Interactions of soluble recombinant integrin alphav beta5 with human adenoviruses.

alphav integrins have been identified as coreceptors for adenovirus (Ad) internalization; however, direct interactions of these molecules with Ad have not been demonstrated. We report here the expression of soluble integrin alphav beta5, which retains the ability to recognize the Ad penton base as well as vitronectin, an Arg Gly Asp (RGD)-containing extracellular matrix protein. Soluble integrin alphav beta5 reacted with seven different Ad serotypes (subgroups A to E) in solid-phase binding assays. The soluble integrin exhibited different levels of binding to each Ad serotype; however, binding to multiple Ad types required the presence of divalent metal cations and was inhibited by a synthetic RGD peptide, indicating that RGD and cation-binding sequences regulate Ad interactions with alphav beta5. Incubation of Ad particles with soluble alphav beta5 integrin also inhibited subsequent Ad internalization into epithelial cells as well as virus attachment to monocytic cells. These findings suggest that soluble alphav integrins or antagonists of these coreceptors could be used to limit infection by multiple Ad types. The generation of soluble alphav integrins should also permit further detailed kinetic and structural analysis of Ad interactions with its coreceptors.

Expression of recombinant HLA-DR2 molecules. Replacement of the hydrophobic transmembrane region by a leucine zipper dimerization motif allows the assembly and secretion of soluble DR alpha beta heterodimers.

Major histocompatibility complex (MHC) class II molecules are membrane-anchored heterodimers that present peptides on the surface of antigen presenting cells to T cells. Soluble HLA-DR2 molecules were expressed for structural and functional characterization of the MHC/peptide/T cell receptor recognition unit. The alpha and beta chains of DR2 (encoded by the DRA, DRB1*1501 genes) did not assemble in mammalian or insect cell lines when the transmembrane regions of one or both chains were truncated. The hydrophobic transmembrane regions of DRalpha and DRbeta facilitate assembly of the heterodimer and were therefore replaced by the leucine zipper dimerization motifs from the transcription factors Fos and Jun, which assemble as a soluble, tightly packed coiled coil structure. The DRalpha-Fos and DRbeta-Jun constructs were expressed in a methyltrophic yeast, Pichia pastoris, using the alpha-mating factor secretion signal to direct expression to the secretory pathway. DR alphabeta heterodimers were purified from supernatants using an antibody specific for the DR alphabeta heterodimer. Kinetic and quantitative peptide binding experiments demonstrated that recombinant DR2 molecules were efficiently loaded with an antigenic peptide. Soluble DR2 molecules can be used to define structural aspects of the MHC/peptide/T cell receptor interaction and to study the signals induced by T cell receptor recognition of soluble DR2.peptide complexes.