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Title: PRODUCTION AND ANALYSIS OF SOLUBLE-SECRETED FOOT-AND-MOUTH DISEASE VIRUS INTEGRIN RECEPTORS

Author
item Baxt, Barry
item Duque, Hernando

Submitted to: American Society for Virology Meeting
Publication Type: Proceedings
Publication Acceptance Date: 1/28/2003
Publication Date: 7/12/2003
Citation: N/A

Interpretive Summary:

Technical Abstract: At least three members of the integrin family of receptors, alphaVbeta1, alphaVbeta3, and alphaVbeta6 have been identified as receptors for foot-and-mouth disease virus (FMDV) in vitro. Integrins are heterodimeric type I membrane proteins, consisting of an alpha and beta subunit, which insert into the cellular membrane via a short hydrophobic transmembrane domain. We have recently shown that the efficiency of receptor usage appears to be related to the viral serotype, which may be influenced by structural differences on the viral surface (Duque and Baxt, J. Virol. 77:2500, 2003). To further examine these differences, we generated soluble-secreted (ss) integrins. cDNA plasmids encoding the individual complete integrin alphaV, beta3, and beta6 subunits were used to amplify sequences, by PCR, encoding the subunits¿ signal peptide and ectodomain, placing two stop codons after the last codon of the ectodomain. This resulted in subunits lacking transmembrane and cytoplasmic domains. The amplicons were reinserted back into pcDNA3.1 and resequenced. Plasmids encoding the (ss)alphaV subunit and either the (ss)beta3 or (ss)beta6 subunits were co-transfected into COS-1 cells, which were labeled with [35S]methionine/cysteine between 24-72 hours post-transfection. The culture media were analyzed for the presence of (ss)integrins by radioimmunoprecipitation (RIP) and SDS-PAGE using either complete integrin or subunit-specific antibodies. In both cases (ss)alphaVbeta3 or (ss)alphaVbeta6 heterodimers were detected in the culture fluid. The interactions of the (ss)integrins with FMDV were analyzed by plaque-reduction neutralization using unlabeled concentrated culture fluids from transfected COS-1 cells. In the presence of divalent cations, plaque formation by either type A12 or O1 virus was significantly inhibited by the (ss)alphaVbeta6 integrin. In contrast, (ss)alphaVbeta3 caused little or no reduction of plaque formation by either virus at the concentrations used in these experiments. That at least one of the integrin receptors can interact with virus in solution opens the possibility of structural analysis of FMDV-integrin interactions.