Pseudopeptide TASP Inhibitors of HIV Entry Bind Specifically to a 95-kDa Cell Surface Protein

Christian Callebaut,Etienne Jacotot,Bernard Krust,Gilles Guichard,Julià Blanco,Agustin Valenzuela,Josette Svab,Sylviane Muller,Jean-Paul Briand,Ara G Hovanessian

doi:10.1074/jbc.272.11.7159

Abstract

The template assembled synthetic peptide constructs (TASP), pentavalently presenting the tripeptide KPR or RPK, are potent and specific inhibitors of human immunodeficiency virus (HIV) infection by preventing viral entry into permissive cells. Here the 5[KPsi(CH2N)PR]-TASP construct, Psi(CH2N) for reduced peptide bond, was used in studies to demonstrate its specific binding to a 95-kDa cell surface protein ligand. Compared to its nonreduced 5[KPR]-TASP counterpart, the pseudopeptide 5[KPsi(CH2N)PR]-TASP manifested higher affinity to bind to its cell surface ligand, increased activity to inhibit HIV infection, and resistance to degradation when incubated in serum from an HIV-1 seropositive individual. In ligand blotting experiments, the biotin-labeled 5[KPsi(CH2N)PR]-TASP identified a single 95-kDa protein in crude cell extracts. This 95-kDa protein (p95) is expressed on the cell surface since surface iodination of cells resulted in its labeling, and moreover, following incubation of cells with the biotin-labeled 5[KPsi(CH2N)PR]-TASP, the p95.TASP complex was recovered by affinity chromatography using avidin-agarose. All anti-HIV TASP constructs but not their control derivatives affected the binding of biotin-labeled 5[KPsi(CH2N)PR]-TASP to p95, thus emphasizing the specific nature of this binding. Since 5[KPsi(CH2N)PR]-TASP does not interact with HIV-envelope glycoproteins, our results suggest that TASP inhibitors mediate directly or indirectly a block in HIV-mediated membrane fusion process by binding to the cell surface expressed p95.

Highlights

HIV1 is an enveloped virus that infects target cells by the fusion of viral and cellular membranes
Since 5[K⌿(CH2N)PR]-template assembled synthetic peptide (TASP) does not interact with human immunodeficiency virus (HIV)-envelope glycoproteins, our results suggest that TASP inhibitors mediate directly or indirectly a block in HIV-mediated membrane fusion process by binding to the cell surface expressed p95
HIV entry monitored by the intracellular concentration of p24 (HIV-1 major core protein), following 1 h incubation of CEM cells with the virus, is inhibited almost completely in the pesence of 5–10 ␮M 5[K⌿(CH2N)PR]-TASP

Summary

MATERIALS AND METHODS

Cells and HIV Infection—CEM cells (clone 13) derived from human lymphoid cell line CEM and MOLT4-T4 clone 8 cells selected for high level of CD4 expression Isolation of the Cell Surface Ligand of TASP—Twenty-four hours after passaging, CEM cells were washed extensively with PBS before incubation (50 ϫ 106 cells/300 ␮l of FACS buffer) at 4 °C for 30 min with different concentrations of the biotin-labeled TASP molecule. Cells were washed in FACS buffer (2 ϫ 15 ml), and nuclear-free cell extracts were prepared using buffer E (150 ␮l) Such extracts were first diluted in PBS (600 ␮l) before the addition of 100 ␮l of avidin-agarose (ImmunoPure immobilized avidin from Pierce) to capture the biotin-labeled TASP complexed to its cell surface ligand. In order to assay for the binding of FITC- or biotin-labeled TASP inhibitors to a cell surface antigen, different cells were washed in PBS, suspended in FACS buffer (5 ϫ 105 cells/100 ␮l) containing 0.5 ␮M FITC-labeled or different concentrations of the biotin-labeled TASP constructs, and incubated at 4 °C for 30 min. It should be noted that under these experimental conditions, the values for the bound virus represent particles bound on the cell surface as well as particles (or cores) entered into cells

AND DISCUSSION

None Trypsin Proteinase K Pronase E

Not tested Not tested