No 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.

LIMPII (lysosomal integral membrane protein II) is one of a family of proteins structurally related to the cell surface glycoprotein CD36. We recently defined a single structural domain on CD36 that mediates binding to adhesive glycoprotein thrombospondin-1 (TSP1). The CD36-TSP1 interaction is known to play a role in platelet-tumor and platelet-monocyte adhesion, angiogenesis, and in monocyte uptake of apoptotic cells. To test whether LIMPII also binds TSP1, a LIMPII peptide corresponding to the TSP1 binding domain of CD36 was expressed as a recombinant glutathione S-transferase (GST) fusion protein. In solid phase binding assays, purified 125 I-TSP1 bound to immobilized GST/LIMPII in a time-dependent and saturable manner. Inhibition by excess unlabeled TSP1 or EDTA demonstrated specificity. LIMPII⅐TSP1 complex formation was specifically blocked by soluble LIMPII fusion protein, by monospecific rabbit IgG directed against the LIMPII peptide and by CD36 fusion proteins containing the TSP1 binding domain. Transfection of Bowes melanoma cells with a chimeric LIMPII cDNA that targets expression to the plasma membrane conferred the ability to bind 125 I-TSP1 and to adhere to TSP1-coated surfaces. This study defines a TSP1 binding site conserved between LIMPII and CD36 and suggests that cell surface LIMPII may function in some circumstances as an adhesion receptor for TSP1. Computer-assisted homology searches suggest that the TSP1 recognition motif identified from study of CD36 family members may be widely expressed in nature.

Extended hyperimmunization of rabbits with Sindbis (SIN) or Semliki Forest (SF) viruses causes the production of antisera that are cross-reactive with virus-infected cells in antibodydependent, complement-mediated cytotoxicity assays but that do not cross-neutralize viruses in vitro. C3H/HeJ mice given y globulin fractionated from the extended hyperimmune antiserum against SIN, but not control sera, were protected from challenge by 100 LDso of SF, a virus which is in a different subgroup than SIN. All mice survived if the y globulin was given 24 hr before challenge virus and partial protection occurred if the globulin was given 24 hr after the virus. Cobra venom factor treatment of normal C3H mice challenged with SF did not reduce the protection, suggesting that complement was not involved. Methyl palmitate (40 mg/mouse) given before y globulin and virus challenge suppressed macrophage activity and reduced the level of protection 23% in females and 70% in males, Silica treatment (3 mg/mouse) reduced the protection equally in both males and females by 92%. In vitro experiments were done to test if it were possible that cross-antibody-dependent cellular cytotoxicity (ADCC) could accout for the passive crossprotection observed in this system. Cross-ADCC could be demonstrated in vitro at high dilutions of antiserum (1 :25,600). On the basis of the in vitro and in vivo results presented, we suggest that 56