RESUMEN
Rotavirus nonstructural protein 4 (NSP4) is known to function as an intracellular receptor at the endoplasmic reticulum (ER) critical to viral morphogenesis and is the first characterized viral enterotoxin. Exogenously added NSP4 induces diarrhea in rodent pups and stimulates secretory chloride currents across intestinal segments as measured in Ussing chambers. Circular dichroism studies further reveal that intact NSP4 and the enterotoxic peptide (NSP4(114-135)) that is located within the extended, C-terminal amphipathic helix preferentially interact with caveola-like model membranes. We now show colocalization of NSP4 and caveolin-1 in NSP4-transfected and rotavirus-infected mammalian cells in reticular structures surrounding the nucleus (likely ER), in the cytosol, and at the cell periphery by laser scanning confocal microscopy. A direct interaction between NSP4 residues 112 to 140 and caveolin-1 was determined by the Pro-Quest yeast two-hybrid system with full-length NSP4 and seven overlapping deletion mutants as bait, caveolin-1 as prey, and vice versa. Coimmunoprecipitation of NSP4-caveolin-1 complexes from rotavirus-infected mammalian cells demonstrated that the interaction occurs during viral infection. Finally, binding of caveolin-1 from mammalian cell lysates to Sepharose-bound, NSP4-specific synthetic peptides confirmed the yeast two-hybrid data and further delineated the binding domain to amino acids 114 to 135. We propose that the association of NSP4 and caveolin-1 contributes to NSP4 intracellular trafficking from the ER to the cell surface and speculate that exogenously added NSP4 stimulates signaling molecules located in caveola microdomains.
Asunto(s)
Caveolas/metabolismo , Caveolina 1/metabolismo , Glicoproteínas/metabolismo , Rotavirus/metabolismo , Toxinas Biológicas/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Células CACO-2 , Línea Celular , Retículo Endoplásmico/metabolismo , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Inmunoprecipitación , Péptidos/metabolismo , Toxinas Biológicas/química , Toxinas Biológicas/genética , Transfección , Técnicas del Sistema de Dos Híbridos , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genéticaAsunto(s)
Enterotoxinas/fisiología , Glicoproteínas/fisiología , Rotavirus/fisiología , Proteínas no Estructurales Virales/fisiología , Animales , Enterotoxinas/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Interferencia de ARN , Virus Reordenados/aislamiento & purificación , Receptores Virales , Proteínas Recombinantes/inmunología , Rotavirus/inmunología , Toxinas Biológicas/genética , Toxinas Biológicas/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación ViralRESUMEN
A cloned cell line that spontaneously polarizes in standard glucose-containing media was derived from a single cell of the adenocarcinoma cell line HT-29. The cloned line, designated HT-29/cl.f8, has remained stable over 2 yr in culture, maintained high transepithelial resistance (300 ohm cm(2) or higher), and correctly sorted influenza virus and vesicular stomatitis virus to apical or basolateral domains, respectively. The newly cloned cells also displayed apical microvilli, tight junctions, and desmosomes, the morphological characteristics of mature epithelia. The cloned HT-29/cl.f8 cells function as epithelial enterocytes as shown by the apical expression of intestinal alkaline phosphatase, the expression of vimentin and cytokeratin, and lack of expression of mucin. We propose that the newly cloned HT-29/cl.f8 cells offer a viable alternative for studies of enterocyte function that will readily yield interpretable data not complicated by cell alterations due to the presence of drugs or chemicals that induce differentiation.