The Rotavirus Enterotoxin NSP4 Directly Interacts with the Caveolar Structural Protein Caveolin-1

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 twohybrid 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.Rotaviruses (RV) cause severe, life-threatening gastroenteritis in children and animals worldwide and in immunocompromised and elderly adults (46). The RV genome is composed of 11 segments of double-stranded RNA that encodes five nonstructural and six structural proteins (17). Nonstructural protein 4 (NSP4), encoded by RV gene 10, initially was identified as an endoplasmic reticulum (ER) transmembrane glycoprotein essential to RV morphogenesis by serving as an intracellular receptor to double-layered particles (DLPs) (5,44,67,66). NSP4 residues 161 to 175 bind the outer coat protein (VP6) of the DLPs, which facilitates translocation into the ER and the addition of two viral proteins, VP7 and VP4, and a transient ER membrane (40,66,67); NSP4 is sufficient for the budding of DLPs into the ER lumen (33). The ER transient viral envelope is eventually removed by an unknown mechanism prior to virus release (40,44). Because the NSP4 sequence lacks classical ER retention signals and does not appear to be retrieved by retrograde transport and the two N-linked, high-mannose glycosylation sites remain sensitive to endoglycosidase H (endo H) digestion, the current tenet is that NSP4 does not enter or traffic through the Golgi (5, 16).In addition to facilitating RV maturation at the ER, NSP4 functions as the first described viral enterotoxin that induces diarrhea in neonatal...

A New N-Terminal Recognition Domain in Caveolin-1 Interacts with Sterol Carrier Protein-2 (SCP-2)

“…SCP-2 and caveolin-1 peptide-specific antibodies were generated in rabbits in our laboratory as described (50)(51)(52)(53). Rabbit anti-human caveolin-1 was purchased from Jackson Immunoresearch Labs Inc. (West Grove, CA) or Transduction Labs (San Diego, CA).…”

“…Total protein in each pellet was quantitated using the BCA protein assay kit (Pierce). Approximately 10 µg of each pellet was separated on a 12% SDS-PAGE, transferred to nitrocellulose and probed with rabbit polyclonal SCP-2 or caveolin-1 antibodies (50,53). The primary antibodies were detected using goat anti-rabbit-HRP antibodies (Pierce) followed by addition of the Super Signal Pico West Chemiluminescent Substrate (Pierce), and bands were visualized using X-OMAT film (Kodak).…”

“…A collection of yeast strains that contain plasmid pairs expressing fusion proteins with a spectrum of interaction strengths [pPC97 (GAL4-DB, LEU2), pPC97-CYH2 s and pPC86 (GAL4-AD, TRP1)] were used as controls (53,(56)(57)(58)(59). The control plasmids pDBIeu and pEXP-AD507 contain only the Gal4 DNA-binding domain (BD) and the Gal4 activating-domain (AD) respectively.…”

“…Due to the high expression level of caveolin-1 in MDCK cells (53), MDCK lysates were prepared as a source of caveolin-1 antigen. Murine L-cells (L arpt-tk-) were cultured as previously described (54).…”

“…SCP-2 and full length caveolin-1 cDNA were cloned into the Invitrogen Gateway™ Destination vectors pDEST22 and pDEST32 (ProQuewst Two hybrid system with Gateway Technology Manual, Invitrogen Life Technologies Inc.) as previously described (53,55). The deletion mutant clones of caveolin-1, Caveolin-1-156, 60-178, Δ60-100 and Δ83-123, were constructed by site-directed mutagenesis using the primers described to the right of the schematic representation of the deletion mutants (Fig.…”