as the leading cause of gastroenteritis in young children under the age of five , rotavirus ( rv ) infections annually are responsible for approximately 600,000 deaths worldwide [ 1 , 2 ] . during infection ,
the rv nonstructural protein 4 ( nsp4 ) functions as a viral enterotoxin by binding an extracellular receptor and activating a signal transduction pathway which increases intracellular calcium ( [ ca]i ) levels through the release of er calcium stores [ 3 , 4 ] .
this increase in [ ca]i induces secretory chloride currents which result in diarrhea , but only when initiated from the exofacial leaflet of the plasma membrane ( pm ) .
the initiated increases in intracellular , calcium levels fail to induce the chloride secretory response [ 46 ] .
traditionally defined as an er glycoprotein , nsp4 contains a single transmembrane domain that serves to anchor the protein into the er membrane , such that a short n - terminal domain ( amino acids [ aa ] 124 ) remains within the lumen of the er while the longer c - terminus ( aa 45175 ) extends into the cytoplasm .
interactions with numerous viral and cellular proteins occur within the extended c - terminal tail of nsp4 [ 812 ] .
also contained within the c - terminal cytoplasmic tail is an amphipathic -helix ( aah ) , coiled - coil domain ( aa 95137 ) [ 13 , 14 ] .
cross - linking and crystallographic experiments reveal that this region of nsp4 primarily oligomerizes into dimers and tetramers and contains a cation - binding site [ 1315 ] . residing within the amphipathic -helix ,
coiled - coil domain is the enterotoxic peptide region ( aa 114135 ) as well as the caveolin-1 ( cav-1 ) binding domain   and a putative cholesterol recognition amino acid consensus ( crac ) sequence .
l / v-(x)(1 - 5)-y-(x)(1 - 5)-r / k- , where ( x)(1 - 5 ) represents between one and five unspecified residues .
however , the presence of such a sequence does not assure an interaction with cholesterol , as this sequence is highly variable and likely is not the only requirement for cholesterol binding .
yet a number of viral proteins bind cholesterol , including the hiv-1 gp41 transmembrane glycoprotein [ 17 , 20 ] , the influenza m2 protein , and the f1 subunit of the fusion protein of sendai virus .
there is compelling evidence that the enterotoxic peptide , nsp4114 - 135 , and the full - length nsp4 protein interact with cav-1 . in vivo laser scanning confocal microscopy ( lscm ) colocalizations , fluorescent energy transfer ( fret ) analyses , and co - immunoprecipitation data from our laboratory verify that nsp4 binds cav-1 , the main constituent protein of caveolae , at multiple sites within the cell , including the pm . yeast two - hybrid and in vitro binding assays confirm an interaction between nsp4 and cav-1 and map the cav-1 binding domain to nsp4114 - 135 . using deletion and site - directed mutagenesis ,
the binding site delineated to three hydrophobic residues in the enterotoxic region indicating that nsp4 and cav-1 associate via a hydrophobic interaction ( unpublished ) .
additional studies reveal that the cav-1 binding site for nsp4 maps , to both the n- and c - termini of cav-1 , residues 231 and 161178 , respectively .
cav-1 is an intracellular , 21  kd protein bound by the inner leaflet of the pm in a hairpin - like structure such that both the n- and c - termini are oriented towards the cytoplasm [ 23 , 25 ] .
numerous signaling molecules , such as those involved in calcium signaling , are localized to caveolae and their function appears to be dependent on interaction with cav-1 [ 16 , 26 , 27 ] .
cav-1 also binds cholesterol , which has been shown to be vital to caveolae biogenesis and the transport of cholesterol to caveolae at the pm [ 2831 ] .
purified nsp4 and nsp4114 - 135 peptides interact with caveolae - like model membranes , that is , those that have a high radius of curvature and are rich in anionic phospholipids and cholesterol [ 32 , 33 ] . upon interaction with model membranes , both the protein and the peptide undergo a change in secondary structure characterized by an increase in -helix formation .
additional secondary structure studies of other nsp4 peptides that overlap the enterotoxic peptide and cav-1 binding domain confirm that this region is important for membrane interactions .
when caveolae are isolated from enriched pm fractions of rv - infected mdck cells , the full - length , fully - glycosylated nsp4 is present in the isolated caveolae fractions verifying transport of nsp4 to the pm and caveolae .
it is possible that the transport of nsp4 to caveolae occurs through an interaction with cav-1 and cholesterol , although additional studies are needed to for verification .
our studies reveal the exposure of the nsp4 c - terminus and the enterotoxic region , but not the n - terminus , on the outer pm leaflet and subsequent release into culture media at a time when the membrane is intact as verified by a lack of exposure of cav-1 and other intracellular markers [ 3436 ] .
in this paper , we have extended these studies using synthetic peptides to address the following questions .
( ii ) do the two termini of cav-1 interact with one another and thus facilitate binding of nsp4 to both termini ?
( iv ) do the overlapping nsp4-cav-1 binding , crac motif , and enterotoxin regions interact with cholesterol ?
( v ) does alteration(s ) of nsp4-cholesterol binding have a biological effect ? to answer these questions , we employed peptide - peptide and -cholesterol direct fluorescence - binding assays to ascertain the interaction and determine the binding affinities ( kd ) .
we also employed circular dichroism ( cd ) to investigate secondary structural changes upon interaction of the peptides in the absence or presence of model membranes .
trifluoroacetic acid ( tfa ) , 1-hydroxy - benzotriazole ( hobt ) , and o - benzotriazole - n , n , n,n-tetramethyluronium - hexafluoro - phosphate ( hbtu ) were purchased from american bioanalytical ( natick , ma ) .
n , n - diisopropylethylamine ( dipea ) and n , n-diisopropylcarbodiimide ( dipcidi ) were purchased from creosalus ( louisville , ky ) .
thioanisole , ethanedithiol , and anisole were purchased from emd chemicals ( gibbstown , nj ) .
cholesterol , sephadex g-25 , and trifluoroethanol ( tfe ) were purchased from sigma ( st . louis , mo ) .
phospholipids dops ( 1,2-dioleoyl - sn - glycero-3-[phosphor - l - serine ] ) and popc ( 1-palmitoyl-2-oleoyl - sn - glycero-3-phosphocholine ) were purchased from avanti polar lipids ( alabaster , al ) .  
antibodies specific to the nsp4 peptide aa 150175 ( nsp4150 - 175 ; deduced from the simian rotavirus sa11 nsp4 sequence ) were generated in rabbits by immunizing with peptide cross - linked to keyhole limpet hemocyanin as previously described . anti - nsp4150 - 175 was affinity purified against the inoculating peptide linked to preactivated cyanogen bromide sepharose 4b beads according to the manufacturer ( amersham pharmacia biotech , piscataway , nj ) .
guinea pig anti - nsp5 , a viroplasm ( virus factory in the cell ) marker , was a gift from dr .
oscar burrone ( international center for genetic engineering and biotechnology , trieste , italy ) .
f(ab)2 fragments of goat anti - guinea pig igg - cy3 and goat anti - rabbit igg - cy2 were purchased from jackson immuno research ( west grove , pa ) .  
ht29.f8 cells are a spontaneously polarizing cell line that was cloned from the human adenocarcinoma ht29 intestinal cell line .
cells were maintained in dulbecco 's modification of eagle 's media ( dmem ) with 4.5  g / l glucose , 2  mm l - glutamine , and 1  mm sodium pyruvate ( mediatech , inc . ,
herndon , va ) and supplemented with 5% fetal bovine sera , 5% serum supreme , and antibiotics ( 100  u / l penicillin , 100  ug / l streptomycin , 0.25  ug / l amphotericin b ) ( cambrex , east rutherford , nj ) . for infection , cells were incubated in the absence of sera exactly as described and exposed to virus for 1  h ( multiplicity of infection ( moi ) = 2 ) [ 23 , 35 , 36 ] .
all peptides ( table 1 ) were synthesized by fluorenylmethoxycarbonyl ( fmoc ) solid - phase chemistry with either 1-hydroxy - benzotriazole ( hobt ) , o - benzotriazole - n , n , n,n-tetramethyluronium - hexafluoro - phosphate ( hbtu ) and n , n - diisopropylethylamine ( dipea ) , or hobt and n , n-diisopropylcarbodiimide ( dipcidi ) activation using the model 90 peptide synthesizer ( advanced chemtech ; louisville , ky ) .
following synthesis , the peptides were cleaved from the solid resin support and all side - chain protecting groups were removed by the addition of reagent r ( 90% trifluoroacetic acid ( tfa ) , 5% thioanisole , 3% ethanedithiol , and 2% anisole ) . the peptide /
reagent r cleavage mixture was incubated at rt for a maximum of 2 hours with gentle mixing . to separate the peptides from the solid support
, the mixture was filtered through a sintered glass filter into a 50  ml conical tube containing cold diethyl ether in a dry ice / ethanol bath . following 2 - 3 rinses of the filter with tfa , the ether solution containing
the supernatant was removed and additional cold diethyl ether was added to the peptide pellet .
the mixture was allowed to cool in the dry ice / ethanol bath for several minutes before repelleting .
this step was repeated an additional two times ; the crude peptide was dried under n2 , resolubilized in 10% acetic acid and lyophilized .
the lyophilized crude peptide was resuspended in 510% acetic acid and purified from organic contaminants and incomplete peptide fragments by gravimetric gel filtration chromatography ( sephadex g25 medium ) .
the eluted peptide was lyophilized and further purified by reverse - phase hplc using either a reverse phase c4 delta pak column ( waters chromatography division , milford , ma ) or a reverse phase c18 column ( beckman - coulter , fullerton , ca ) depending on the hydrophobicity of the peptide .
eluted peaks were lyophilized and characterized by matrix - assisted laser desorption / ionization ( maldi ) mass spectrometry ( laboratory for biological mass spectrometry , department of chemistry , texas a&m university , college station , tx ) .  
small unilamellar vesicles ( suvs ) composed of the neutral lipid popc , cholesterol , and the anionic phospholipid dops in the molar ratio 55  :  35  :  30 were prepared following a previously published protocol .
this membrane composition was chosen as a result of previous data showing that this composition and curvature promote an interaction with nsp4-specific peptides as observed by an increase in -helix formation [ 32 , 33 ] .
stock solutions of the lipids dissolved in chloroform were mixed together in the proper ratio in an amber glass vial .
the solvents were removed under n2 with constant rotation so that the dried lipids formed a thin film on the wall of the glass vial .
the vial containing the dried lipids was further dried under vacuum for a minimum of 4  h. the dried lipids were resuspended in 10  mm mops buffer , ph 7.4 ( filtered through a 0.45  m filter ; millipore , bedford , ma ) , vortexed and bath sonicated .
the resulting multilamellar membrane suspension was sonicated with a microprobe under n2 at 4c at 2  min intervals , followed by 1  min pauses to prevent overheating of the lipid solution .
the sonicated lipid solution then was centrifuged at 110,000  g for 4  h to remove any multilamellar vesicles and titanium debris from the sonicator probe .
the lipid concentration of the suv solution was determined by a standard phosphate assay as described .
briefly , the suv solutions were mixed with a 10% magnesium nitrate ( mg(no3)2 ) solution and heated for 30  min at 120c .
each sample was ashed in a flame , 600  l of 0.5  n hcl was added , and the samples were heated in boiling water for 15  min .
each sample was mixed with 1400  l of a 10% ascorbic acid solution that contained 6 parts of a 0.42% ammonium molybdate solution in 1  n sulfuric acid ( h2so4 ) and incubated for 20  min at 45c before reading the absorbance at 660  nm ( a660 ) .
lipid concentrations of the suvs were determined by comparison to a standard curve of 0200  nmol phosphate .
a fluorescence - binding assay was utilized to investigate direct binding of the nsp4112 - 140 peptide with each of the cav-1 peptides ( n - cav2 - 20 , n - cav19 - 40 , cav68 - 80 , and c - cav ) [ 20 , 39 ] .
all of the cav-1 peptides directly were labeled with a cy3 fluorescent dye , using the cy3 monoreactive dye pack from amersham biosciences according to the manufacturer 's protocol .
briefly , each peptide was dissolved in 50  nm phosphate buffer , ph 7.0 , at a concentration of 1  mg / ml , and added to a vial containing lyophilized cy3 dye .
the solution gently was mixed and incubated at 25c for at least 2  h with intermittent mixing .
labeled peptides were separated from unlabeled peptides by sephadex g25 gel filtration chromatography . to demonstrate a direct peptide - peptide interaction between the nsp4112 - 140 peptide and each of the cav-1 peptides , increasing concentrations of nsp4112 - 140 ( 10500  nm ) were added to 25100  nm of the cy3-labeled cav-1 peptides in 2  ml of phosphate - buffered saline ( pbs , ph 7.4 ) in either the absence or presence of small unilamellar vesicles ( suvs , 1  m ) .
the cy3 fluorophore was excited at 550  nm and emission spectra were scanned from 560 to 700  nm .
fluorescence emission spectra were obtained at 25c with a pc1 photon - counting spectrofluorometer with excitation and emission slit widths of 1.0 ( iss , inc . ) .
calculation of the dissociation constants ( kd ) was performed from the titration curves plotted as quenching in cy3-peptide fluorescence intensity ( f0  f , where f0 and f represented the cy3-cav-1 peptide fluorescence intensities in the absence and presence of nsp4112 - 140 or cav-1 peptide , resp .
the kd also was obtained by a reciprocal plot of 1/(1  f / fmax ) and cl / f / fmax according to the following equation : y = bx + y0 , where f is the fluorescence intensity at a given concentration of ligand , fmax is the maximal fluorescence obtained , and cl is the ligand concentration .
similarly , the nsp4112 - 140-cholesterol interaction and binding affinity were determined by a direct fluorescent binding assay in which the peptide was labeled with a cy5 fluorophore using the cy5 monoreactive dye pack from amersham biosciences ( piscataway , nj ) .
a concentrated cholesterol stock solution ( 20  um ) was prepared by dissolving the cholesterol in ethanol . to analyze a binding interaction between nsp4112 - 140 and cholesterol , an increasing quantity of cholesterol ( 535  nm )
was added to 10  nm of the cy5-labeled nsp4112 - 140 in 2  ml of pbs ( ph 7.4 ) .
the cy5 fluorophore was excited at a wavelength of 649  nm , and the emission spectra were scanned from 655 to 720  nm using a pci photon - counting spectrofluorometer ( iss , inc .
fluorescence emission spectra were obtained at 25c with excitation and emission slit widths of 1.0 .
as with the peptide - peptide interactions , spectra were corrected for background and maximal fluorescence intensities were measured .
calculation of the kd was determined from the plotted titration curves whereby the cy5-peptide fluorescence intensity was quenched and measured as a function of cholesterol concentration ( f0f , where f0 and f represented the cy5- nsp4112 - 140 fluorescence intensities in the absence and presence of cholesterol , resp .
the sigmoidal curves also were fitted to a hill plot according to the following equation : y = ax/(c + x ) , where y and x correspond to ( f0f ) and the ligand concentration at each point , while a , b , and c represent the maximum binding ( bmax ) , the number of binding sites ( n ) , and the kd value , respectively .
kd values were calculated with the sigmoidal function of sigma plot ( spss , chicago , il ) utilizing the hill plot feature .
the secondary structures of the nsp4 and cav-1 specific peptides were determined by circular dichroism following a previously published protocol [ 32 , 33 ] .
briefly , each peptide ( 1535  m ) was suspended in 10  mm potassium phosphate buffer , ph 7.4 , or 50% trifluoroethanol ( tfe ) to promote a hydrophobic environment and folding of the peptide , in the presence or absence of lipid vesicles ( 1  mm )
peptide concentrations were determined by amino acid analysis ( protein chemistry laboratory , department of biochemistry , texas a&m university , college station , tx ) .
cd spectra were obtained in a 1  mm circular quartz cell using a model j-710 jasco spectropolarimeter ( jasco , easton , md ) or in a 1  mm rectangular quartz cell in a model 202 aviv spectrometer ( aviv biomedical , lakewood , nj ) .
the spectra for each of the peptides were recorded from 185  nm to 260  nm , with a step resolution of 1  nm , speed of 50  nm / min , response of 1  sec , bandwidth of 2.0  nm , and sensitivity of 10  mdeg .
data were averaged from 5 scans , background subtracted , smoothed , and converted into mean residue molar ellipticity [  ] ( deg  cm / dmol ) . to determine the percent -helix for each of the nsp4- and cav-1 specific peptides ,
the following equation was used : 222 = ( fh  /n)[h  222 ] [ 12 , 21 , 22 ] . in this equation
, 222 is the mean residue molar ellipticity at 222  nm , fh is the fraction in -helical form ,  is the number of helices ,  is a wavelength - specific constant with a value of 2.6 at 222  nm , n is the number of residues in the peptide , and h  222 is the molar ellipticity for a helix of infinite length at 222  nm , that is , 39,500  deg  cm / dmol .
mdck cells were infected with rv at a multiplicity of infection ( moi ) of 2 and incubated for 12 hpi .
the cells were washed and treated in triplicate with the cholesterol altering drugs , fillipin , and nystatin   exactly as described .
chlorpromazine [ 40 , 42 ] and nocodazole   were utilized as negative and positive controls , respectively .
cells were fixed with methanol  :  acetone ( 1  :  1 ) at 20c for 10  min , blocked with 5% dry milk for 20  min , incubated with anti - nsp4150 - 175 and anti - rabbit - igg - fitc , and examined under a uv microscope ( zeiss ) .
the cell surface carefully was observed and the presence or absence of fluorescence carefully noted .
primary and secondary antibody controls were negative . to ensure the lack of drug toxicity , a separate set of cells were treated and then stained with trypan blue .
we previously demonstrated that nsp4114 - 135 interacts with both the n- and c - termini of cav-1 using yeast-2-hybrid and pull - down assays , which is unusual . to further investigate this finding and determine if there was a preferential binding of nsp4 to the n- or c - terminus of cav-1 , the nsp4112 - 140 peptide that encompasses aa 114135 and various cav-1 peptides ( n - cav2 - 20 , n - cav19 - 40 , and c - cav161 - 178 ) were utilized in a direct fluorescence - binding assay .  
upon titration with increasing concentrations of nsp4112 - 140 , each of the cy-3-labled n- or c - terminal cav-1 peptides showed an increase in fluorescence intensity at 565  nm , indicative of an interaction .
plots of nsp4112 - 140 concentrations versus maximum fluorescence intensities revealed saturated binding curves for each of the cav-1 peptides ( figure 1 ) .
linear reciprocal plots were used to calculate the kd for each peptide - peptide pair ( table 2 ) .
the calculated kd values indicated that nsp4112 - 140 bound the n - cav19 - 40 peptide with the strongest affinity ( 40  10  nm ) , while it bound the c - cav161 - 178 peptide with the weakest affinity ( 217  35  nm ) . the n - cav2 - 20 peptide bound with intermediate affinity ( 85  6  nm ) .
none of the cav-1 peptides bound to the control peptide corresponding to the c - terminus of nsp4 ( nsp4150 - 175ala ) , which is known to not bind cav-1 .
we now show that there is a preferential interaction of nsp4112 - 140 with the n - terminus of cav-1 , which , to our knowledge , is the first report of a preferential binding to one termini of cav-1 .
previous cd analyses of nsp4 peptides corresponding to the cav-1 binding domain show that aa 114135 interacts with model membranes ( suvs ) [ 32 , 33 ] .
therefore , nsp4-cav-1 peptide - peptide interactions also were investigated in the presence of the same suv membranes .
the suvs were mixed with each of the cy3-labeled cav-1 peptides in pbs and then titrated with increasing concentrations of nsp4112 - 140 ( figure 2 ) .  
the presence of lipid vesicles had no effect on the interaction of nsp4112 - 140 with either n - cav19 - 40 or c - cav161 - 178 ( figures 2(b ) and 2(c ) ) .
binding affinities in the presence of membranes were similar to those calculated in the absence of membranes ( table 2 ) .
however , the presence of the suvs resulted in an increased affinity between nsp4112 - 140 and n - cav2 - 20 .
the calculated kd value was determined to be 26  4  nm , an approximate 3-fold increase over that observed in the absence of membranes ( 85  6  nm ) and about a1.5-fold increase over the kd of n - cav19 - 40 in the absence of membranes ( 40  10  nm ) .
these results strongly suggest that the presence of lipid vesicles enhances binding between nsp4112 - 140 and the extreme n - terminus of cav-1 ( aa 2 - 20 ) , but not the adjacent n - terminal cav-1 peptide , n - cav19 - 40 .
to evaluate the mechanism of the interaction of nsp4 with both cav-1 termini , we examined whether the two cav-1 termini bound one another to facilitate the interaction with nsp4 by employing the fluorescence - binding assay .
c - cav161 - 178 was labeled with a cy3-fluorophore and titrated with either the n - cav2 - 20 or n - cav19 - 40 peptide .
titration of c - cav161 - 178 with increasing concentrations of either n - terminal peptide resulted in no change in fluorescence intensity at 565  nm , indicative of a lack of association ( figure 3 ) .
hence , we propose that the n- and c- termini of cav-1 do not bind one another and the interaction between nsp4 and the cav-1 termini does not result from an initial binding between the cav-1 n- and c - termini . to our knowledge , this is the first report of the cav-1 termini not interacting .
additional studies are needed to dissect the mechanism and implications of both cav-1 termini binding nsp4 .
the structure of the nsp4112 - 140 peptide was first determined in an aqueous buffer ( 10  mm potassium phosphate buffer , ph = 7.4 ) ( figure 4 , dark circles ) .
the cd spectrum showed double minima at 208 and 222  nm and a single maximum peak at 190  nm , indicative of some -helical secondary structure . using the molar ellipticity value at 222  nm , the -helical content of the nsp4112 - 140 peptide was calculated to be 30.6  2.4% .   to enhance innate peptide structure , the secondary structure of nsp4112 - 140
was determined in 50% tfe , a hydrophobic solvent that promotes intramolecular hydrogen bonding ( figure 4 , open circles ) .
the cd spectrum showed a dramatic increase in -helix formation of the peptide , as evidenced by an increase in both the negative molar ellipticity at 208 and 222  nm and the positive molar ellipticity at 190  nm .
the -helical content was calculated using the molar ellipticity value at 222  nm and determined to be 79.6  4.6% ( table 3 ) .  
previous cd experiments with the enterotoxic peptide , nsp4114 - 135 demonstrate that the -helical content increases in the presence of suvs containing popc , cholesterol , and dops at a 55  :  35  :  10 molar ratio [ 32 , 33 ] . to determine the secondary structural changes that occur in the presence of membranes , nsp4112 - 140 was mixed with suv membranes and changes in -helical content
the cd spectrum of the nsp4112 - 140 peptide in the presence of 1  mm suv showed nearly a 2-fold increase in -helix formation over that in aqueous buffer , as evidenced by the increase in both the negative molar ellipticity as 208 and 222  nm and the positive molar ellipticity at 190  nm ( figure 5 ) .
the -helical content was calculated using the molar ellipticity value at 222  nm and determined to be 57.9  1.6% ( table 3 ) .
the secondary structures of the cav-1 peptides were determined and evaluated in the presence of 50% tfe and 1  mm suvs ( 55  :  35  :  10 ; popc  :  cholesterol  :  dops ) by cd ( table 3 ) . in aqueous buffer ,
the -helical content of n - cav2 - 20 , n - cav19 - 40 , and cav68 - 80 was quite similar , at 18.9% , 20.7% , and 23.4% , respectively ( figures 6(a)6(c ) , dark circles ) .
the -helical contents of the c - terminal peptide ( c - cav161 - 178 ) was approximately twice ( 43.4% ) that of the n - terminal peptides ( table 3 ) .
when placed in 50% tfe , the -helical content of n - cav2 - 20 , n - cav19 - 40 , cav68 - 80 , and c - cav161 - 178 peptides were calculated to be 16.5% , 33.4% , 23.1% , and 67.1% respectively ( figure 6 , open circles ) . only n - cav19 - 40 and c - cav161 - 178 showed an increase in -helix formation in the presence of 50% tfe .
when mixed with the suvs , none of the cav-1 peptides demonstrated a change in -helix formation over that seen in aqueous buffer ( figure 6 , dark triangles ) . this result was anticipated as none of these peptides corresponded to a cav-1 membrane interacting or transmembrane domain .
analyses by direct fluorescence - binding assays revealed that n - cav19 - 40 bound nsp4112 - 140 with a stronger affinity than c - cav161 - 178 .
we therefore investigated whether structural changes occurred upon the association of the nsp4 and cav-1 peptides , in particular with the amino terminus . each of the cav-1 peptides ( n - cav2 - 20 , n - cav19 - 40 , cav68 - 80 , and c - cav161 - 178 ) was mixed with the nsp4112 - 140 peptide and was analyzed by cd .
cav68 - 80 was used as a negative control , as previous studies have shown that nsp4 does not interact with this region of cav-1 [ 23 , 24 ] .
analyses of the mixing experiments were achieved by comparing the spectrum of each peptide - peptide mixture ( observed ) with the sum of the individual spectra of each peptide ( theoretical ) . the -helical content was calculated and significant differences ( student 's t - test , p < 0.05 ) between the observed and theoretical values indicated a change in secondary structure and binding between the two peptides .
however , a lack of secondary structural alterations upon mixing the peptides did not negate binding between the nsp4112 - 140 and the cav-1 peptide being tested . while direct fluorescent binding assays revealed the affinity of each of the peptide - peptide interactions , the cd experiments disclosed the extent to which the interaction(s ) caused a change in the secondary structure .  
when each of the cav-1 peptides was mixed with nsp4112 - 140 in an aqueous buffer , only the nsp4112 - 140-c - cav161 - 178 mixture showed a significant difference between the observed and theoretical values ( 25.0  1.7% versus 35.3  1.6%  -helix ) ( figure 7(d ) ; table 4 ) .
no change in secondary structure was observed for the nsp4112 - 140-n - cav2 - 20 ( 21.7  0.8% versus 23.6 
2.6% ) , the nsp4112 - 140-n - cav19 - 40 ( 23.8  1.2% versus 24.4  0.6% ) , or the nsp4112 - 140-cav68 - 80 negative control ( 24.2  0.3% versus 23.9  0.7% ) mixtures even though n - cav19 - 40 had the lowest kd ( figures 7(a)7(c ) ; table 4 ) . similarly ,
when each peptide - peptide pair was placed in 50% tfe , only the nsp4112 - 140-c - cav161 - 178 mixture showed a significant change in secondary structure ( 44.2  10.6% versus 67.8  3.2% ) ( table 4 ) .
these data confirmed the weak binding between nsp4112 - 140 and c - cav161 - 178 and verified that secondary structure alterations do not always occur upon an interaction .
cav-1 is critical to both the structure and function of caveolae , is the major constituent of caveolae , and is controller of caveolae formation .
nsp4 is present in isolated caveolae from rv - infected cells , and together with nsp4114 - 135 , preferentially interacts with caveolae - like model membranes .
therefore we evaluated the extent the suvs influenced secondary structure alterations of the nsp4-cav-1 peptide - peptide interactions .
even though the nsp4112 - 140-c - cav161 - 178 peptide pair showed the most change in -helical content in aqueous buffer and 50% tfe , in the presence of suvs there was not a significant conformational change indicating that this interaction was not enhanced by the presence of model membranes ( table 4 ) .
similarly , the nsp4112 - 140-n - cav19 - 40 showed the strongest affinity by direct fluorescent binding assays ( table 2 ) , yet this interaction failed to result in a significant change in secondary structure when mixed with membranes . the observed
-helical content did not vary from the expected theoretical value , regardless of whether the peptide mixture was in aqueous buffer , 50% tfe , or mixed with suvs .
these results suggest that the strong binding between nsp4112 - 140 and n - cav19 - 40 required neither a change in structure nor the presence of specific lipids . only the nsp4112 - 140-cav68 - 80 ( negative control ) mixture showed a significant change in the -helical content in the presence of the suvs ( 31.3  0.2% versus 37.5  1.9% ) ( figure 8(c ) ; table 4 ) .
it is currently unclear why the control peptide mixture resulted in an alteration in helical structure and requires further study .
the nsp4112 - 140 peptide , which contains the crac motif , was labeled with a cy5-fluorophore and titrated with cholesterol in a direct fluorescence - binding assay .
the maximum solubility of cholesterol in water is 4.7  m and the critical micellar concentration ( cmc ) is 2540  nm , so cholesterol concentrations were kept below these limits in the binding assays .   in the absence of cholesterol ,
the cy5-nsp4112 - 140 peptide showed maximum fluorescence at 665  nm ( figure 9 ) .
upon titration with increasing concentrations of cholesterol ( 535  nm ) , cy5-nsp4112 - 140 showed a decrease in fluorescence intensity , indicative of an interaction .
measurements of fluorescent intensities at 665  nm of cy5-nsp4112 - 140 in the presence of increasing concentrations of cholesterol were corrected for background ( cy5-nsp4112 - 140 in buffer alone ) and plotted as a function of cholesterol concentration , demonstrating a saturable binding curve ( figure 9(b ) ) .
the kd of cholesterol for nsp4112 - 140 was calculated to be 8  1  nm .
a hill plot revealed a single cholesterol binding site ( n = 1 ) in the 28 residue peptide . a control peptide ( nsp4150 - 175ala ) , corresponding to the c - terminus of nsp4 and lacking the crac motif , failed to demonstrate a change in fluorescence intensity in the presence of cholesterol , indicative of a lack of binding ( figures 9(c ) and 9(d ) ) .
we evaluated the effect(s ) of drugs known to disrupt cholesterol ( fillipin , nystatin , lovastatin ) [ 40 , 41 , 45 ] , hence caveolin / caveolae transport properties , an inhibitor of clathrin - coated pit processes ( chloropromazine ) [ 40 , 42 ] , and a disruptor of the cytoskeleton ( nocodazole ) .
cells were treated at the recommended effective dose , evaluated with trypan blue to ensure a lack of toxicity , and nsp4 transport to the cell periphery was noted . whenever cholesterol trafficking was disrupted , nsp4 likewise failed to transport to the cell surface as evidenced by a lack of peripheral staining ( table 5 ) .
several reports highlight the use of peptides as a means of investigating the interactions of full - length proteins [ 3 , 4750 ] .
this study similarly demonstrated the utility of synthetic peptides by showing differential binding of nsp4112 - 140 to the cav-1 termini and disclosing secondary structural changes that were not necessary for the interaction between the nsp4 and cav-1 binding domains .
the kd was calculated as 8.0  1  nm and a hill plot revealed a single binding site for cholesterol when reacted with nsp4112 - 140 . an interaction between nsp4114 - 135 and the cellular protein cav-1 [ 2325 ] as well as caveolae is well established .
additionally , we report the interaction of nsp4 with both the n- and c - termini of cav-1 ( aa 231 and 161178 , resp . ) based on yeast two - hybrid analyses and in vitro peptide binding assays . herein , we confirmed the interaction between nsp4 and the n- and c - termini of cav-1 and calculated the binding affinities of those interactions using peptides and direct fluorescence - binding assays .
nsp4112 - 140 , which encompasses the cav-1 binding domain and overlaps the aah and enterotoxic peptide , was utilized with cav-1 peptides corresponding to the n- ( n - cav2 - 20 and n - cav19 - 40 ) and c- ( c - cav161 - 178 ) termini and a central region ( cav68 - 80 ) . calculations of the binding affinities revealed that nsp4112 - 140 bound the n - terminus of cav-1 ( n - cav19 - 40 ) with an affinity over 5 times stronger than that of the c - terminus ( c - cav161 - 178 ) ( 40  10  nm versus 217  35  nm , resp . ) . when mixed with suvs
, the kd values showed an increase in binding affinity for the nsp4112 - 140-n - cav2 - 20 interaction such that this peptide - peptide interaction occurred with similar affinity ( 26  4  nm ) as that observed between nsp4112 - 140 and n - cav19 - 40 in the absence of membranes ( 40  10  nm ) .
no change , however , was observed in the binding affinities between nsp4112 - 140 and n - cav19 - 40 or c - cav161 - 178 in the presence of the lipid vesicles indicating that lipids were not involved in these interactions .
the functional significance of the preferential binding to n - terminal cav-1 currently is unknown . while we did not specifically analyze the oligomerization of the nsp4112 - 140 peptide in this study , our previous data on a similar and overlapping peptide ( nsp4114 - 135 ) showed oligomerization / aggregation at a peptide concentration of ~50  um .
the peptide concentration utilized in the binding assays stayed within the nm range , well below the 50  um concentration at which the oligomerization occurred with the nsp4114 - 135 peptide .  
following confirmation of an interaction between nsp4 and both termini of cav-1 , we analyzed potential secondary structural changes upon peptide - peptide association by cd .
previous secondary structure analysis of nsp4114 - 135 yielded an estimate of 37%  -helix for the peptide [ 32 , 33 ] .
analyses of the secondary structure of the full - length nsp4 protein showed ~26%  -helix when analyzed by cd . to our knowledge , all other structural studies of nsp4 were completed on specific fragments of nsp4 and not the full - length protein .
cd analysis of nsp4112 - 140 showed 30%  -helix , which is within the range calculated for the enterotoxic peptide and the full - length protein . when placed in the presence of suvs , which mimics the more biologically relevant caveolae environment , nsp4112 - 140 demonstrated an increase in -helical content .
this increase in -helix formation indicated that this peptide behaved similarly to the nsp4114 - 135 peptide and the presence of membranes influenced the structure .
to date the crystallographic structure of cav-1 has not been resolved ; however , limited secondary structural studies and bioinformatics analyses have provided insight into its folded conformation .
cd analysis of aa 1101 reveals that the n - terminus contains 20%  -helix , with aa 7996 constituting the -helix portion , while aa 178 likely lacks significant secondary structure .
an additional study , which used a bioinformatics approach , notes that aa 95101 contains -helical structure , while aa 8494 forms two -strands .
further , cav-1 is known to form high molecular weight oligomers that requires the cav-1 scaffolding domain ( csd , aa 82101 ) [ 5254 ] .
additionally , the c - terminus is palmitoylated on three cysteines , which helps anchor the protein to the membrane and may thus explain the preferential binding of nsp4 to the n - terminus of cav-1 [ 5456 ] .
peptides corresponding to the n - terminus of cav-1 displayed ~20%  -helix , whereas the cav-1 c - terminal peptide unexpectedly showed 43%  -helix , twice that of the n - terminus when peptides were analyzed by cd .
this was surprising as the bioinformatics study predicted that the c - terminus would contain primarily random secondary structure .
hence , our data show that the cav-1 c - terminus contains helical structure , suggesting that it may not exclusively be random , and supports the consensus structural predictions of cav-1 aa 134167 .  
cd analysis of nsp4-cav-1 peptide - peptide interactions revealed that there was no significant change in -helix formation upon binding of nsp4112 - 140 and the n - cav19 - 40 peptide .
however , a structural change was noted upon interaction of nsp4112 - 140 with n - cav2 - 20 and c - cav161 - 178 . taken together with the binding assays , these results reveal that while the interaction between nsp4112 - 140 and n - cav19 - 40 occurs with a strong affinity , binding does not require a conformational change .
however , the secondary structure change observed when the nsp4112 - 140 and c - cav161 - 178 peptides were mixed likely occurred to allow the two peptides to interact , albeit more weakly .
a statistically significant conformational change was observed when the nsp4112 - 140-cav68 - 80 mixture was combined with model membranes . since cav-168 - 80 is located near the n - terminal membrane attachment domain ( n - mad ; aa 82101 ) , as well as the transmembrane domain ( aa 102134 ) of cav-1 , it was postulated that the observed conformational change was due to the interaction of cav68 - 80 with the lipid vesicles .
however , when cav68 - 80 was mixed with suvs in the absence of nsp4112 - 140 , no change in secondary structure was observed .
extensive studies are needed to dissect how the suvs alter the structure of the nsp4112 - 140 and cav68 - 80 mixture .  
the interaction of nsp4 with both termini of cav-1 is unique , as most cav-1 binding proteins interact with the csd [ 57 , 58 ] . since nsp4 ( aa 112140 )
interacted with the n - terminus of cav-1 more strongly than the c - terminus , it is possible that the interaction with the c - terminus is transitory .
nsp4 activates signaling pathways that mobilize [ ca]i , requiring the protein to interact with membrane - associated signaling molecules [ 59 , 60 ] .
the weak binding between nsp4 and the c - terminus of cav-1 may serve to present and/or orient the viral protein for interactions with signaling molecules or lipids , such as the phosphoinositides .
once nsp4 is properly presented to signaling molecules within the membrane , the c - terminus of cav-1 may then quickly disassociate thus allowing nsp4 to carry out its biological function(s ) .
since the c - terminus of the cav-1 protein is closely associated with membranes and does not extend into the cytoplasm to the same extent as the n - terminus , it likely has less flexibility and/or accessibility for interactions with other proteins .
this lack of freedom is another potential explanation for the weak binding observed between nsp4 and the c - terminus of cav-1 .
it has been shown that nsp4 is released from rv - infected cells in different forms dependent on the cell type , virus strain , and moi [ 36 , 61 , 62 ] .
the differential binding of nsp4 to the n- and c - termini of cav-1 could also contribute to the presentation and transport of nsp4 across the pm and provide a key target to block nsp4 from exiting the cell .
results of the fluorescence - binding assay showed that the n- and c - termini of cav-1 do not interact , so this possibility can be ruled out .
lastly , we demonstrated that an rv nsp4 peptide directly interacted with cholesterol and this interaction overlapped the amphipathic -helix , the enterotoxic peptide , and cav-1 binding domains ( aa 112140 ) , as well as a putative crac motif .
the interaction between cholesterol and the nsp4 crac sequence has implications for the trafficking of nsp4 in rv - infected cells .
given that caveolae are involved in de novo cholesterol transport from the er to pm caveolae [ 30 , 39 ] and cav-1 assists in cholesterol transport [ 29 , 30 , 63 , 64 ] , contains a crac motif , and binds cholesterol in a 1  :  1 ratio , it is reasonable to propose that cav-1 and cholesterol function in nsp4 intracellular localization and transport [ 65 , 66 ] . indeed
the interaction between nsp4 and cholesterol supports the hypothesis that nsp4 traffics via a cholesterol transport pathway in rv - infected cells .
likewise , nsp4 interacts with cav-1 , has a proposed crac motif , traffics to the cell surface via a golgi - bypassing , unconventional pathway [ 7 , 34 , 36 , 67 ] , and , as we show herein , interacts with cholesterol with a kd of 8  1  nm . we also showed that the nsp4-cholesterol interaction was specific to aa 112140 , as there was no binding to a c - terminal nsp4150 - 175ala peptide .
based on these results using synthetic peptides , we hypothesize that nsp4 traffics with cholesterol and cav-1 from the er to the pm , and to viroplasms via association to cholesterol .
additional studies are needed to verify this hypothesis , dissect nsp4 transport to the pm , and resolve viroplasm localization , all of which may constitute novel targets of reducing nsp4 effects .
 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 . 
 nsp4112140 that overlaps the caveolin-1 binding domain and a cholesterol recognition amino acid consensus ( crac ) motif and both termini of caveolin-1 ( n - caveolin-1220 ,  1940 and c - caveolin-1161180 ) 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 nsp4112140-n - caveolin-11940 and cholesterol interactions may block nsp4 intracellular transport , hence enterotoxicity . 
1. Introduction
2. Materials and Methods
3. Discussion