Article

The rotavirus enterotoxin NSP4 directly interacts with the caveolar structural protein caveolin-1.

Department of Pathobiology, Texas A&M University 4467, College Station, Texas 77843, USA.
Journal of Virology (Impact Factor: 5.08). 04/2006; 80(6):2842-54. DOI: 10.1128/JVI.80.6.2842-2854.2006
Source: PubMed

ABSTRACT 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.

0 Bookmarks
 · 
65 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Rotavirus (RV) nonstructural protein 4 (NSP4) is the first described viral enterotoxin, which induces early secretory diarrhea in neonatal rodents. Our previous data show a direct interaction between RV NSP4 and the structural protein of caveolae, caveolin-1 (cav-1), in yeast and mammalian cells. The binding site of cav-1 mapped to the NSP4 amphipathic helix, and led us to examine which helical face was responsible for the interaction. A panel of NSP4 mutants were prepared and tested for binding to cav-1 by yeast two hybrid and direct binding assays. The charged residues of the NSP4 amphipathic helix were changed to alanine (NSP446-175-ala6); and three residues in the hydrophobic face were altered to charged amino acids (NSP446-175-HydroMut). In total, twelve mutants of NSP4 were generated to define the cav-1 binding site. Synthetic peptides corresponding to the hydrophobic and charged faces of NSP4 were examined for structural changes by circular dichroism (CD) and diarrhea induction by a neonatal mouse study. Mutations of the hydrophilic face (NSP446-175-Ala6) bound cav-1 akin to wild type NSP4. In contrast, disruption of the hydrophobic face (NSP446-175-HydroMut) failed to bind cav-1. These data suggest NSP4 and cav-1 associate via a hydrophobic interaction. Analyses of mutant synthetic peptides in which the hydrophobic residues in the enterotoxic domain of NSP4 were altered suggested a critical hydrophobic residue. Both NSP4HydroMut112-140, that contains three charged amino acids (aa113, 124, 131) changed from the original hydrophobic residues and NSP4AlaAcidic112-140 that contained three alanine residues substituted for negatively charged (aa114, 125, 132) amino acids failed to induce diarrhea. Whereas peptides NSP4wild type 112 -140 and NSP4AlaBasic112-140 that contained three alanine substituted for positively charged (aa115, 119, 133) amino acids, induced diarrhea. These data show that the cav-1 binding domain is within the hydrophobic face of the NSP4 amphipathic helix. The integrity of the helical structure is important for both cav-1 binding and diarrhea induction implying a connection between NSP4 functional and binding activities.
    Virology Journal 11/2013; 10(1):336. · 2.09 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Caveolin-1 (CAV-1) is a key structural component of caveolae that regulates cholesterol. Employing transgenic techniques to regulate the cholesterol content of pork through CAV-1 is hindered by our lack of knowledge about its regulation. To investigate the regulatory mechanism of porcine CAV-1, a DNA segment containing the 5'-flanking region of CAV-1 was isolated from porcine genomic DNA and sequenced. The luciferase reporter assay detected five cis-acting elements for efficient expression of the CAV-1 gene at the region spanning nucleotides -213 to -20 with serially deleted 5'-flanking sequences and site-directed mutants, -123 to -114 was the core promoter. The electrophoretic mobility shift assay demonstrated potential binding of Sp1 protein to this core promoter. The purpose of this study is to systematically elucidate the transcriptional regulation mechanism of porcine CAV-1 and to contribute to the investigation of the interaction between CAV-1 and cholesterol.
    DNA and cell biology 06/2011; 30(7):491-7. · 2.28 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Progestin and adipoQ receptor family member VI (PAQR6) is a new member of the Progestin and adipoQ receptors (PAQRs) family that functions as an important factor in the nongenomic actions of rapid steroid response. Considering the important role of PAQR6 in these nongenomic actions, we determined porcine PAQR6 expression and regulation. We cloned the cDNA of porcine PAQR6 and analyzed its genomic structure. Subcellular localization analysis showed that PAQR6-green fluorescent protein fusion protein was distributed in cytoplasm. Spatial expression patterns analysis revealed that porcine PAQR6 was more highly expressed in small intestinal than in other tissues. To investigate the regulatory mechanism of porcine PAQR6, we cloned approximately 1.5 kb of porcine PAQR6 5'-regulatory region and generated sequential deletion constructs and evaluated their activity in a dual-luciferase reporter assay. The results suggested that the core promoter region of this gene is located in the first exon region from +90 to +241. Site-directed mutagenesis indicated that adiponectin receptor protein 1, E2F transcription factor 1, and Sp1 transcription factor might be important transcription factors for porcine PAQR6. This study is the first attempt to elucidate the transcriptional regulation of porcine PAQR6, which established a foundation for further study and contributed to the deeper investigation of the regulation and function of PAQR6.
    DNA and cell biology 11/2011; 30(11):947-54. · 2.28 Impact Factor

Full-text

View
0 Downloads
Available from