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...

HDL-mediated reverse-cholesterol transport as well as phosphoinositide signaling are mediated through plasma membrane microdomains termed caveolae/lipid rafts. However, relatively little is known regarding mechanism(s) whereby these lipids traffic to or are targeted to caveolae/lipid rafts. Since sterol carrier protein-2 (SCP-2) binds both cholesterol and phosphatidylinositol, the possibility that SCP-2 might interact with caveolin-1 and caveolae was examined. Double immunolabeling and laser scanning fluorescence microscopy showed that a small but significant portion of SCP-2 colocalized with caveolin-1 primarily at the plasma membrane of L-cells and more so within intracellular punctuate structures in hepatoma cells. In SCP-2 overexpressing L-cells, SCP-2 was detected in close proximity to caveolin, 48 +/- 4 A, as determined by fluorescence resonance energy transfer (FRET) and immunogold electron microscopy. Cell fractionation of SCP-2 overexpressing L-cells and Western blotting detected SCP-2 in purified plasma membranes, especially in caveolae/ lipid rafts as compared to the nonraft fraction. SCP-2 and caveolin-1 were coimmunoprecipitated from cell lysates by anti-caveolin-1 and anti-SCP-2. Finally, a yeast two-hybrid assay demonstrated that SCP-2 directly interacts with caveolin-1 in vivo. These interactions of SCP-2 with caveolin-1 were specific since a functionally related protein, phosphatidyinositol transfer protein (PITP), colocalized much less well with caveolin-1, was not in close proximity to caveolin-1 (i.e., >120 A), and was not coimmunoprecipitated by anti-caveolin-1 from cell lysates. In summary, it was shown for the first time that SCP-2 (but not PITP) selectively interacted with caveolin-1, both within the cytoplasm and at the plasma membrane. These data contribute significantly to our understanding of the role of SCP-2 in cholesterol and phosphatidylinositol targeted from intracellular sites of synthesis in the endoplasmic reticulum to caveolae/lipid rafts at the cell surface plasma membrane.

Over 20 years ago, it was reported that liver cytosol contains at least two distinct proteins that transfer phosphatidylinositol in vitro, phosphatidylinositol transfer protein (PITP) and a pH 5.1 supernatant fraction containing sterol carrier protein-2 (SCP-2). In contrast to PITP, there has been minimal progress on the structural and functional significance of SCP-2 in phosphatidylinositol transport. As shown herein, highly purified, recombinant SCP-2 stimulated up to 13-fold the rapid (s) transfer of radiolabeled phosphatidylinositol (PI) from microsomal donor membranes to highly curved acceptor membranes. SCP-2 bound to microsomes in vitro and overexpression of SCP-2 in transfected L-cells resulted in the following: (i) redistribution of phosphatidylinositols from intracellular membranes (mitochondria and microsomes) to the plasma membrane; (ii) enhancement of insulin-mediated inositol-triphosphate production; and (iii) 5.5-fold down regulation of PITP. Like PITP, SCP-2 binds two ligands required for vesicle budding from the Golgi, PI, and fatty acyl CoA. Double immunolabeling confocal microscopy showed SCP-2 significantly colocalized with caveolin-1 in the cytoplasm (punctate) and plasma membrane of SCP-2 overexpressing hepatoma cells (72%), HT-29 cells (58%), and SCP-2 overexpressing L-cells (37%). Taken together, these data show for the first time that SCP-2 plays a hitherto unrecognized role in intracellular phosphatidylinositol transfer, distribution, and signaling.

No abstract

The infectious bronchitis virus (IBV) nucleocapsid protein was expressed as a bacterial fusion protein which differed from the native protein only in the addition of six amino terminus histidine residues. Using RNA overlay protein blot assays, the recombinant protein was shown to bind to RNA fragments specific for the positive sense 3' noncoding end of the IBV genome. At greater concentrations of sodium chloride, the native and fusion nucleocapsid proteins similarly bound to G RNA, representing the terminal 1805 3' nt of the genome, whereas bovine serum albumin and allantoic fluid protein did not bind to labeled G RNA. Competitive gel shift assays with labeled G RNA indicated that the protein interacted with several unlabeled RNA representing sequences at the 3' noncoding end of the IBV genome. Cache Valley virus (a bunyavirus) mRNA transcribed from the small segment cDNA also inhibited the interaction with IBV G RNA to approximately the same extent as homologous unlabeled G RNA, whereas reactions with bovine liver RNA and yeast tRNA were considerably weaker. Whereas yeast tRNA did not inhibit the interaction with the labeled large G RNA, interactions of the fusion protein with EF, a region from 78 to 217 nt from the 3' terminus of the IBV genome, were also apparently weaker than interactions with fragment CD which consisted of the 3' terminal 155 nt. On a molar basis, the latter interacted in an identical nature to a RNA consisting of CD and an additional 1053 nt of plasmid sequences. Compared to bovine liver RNA, unlabeled G specifically inhibited binding to the two smaller labeled IBV fragments in gel shift assays. The binding of IBV nucleocapsid protein with RNA probably requires specific sequences and/or structures that are present on the genome, and may represent a common mechanism used by similar viral nucleoproteins whose functions depend on binding to RNA.

The nucleocapsid protein of the Gray strain of infectious bronchitis virus (IBV) is highly immunogenic and cross-reactive among various distinct serotypes. Recombinant nucleocapsid polypeptide expressed in bacteria with a histidine tag at the amino terminus has been used as antigen for developing an assay to detect IBV-specific antibody. This fusion protein was produced readily in bacteria and easily purified with a nickel column which bound to the histidine tag. Conditions were optimized for using these preparations for an IBV-specific ELISA. Although differences in optical densities could be detected between pre-immune and positive sera for the Ark, Mass, and Gray strains with antigen concentrations between 50 and 0.1 microg per well, the greatest differences could be detected with 3 and 1.5 microg of protein per well. Maximum differences in optical densities between pre-immune and positive sera were obtained using 2.4 microg per well of protein and sera diluted between 1:80 and 1:160. In addition, as little as 30 ng/dot of recombinant nucleocapsid consistently detected IBV-specific sera in immunoblot assays which have convenient field applications.

Previous studies showed that the N-terminal 32 amino acids of sterol carrier protein-2 ((1-32)SCP(2)) comprise an amphipathic alpha-helix essential for SCP(2) binding to membranes [Huang et al. (1999) Biochemistry 38, 13231]. However, it is unclear whether membrane interaction of the (1-32)SCP(2) portion of SCP(2) is in itself sufficient to mediate intermembrane sterol transfer, possibly by altering membrane structure. In this study a fluorescent sterol exchange assay was used to resolve these issues and demonstrated that the SCP(2) N-terminal peptide (1-32)SCP(2) did not by itself enhance intermembrane sterol transfer but potentiated the ability of the SCP(2) protein to stimulate sterol transfer. Compared with SCP(2) acting alone, (1-32)SCP(2) potentiated the sterol transfer activity of SCP(2) by increasing the initial rate of sterol transfer by 2.9-fold and by decreasing the half-time of sterol transfer by 10-fold (from 11.6 to 1.2 min) without altering the size of the transferable fractions. The ability of a series of SCP(2) mutant N-terminal peptides to potentiate SCP(2)-mediated sterol transfer was directly correlated with membrane affinity of the respective peptide. N-Terminal peptide (1-32)SCP(2) did not potentiate intermembrane sterol transfer by binding sterol (dehydroergosterol), altering membrane fluidity (diphenylhexatriene) or membrane permeability (leakage assay). Instead, fluorescence lifetime measurements suggested that SCP(2) and (1-32)SCP(2) bound to membranes and thereby elicited a shift in membrane sterol microenvironment to become more polar. In summary, these data for the first time showed that while the N-terminal membrane binding domain of SCP(2) was itself inactive in mediating intermembrane sterol transfer, it nevertheless potentiated the ability of SCP(2) to enhance sterol transfer.

Previous studies indicated that the nucleocapsid (N) protein of infectious bronchitis virus (IBV) interacted with specific sequences in the 3' non-coding region of IBV RNA. In order to identify domains in the N protein that bind to RNA, the whole protein (409 amino acids) and six overlapping fragments were expressed as fusion polypeptides with six histidine-tags. Using gel shift assays, the intact N protein and amino polypeptides, from residues 1 to 171 and residues 1 to 274, and carboxyl polypeptides, extending from residues 203 to 409 and residues 268 to 407, were found to interact with positive-stranded IBV RNA representing the 3' end of the genome. The two 32P-labeled probes that interacted with N and the amino and carboxyl fragments of N were RNA consisting of the IBV N gene and adjacent 3' non-coding terminus, and RNA consisting of the 155-nucleotide sequences at the 3' end of the 504-nt 3' untranslated region. In contrast, the polypeptide fragment from the middle region, residues 101-283, did not interact with these 3' IBV RNAs. The binding site in the amino region of N was either not present or only partially present in the first 91 residues because no interaction with RNA was observed with the polypeptide incorporating these residues. Cache Valley virus N expressed with a histidine tag, bovine serum albumin, and the basic lysozyme protein did not shift the IBV RNA. The lower molarities of the carboxyl fragment compared with residue 1-274 fragment needed for equivalent shifts suggested that the binding avidity for RNA at the carboxyl domain was greater than the amino domain.