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: 4.44). 04/2006; 80(6):2842-54. DOI: 10.1128/JVI.80.6.2842-2854.2006
Source: PubMed


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.

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Available from: Rebecca Parr, Nov 20, 2014
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    • "The interactions of these FLNSP4 mutants with cav-1 were evaluated by yeast-two-hybrid (Y2H) assays by reacting the Gal4 activation domain fusion protein with the NSP4:Gal4 binding domain fusion proteins. The plasmids were co-transformed into the yeast strain MaV203 as previously described [25]. The growth patterns of the co-transformed yeast confirmed the activation of the three reporter genes of the Y2H assay and established FLNSP4-Ala6 as positive for interacting with cav-1. "
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    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. DOI:10.1186/1743-422X-10-336 · 2.18 Impact Factor
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    • "A ProQuest Two-Hybrid system with Gateway technology (Life Technologies) was used (Parr et al., 2006; Wang and Chory, 2006). The DNA fragment encoding amino acids 1-535 of UL21 was subcloned in-frame into the pDBLeu vector and used as a bait. "
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    ABSTRACT: UL21 of herpes simplex virus type 1 (HSV-1) is an accessory gene that encodes a component of the tegument. Homologs of this protein have been identified in the alpha, beta, and gamma herpesvirus subfamilies, although their functions are unclear. To clarify the functions of UL21, we generated a UL21-null HSV-1 mutant. Growth analysis showed that the synthesis of infectious UL21-null HSV-1 in glial cells was delayed and that the overall yield was low. The plaque sizes of the UL21-null mutant were smaller than those of wild-type HSV-1. We identified several candidate UL21-interacting proteins, including intermediate filaments, by yeast two-hybrid screening. The distribution of glial fibrillary acidic protein (GFAP), which is the main component of intermediate filaments, was altered in UL21-null mutant-infected glial cells compared to wild-type virus-infected cells. These results will help clarify the function of UL21 and broaden our understanding of the life cycle of HSV.
    Frontiers in Microbiology 11/2012; 3:394. DOI:10.3389/fmicb.2012.00394 · 3.99 Impact Factor
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    • "An interaction between NSP4 114−135 and the cellular protein cav-1 [23] [24] [25] as well as caveolae is well established [34]. Additionally, we report the interaction of NSP4 with both the N-and C-termini of cav-1 (aa 2–31 and 161–178, resp.) based on yeast two-hybrid analyses and "
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    ABSTRACT: Rotavirus (RV) NSP4, the first described viral enterotoxin, is a multifunctional glycoprotein that contributes to viral pathogenesis, morphogenesis, and replication. NSP4 binds both termini of caveolin-1 and is isolated from caveolae fractions that are rich in anionic phospholipids and cholesterol. These interactions indicate that cholesterol/caveolin-1 plays a role in NSP4 transport to the cell surface, which is essential to its enterotoxic activity. Synthetic peptides were utilized to identify target(s) of intervention by exploring the NSP4-caveolin-1 and -cholesterol interactions. NSP4(112-140) that overlaps the caveolin-1 binding domain and a cholesterol recognition amino acid consensus (CRAC) motif and both termini of caveolin-1 (N-caveolin-1(2-20),  (19-40) and C-caveolin-1(161-180)) were synthesized. Direct fluorescence-binding assays were employed to determine binding affinities of the NSP4-caveolin-1 peptides and cholesterol. Intracellular cholesterol alteration revealed a redistribution of NSP4 and disintegration of viroplasms. These data further imply interruption of NSP4(112-140)-N-caveolin-1(19-40) and cholesterol interactions may block NSP4 intracellular transport, hence enterotoxicity.
    03/2012; 2012:575180. DOI:10.1155/2012/575180
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