.2 μm filters (+). PMA-activated primary PBMC, SK23 (T cell line) or U937 (promonocytic line) were inoculated with the mois indicated. After an additional 1 h at 37°C, infected cells were washed, cultured for 7 d, and HIV-1 p24 antigen was measured by ELISA. Inhibition is expressed for a single data set as percentage of maximum p24 detected in the absence of TSP1.
Mentions: To investigate whether purified TSP1 could inhibit HIV-1 infection similarly to saliva, viral isolate IIIB was added to target cells after preincubation with TSP1. Many studies have shown that filtration of virus–saliva mixtures is required for maximum inhibition (10, 11, 18, 24). The physiologic equivalent of such filtration is thought to be the constant cleansing of the oral cavity by salivary flow. Therefore, in some experiments preincubated virus-TSP1 mixtures were passed through 0.2-μm filters. Different multiplicities of infectious virus per target cell were tested in both systems. At concentrations found in saliva (2–10 μg/ml), TSP1 reduced HIV-1 infection of PHA-activated donor PBMCs by >83% when prefiltered, as measured by ELISA detection of p24 viral antigen (Fig. 7). This was comparable to a 1:2 dilution of whole saliva. In contrast, fibrinogen, another high molecular weight adhesive glycoprotein in saliva, had no effect in this system. Specificity was documented by abrogation of the inhibitory effect in the presence of a specific anti-TSP1 polyclonal antibody (107% of control p24), but not control IgG (data not shown). Prolonged incubation of the TSP–virion mixture was unnecessary, as exposures as brief as 5 min appeared sufficient to reduce infectivity by ⩾50% at 1 μg/ml TSP1 (data not shown). The TSP1 effect also was apparent for HIV-1 IIIB infection of CD4+ T-lymphoblastoid and monocytoid cell lines (SK23 ⩾ 90.7% and U937 ⩾83.0% inhibition, respectively). In addition, two monocytotropic strains of HIV-1, p13 and HA593, representing patient isolates obtained from the NIH AIDS Retroviral Repository (Bethesda, MD), were susceptible to inhibition by purified TSP1. At viral mois of 0.8, 100 μg/ml TSP1 inhibited HIV infectivity by ⩾98 ± 1% (data not shown). In contrast to prefiltration experiments, direct addition of TSP1 was able to inhibit HIV-1 by 50–75% only when a high concentration of TSP1 and a low moi of inoculum was used (Fig. 7). In parallel assays, saliva could inhibit HIV-1 as a direct addition only when added in dilutions of <1:4. However, preincubation of virus with saliva permitted dilutions >1:10. The results again are consistent with levels of TSP1 found in these dilutions of saliva.

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