noroviruses ( novs ) , 
 a group of small , round - structured rna viruses 
 constituting the norovirus genus in 
 the family caliciviridae , infect both 
 humans and animals . human novs cause epidemic acute gastroenteritis , 
 affecting millions of people and claiming over 200,000 lives annually 
 worldwide . at present
structurally , novs are nonenveloped , 
 containing an outer protein capsid that encapsulates the single - stranded , 
 positive sense rna genome of 7.7 kb .
the nov capsid is made 
 from a single major structural viral protein , vp1 .
crystallography 
 of recombinant nov - like particles ( vlps ) reveals a t = 3 icosahedral symmetry consisting of 180 copies of vp1 organized 
 into 90 dimers .
vp1 is divided into two 
 major domains , the shell ( s ) and the protruding ( p ) domains .
the s 
 domain forms the interior , icosahedral shell ; while the p domain forms 
 the dimeric protrusions extending outward from the shell . the p domain can be further divided into p1 and 
 p2 subdomains , corresponding to the legs and the head of the arch - like 
 protrusion , respectively .
the p2 subdomain forms the outermost surface 
 of the capsid with highly variable sequence , responsible for the virus 
human novs are difficult to study due to the 
 lack of an efficient 
 cell culture system and a small animal model .
currently , research 
 into nov  host interactions relies on various nov subviral particles . 

expression of full - length vp1 results in vlps that are structurally 
 similar to an authentic virus . furthermore , 
 expression of various subdomains results in smaller subviral particles 
 or complexes . for example , production of the s domain forms s particles , corresponding to the interior shell of the capsid , while expressions 
 of the p domains with or without modifications can form p dimers , 12-mer small p particles , or 24-mer 
 p particles .
in addition , various glutathione 
 s - transferase ( gst)-p domain fusion proteins have been shown to form 
 polyvalent complexes owing to the dimeric and oligomeric features 
 of the gst and the p domain .
these vlps , p particles 
 and p complexes retain the basic structures of the capsid or p dimer , 
 recognize host ligands and , thus , have been used as tools or models 
 for the study of nov  host interactions .
human novs recognize 
 histo - blood group antigens ( hbgas ) as attachment 
 factors or receptors , which play an important role in the host susceptibility 
 of nov infection , as shown by both human challenge studies and outbreak 
 investigations .
hbgas are oligosaccharides linked to membrane proteins 
 or lipids as glycoprotein or glycolipid that are distributed extensively 
 on the surfaces of red blood cells and mucosal epithelia .
they are also present as free oligosaccharides 
 in biological fluids , such as saliva or milk .
human novs interact with hbgas in a strain - specific manner , whereby 
 a number of nov - hbga binding patterns involved in all abo , lewis and 
 secretor / nonsecretor types have been identified .
the structural basis of these interactions have been elucidated 
 by x - ray crystallography of nov p dimers in complex with hbga oligosaccharides .
however , it has been observed 
 that some human novs , such as va115 ( gi.3 ) , desert shield virus ( gi.3 ) and noda485 
 ( gii.1 ) , do not bind any hbgas .
a human 
 challenge study of snow mountain virus ( smv , gii.2 ) did not reveal 
 a dependence of host susceptibility on hbga type , despite the fact 
 that the smv vlp recognizes only the b antigen .
in addition , a recent study showed that nov vlps of ueno 
 7k ( gii.6 ) and noda485 binds caco-2 cells and human small intestinal 
 epithelium biopsy in a hbga - independent manner .
these data suggest that hbgas may not be the only receptors 
 for human novs .
for example , using thin - layer chromatography 
 and quartz crystal microbalance with dissipation monitoring , larson 
 et al . reported binding of gii.4 vlps to galactosylceramide and hbga 
 glycosphingolipids that were purified from human meconium samples .
takeda and co - workers demonstrated that vlps of gii novs bound heparan 
 sulfate on the cell surface , while belliot 
 and co - workers showed that gii.4 vlps recognized sialic acid - containing 
 carbohydrates , such as sialyl lewis x ( le ) , sialyl - lacto - n - fucopentaose , sialyl - lacto - n - tetraose , 
 and sialyl - lacto - n - neotetraose , with affinities comparable 
 to those of hbga ligands . using saturation 
 transfer difference nuclear magnetic resonance spectroscopy , peters 
 and co - workers detected the interactions between gii.4 vlps and the 
 sialic acid moiety of sialyl le and sialyl le .
however , they also found that carbohydrates 
 containing sialic acid , but not fucose , e.g. 3-sialyllactose 
 and 6-sialyllactose , do not exhibit detectable binding with 
 the vlp .
the results of these studies , 
 taken together , imply that sialic acid - containing oligosaccharides 
 could also be ligands of human novs .
in fact , sialic acid - containing 
 oligosaccharides have been shown to be ligands or receptors for some 
 animal caliciviruses ( cvs ) , including murine nov ( mnv1 ) , feline calicivirus ( fcv ) and a porcine sapovirus ( psav , cowden strain ) . however , solid evidence to establish the ligand 
 status of sialic acid for human cvs ( human novs and human sapoviruses ) 
 is lacking . here
, we report the first experimental evidence 
 that human novs 
 recognize sialic acid - containing glycosphingolipids ( gangliosides ) . 
 the catch - and - release electrospray ionization mass spectrometry ( car - esi - ms ) 
 assay was used to screen a library of 
 gangliosides against the p particle of human nov va387 ( gii.4 ) .
the 
 affinities of 13 gangliosides for the p dimer of va387 and of a second 
 human nov strain , va115 ( gi.3 ) , were measured using the direct esi - ms 
 assay . using a competitive esi - ms assay , 
 the proxy protein method , the highest 
 affinity ligand , gm3 ,
was subjected to additional binding measurements 
 and the affinities for both the va387 p particle and vlp were determined . 

notably , the ganglioside affinities measured for nov va387 are comparable 
 to those of known hbga oligosaccharide receptors .
enzyme - linked immunosorbent assays ( elisa ) provided additional 
 evidence that both strains of novs exhibit binding to sialic acid - containing 
 oligosaccharides .
the vlps of va387 ( gii.4 ) were produced in 
 insect cells ( sf9 ) through a recombinant baculovirus containing the 
 gene encoding va387 vp1 ( genbank accession number ay038600 , molecular 
 weight ( mw ) of monomer 58,887 da ) as described previously .
the resulting vlps were purified by sucrose 
 gradient . va387 p particles ( 24-mer , mw 865,036 da ) , p dimers ( mw 
 69,312 da ) , and gst - p domain fusion proteins were produced based on 
 the p domain sequences ( residues 222539 ) of vp1 via e. coli as reported in our previous studies .
the gst gene fusion system ( ge healthcare life sciences , 
 piscataway , nj ) with plasmid vector pgex-4t-1 was used for the p proteins 
 expression .
preparations of va115 ( gi.3 ) vlps and p particles were 
 attempted based on the vp1 sequences ( genbank accession number ay038598 ) and 
 the established procedure described above , but the yields for both 
 particles were found to be very low .
the p dimers ( mw 67,712 da ) and 
 the gst - p fusion proteins of va115 were produced in high yield ( > 20 
 mg l bacteria ) through the same procedure as used 
 for the production of the p proteins of va387 . formations of the 24-mer 
 p particles , p dimers , and the gst - p polymers were analyzed by gel - filtration 
 chromatography via a superdex 200 size exclusion column ( ge healthcare 
 life sciences ) controlled by an akta fast performance liquid chromatography 
 system ( fplc , model 920 , ge healthcare life sciences ) .
a single 
 chain fragment ( scfv , mw 26,539 da ) of the monoclonal antibody se1554 
 was produced using recombinant technology as described elsewhere .
a recombinant fragment of the c - terminus of 
 human galectin-3 ( gal-3c , mw 16,330 da ) was generously provided by 
 prof . c. cairo ( university of alberta ) .
bovine ubiquitin ( ubq , mw 
 8565 da ) was purchased from sigma - aldrich canada ( oakville , canada ) . 
 the proteins were concentrated and exchanged into an aqueous 200 mm 
 ammonium acetate ( ph 7 ) using vivaspin 0.5 ml centrifugal filters 
 ( sartorius stedim biotech , gttingen , germany ) with a mw cutoff 
 of 10 kda and stored at 80 c until use .
the structures of the oligosaccharides and 
 glycoconjugates used in this study are shown in figure s1 ( supporting information ) .
the 17 ganglioside and 
 globoside oligosaccharides ( gm3 , gm2 , gm1a , gm1b , gd3 , gd2 , gd1a , 
 gd1b , gt3 , gt2 , gt1a , gt1c , fucosyl - gm1 , asialo - gm2 , asialo - gm1 , gb3 
 and gb4 ) were purchased from elicityl sa ( crolles , france ) .
h type 
 3 trisaccharide , a type 3 tetrasaccharide , and b type 3 tetrasaccharide 
 were a gift from prof .
each solid compound was dissolved in ultrafiltered 
 milli - q water ( millipore , ma ) to give a 1 mm stock solution .
polyacrylamide 
 ( paa)-conjugated neu5ac , 6-sialylacnac , and gm3 trisaccharide 
 were purchased from vector lab ( burlingame , ca ) .
all of the esi - ms assays were carried 
 out on a synapt g2s quadrupole - ion mobility separation - time - of - flight 
 ( q - ims - tof ) mass spectrometer ( waters , manchester , u.k . )
the car - esi - ms and direct esi - ms 
 assays were performed in negative ion mode , whereas the proxy protein 
 esi - ms assay was implemented in positive ion mode .
, 0.68 mm i.d . ) 
 pulled to 5 m using a p1000 micropipette puller 
 ( sutter instruments , novato , ca ) .
a platinum wire was inserted into 
 the nanoesi tip , and a capillary voltage was applied to carry out 
 esi .
the source parameters for both negative and positive ion modes 
 were : capillary voltage 0.8 kv ( negative ion mode ) or 1.0 
 kv ( positive ion mode ) , source temperature 60 c , cone voltage 
 60 v ( negative ion mode ) or 35 v ( positive ion mode ) , trap voltage 
 5 v , and transfer voltage 2 v. data acquisition and processing were 
 performed using masslynx software ( version 4.1 ) .
the car - esi - ms assay was performed 
 to identify carbohydrate ligands of the nov va387 p particle .
ions 
 corresponding to ligand - bound p particle were isolated using the quadrupole 
 mass filter .
the quadrupole was set to transmit a broad mass - to - charge 
 ratio ( m / z ) window ( approximately 
 200 m / z units ) , which allows for 
 the simultaneous passage of free and ligand - bound p particle complexes 
 at a given charge state . protein 
ligand complexes were subjected 
 to collision - induced dissociation ( cid ) in the trap region of the 
 synapt g2s by increasing the trap voltage from 5 to 200 v. argon ( 1.42 
  10 mbar ) was used to carry out cid in the 
 trap region . in most instances , the deprotonated ligands released 
 from the complexes could be identified from their mws . where required , 

ims was used to separate the released isomeric ligands . for ims separation 
 a wave height of 35
v was used , and the wave velocity was ramped from 
 2000 to 500 m s. in all cases a helium flow rate 
 of 150 ml min and a nitrogen flow rate of 40 ml 
 min were used .
the arrival time distributions 
 ( atds ) for the released ligands were compared to reference atds , which 
 were measured for the deprotonated carbohydrates produced directly 
 from solution . the direct esi - ms 
 assay was used 
 to quantify the affinities of the carbohydrate ligands for the nov 
 p dimers of va387 and va115 .
at least four different initial ligand 
 concentrations were used for each oligosaccharide tested , and the 
 binding measurements were carried out in triplicate .
a complete description 
 of the data analysis method employed to calculate the intrinsic association 
 constants ( ka , int ) can be found elsewhere .
briefly , the abundance ratio ( ri ) of the ligand - bound protein ( pli ) , bound to i molecules of l , to free protein ( p ) 
 measured by esi - ms ( after correction for nonspecific ligand - protein 
 binding ) is taken to be equal to the equilibrium concentration ratio 
 in solution , eq 1:1 assuming 
 the protein has h independent and identical binding 
 sites , ka , int can be expressed by eq 2:2where [ p]0 and [ l]0 are 
 the initial concentrations of the protein and ligand , respectively , 
 and f is the fraction of occupied binding sites , 
 eq 3:3 in the case of the p dimer , which has two 
 equivalent binding sites , ka , int can be 
 found using eq 4:4 the proxy protein esi - ms 
 assay was used to quantify the affinities of gm3 trisaccharide for 
 nov va387 p particle and vlp . a complete description of the data analysis 
 method employed to calculate ka , int can 
 be found elsewhere .
briefly , a proxy 
 protein ( pproxy ) , which binds to l with a known affinity , 
 is used to monitor the extent of l binding to p. specifically , in 
 the presence of p , the abundance ratio rproxy (= [ pproxyl]/[pproxy ] ) will quantitatively 
 reflect the concentration of l bound to p and ka , int can be evaluated using eq 5:5where the initial concentrations 
 of target 
 protein ( [ p]0 ) , proxy protein ( [ pproxy]0 ) and ligand ( [ l]0 ) as well as the association 
 constant for binding of pproxy to the ligand ( ka , pproxy ) are known ; [ p]m,0 is the 
 initial concentration of binding sites in the target protein , i.e. , 
 [ p]m,0 = h  [ p]0 .
paa - conjugated 
 neu5ac , 6-sialylacnac and gm3 trisaccharide were dissolved 
 in 1x pbs ( ph 7.4 ) .
they were diluted and coated on a 96-well microtiter 
 plate at concentration of 2 g ml and stored 
 at 4 c overnight . after blocking with 5% nonfat dry milk , nov 
 vlp ,
p particle , or gst - p domain fusion proteins as well as gst ( negative 
 control ) at 50 ng l were added and incubated 
 for 2 h at 37 c .
the ligand - bound nov vlp and p proteins were 
 detected by homemade guinea pig hyperimmune serum against va387 vlp 
 and va115 p protein ( 1:3000 ) , respectively , followed by horseradish 
 peroxidase ( hrp)-conjugated goat antiguinea pig immunoglobulin g ( igg , 
 1:3000 ; icn , aurora , oh ) .
the signals were displayed using a tmb kit ( thermo fisher 
 scientific , rockford , il ) .
evidence 
 of ganglioside binding to novs was initially revealed through the 
 screening of a small ( 20 components ) carbohydrate library against 
 the p particle ( 24-mer , mw 865,036 da ) of nov va387 ( gii.4 ) using 
 the car - esi - ms assay .
the library consisted 
 of the oligosaccharides of 17 glycosphingolipids , gm1a , gm1b , gm2 , 
 gm3 , gd1a , gd1b , gd2 , gd3 , gt1a , gt1c , gt2 , gt3 , fucosyl - gm1 ( referred 
 to as fuc - gm1 ) , asialo gm1 , asialo gm2 , gb3 and gb4 as well as three 
 known hbga oligosaccharide ligands , h type 3 trisaccharide ( referred 
 to as h3 ) , a type 3 tetrasaccharide ( a3 ) , and b type 3 tetrasaccharide 
 ( b3 ) .
the intrinsic affinities of the hbga ligands range from 700 
 to 1500 m. the car - esi - ms 
 assay was carried out by first incubating the p particle with the 
 carbohydrate library , followed by direct esi - ms analysis of the mixture . 

because of the high mw of the p particle , the identity of the bound 
 ligands could not be established directly from the mass spectrum . 
 instead , using a quadrupole mass filter set to pass a range of mass - to - charge - ratio 
 ( m / z ) ions , all of the ligand - bound 
 p particle ions at a given charge state were isolated and then activated 
 ( heated ) using cid to release the ligands ( as ions ) from the complex . 
 given that carbohydrates have relatively low gas - phase acidities and 
 are able to effectively compete with proteins for negative charge , 

accurate mass analysis , alone or in combination 
 with ion mobility separation ( ims ) , which separates ions based on 
 size and shape , allowed for positive ligand identification . shown in figure 1a is a representative esi 
 mass spectrum acquired in negative ion mode for an aqueous ammonium 
 acetate ( 200 mm , ph 7 , 25 c ) solution of p particle ( 3 m ) 
 and the carbohydrate library ( 10 m each ) . from the mass spectrum 

it can be seen that the p particle exists predominately as a 24-mer , 
 with a charge state distribution ranging from 60 to 65 . 

signal corresponding to an 18-mer is also present , although at lower 
 abundance , with a charge state distribution of 51 to 54 . 
 due to the high mw of the p particle and the formation of adducts 
 during the esi process
, it was impossible to resolve the ions corresponding 
 to free p particle and its complexes with one or more oligosaccharide 
 ligands .
however , cid , performed using a 200 m / z wide isolation window centered at 14,350 to pass ions 
 corresponding to the 61 charge state of the p particle , led 
 to the appearance of singly deprotonated ions of the three hbga oligosaccharides 
 as well as gm3 ( m / z 632.2 ) , gm2 
 ( m / z 835.3 ) , gd3 ( m / z 923.3 ) , gm1a and/or gm1b ( m / z 997.3 ) , and fuc - gm1 ( m / z 1143.4 ) ( figure 1b ) .
ions corresponding to 
 the singly deprotonated gd2 ( m / z 1126.4 ) and the doubly deprotonated ions of gd1a and/or gd1b ( m / z 644.1 ) were also detected , although 
 at low abundance ( figure 1b ) .
abundant multiply 
 charged protein monomer ions , pm at n = 1023 , were also evident 
 ( figure 1b ) .
implementation of the car - esi - ms 
 assay using other charge states of the p particle complexes produced 
 similar results ( figure s2 , supporting information ) .
ion mobility separation of the released ligands revealed evidence 
 that both gm1a and gm1b are released from the p particle , with gm1a 
 being more abundant ( figure s3a , supporting information ) .
the doubly deprotonated ions of gd1a and gd1b could not be differentiated 
 using optimized ims conditions ( figure s3b , supporting 
 information ) , and therefore , it was not possible to establish 
 whether one or both oligosaccharides bind to the p particle directly 
 from these measurements . instead
, the car - esi - ms assay was applied 
 to solutions containing p particle ( 3 m ) and 10 m of 
 gd1a or gd1b .
these data revealed that only gd1a binds to the p particle 
 under these solution conditions ( figure s4 , supporting 
 information ) .
the car - esi - ms results provide compelling evidence 
 that the p particle of va387 exhibits a broad specificity for mono- 
 and disialylated gangliosides .
however , there is a clear preference 
 for gm3 , and the addition of saccharides to gal ( e.g. , gm1 or gm2 ) 
 or sia ( e.g. , gd3 , gd2 or gd1b ) decreases binding , compared to gm3 . 

these data , combined with affinities measured for ganglioside oligosaccharides , vide infra , suggest that the sia - gal - glc moiety represents 
 the dominant recognition epitope for this nov .
( a ) esi mass spectrum 
 acquired in negative ion mode for an aqueous 
 ammonium acetate solution ( 200 mm , ph 7 and 25 c ) of nov va387 
 p particle ( 3 m ) and a 20-component ( 10 m each ) carbohydrate 
 library consisting of the oligosaccharides of gm1a , gm1b , gm2 , gm3 , 
 gd1a , gd1b , gd2 , gd3 , gt1a , gt1c , gt2 , gt3 , fuc - gm1 , asialo gm1 , asialo 
 gm2 , gb3 , and gb4 as well as the h3 , b3 , and a3 oligosaccharides . 

( b ) cid mass spectrum measured for the 61 charge state of 
 the free and ligand - bound p particle . based 
 on the relative abundances of the released oligosaccharide 
 ligands measured by car - esi - ms ( figure 1 )
it 
 would appear that the affinities of the ganglioside ligands are similar 
 to those of the highest affinity hbga oligosaccharides .
however , this conclusion is predicated on the 
 assumption that the release efficiency of the bound - ligands is essentially 
 independent of structure . because of the presence of the sialic acid , 
 it is possible that gangliosides ( which are likely deprotonated in 
 the gaseous complexes ) are preferentially released from the p particle 
 due to a lower activation energy resulting from coulombic repulsion .
therefore , it was important to measure the affinities directly . 
 in order to do this ,
the affinities were measured using the direct esi - ms assay , which 
 has been shown to provide reliable ka values 
 for many protein  carbohydrate interactions .
affinities were measured for the oligosaccharides of 13 
 gangliosides ( gm3 , gm2 , gm1a , gm1b , gd3 , gd2 , gd1a , gd1b , gt3 , gt2 , 
 gt1a , gt1c , and fuc - gm1 ) for the va387 p dimer ( mw 69,312 da ) .
a reference 
 protein ( pref ) was used in all cases to correct the mass 
 spectra for the occurrence of nonspecific carbohydrate 
a representative 
 esi mass spectrum acquired for an aqueous ammonium acetate solution 
 ( 200 mm , ph 7 , 25 c ) of va387 p dimer ( 12 m ) and gm3 
 trisaccharide ( 80 m ) is shown in figure 2a as well as the distribution of ligand - bound p dimer after correction 
 for nonspecific binding . from the esi - ms data , ka , int values were calculated for each oligosaccharide ( table 1 ) .
affinities were also measured for a3 , b3 , and 
 h3 and shown to agree well with the reported values ( table s1 , supporting information ) .
inspection of the ka , int values reveals 
 that , of the tested gangliosides , gm3 exhibits the highest affinity 
 for the va387 p dimer , which is consistent with the results of the 
 car esi - ms measurements , vide supra .
moreover , the ka , int ( 1500 m ) is identical , 
 within experimental error , to that of b3 ( 1500  150 m ) . of the 12 other gangliosides investigated , 
 nine
bind weakly ( ka , int < 500 m ) and three ( gd1b , gt3 and gt1a ) do not show any detectable 
 binding .
notably , the quantitative binding data obtained for the p 
 dimer agree qualitatively with the relative affinities inferred from 
 the car - esi - ms measurements performed on the p particle . moreover , 

all ligands with affinities > 100 m were detected 
 in the car - esi - ms measurements ( table s2 , supporting 
 information ) .
( a ) esi mass spectrum acquired in negative ion mode for 
 aqueous 
 ammonium acetate solution ( 200 mm , ph 7 and 25 c ) of nov va387 
 p dimer ( p2 , 12 m ) , gm3 trisaccharide ( 80 m ) 
 and pref ( 4 m ) . another minor form of p dimer ( p2 , mw 74,080 da ) was also detected with lower abundance .
inset , 
 normalized distribution of gm3 bound to p2 after correction 
 for nonspecific ligand binding .
( b ) esi mass spectrum acquired in 
 negative ion mode for aqueous ammonium acetate solution ( 200 mm , ph 
 7 and 25 c ) of nov va115 p dimer ( p2 , 12 m , 
 mw 67,712 da ) , gm3 trisaccharide ( 80 m ) , and pref ( 4 m ) .
inset , normalized distribution of gm3 bound to p2 after correction for nonspecific ligand binding .
nb = no binding 
 detected . to demonstrate 
 the relevance of the affinity data acquired for 
 the p dimer
, affinity measurements were also carried out for gm3 trisaccharide 
 binding to the va387 p particle and vlp ( 180-mer , mw 10.5 
 mda ) .
an adaptation of the proxy protein esi - ms method , which combines 
 direct esi - ms binding measurements and competitive protein binding , 
 was used to evaluate the affinities . a 
 recombinant fragment of the c - terminus of human galectin-3 ( gal-3c , 
 mw 16,330 da ) , which contains a carbohydrate recognition domain and 
 interacts with a -galactoside moiety , served as the proxy protein ( pproxy ) .
importantly , gal-3c 
 binds to gm3 trisaccharide with an affinity of ( 1.20  0.02 ) 
  10 m. the extent of binding 
 of gm3 trisaccharide to gal-3c , as determined by esi - ms , in the presence 
 of known concentrations of the target protein ( p particle or vlp ) 
 allowed for a quantitative measure of gm3 binding to the target .
esi - ms measurements were performed on aqueous ammonium acetate 
 solutions ( 160 mm , ph 7 and 25 c ) of pproxy ( 3.0 
 m ) , pref ( 1.0 m ) , gm3 trisaccharide ( 40 m ) , 
 and either p particle , at concentrations ranging from 0 to 7.2 m 
 ( corresponds to monomer concentration of 0172.8 m ) , 
 or vlp , at concentrations ranging from 0 to 570 nm ( monomer concentration 
 of 0102.6 m ) .
representative esi mass spectra acquired 
 in positive ion mode in the absence and presence of nov vlp ( 570 nm ) 
 are shown in figure 3a and 3b , respectively .
the distributions of ligand - bound pproxy , following correction for nonspecific ligand binding , are also given . 

inspection of the distributions reveals a measurable decrease in the 
 extent of gm3 trisaccharide binding to gal-3c upon addition of vlp . 

the 
 dependence of the extent of gm3 trisaccharide binding to pproxy on vlp concentration is shown in figure 3c .
binding measurements performed on solutions containing p particle 
 yielded qualitatively similar results ( figure s5 , supporting information ) .
analysis of the pproxy binding data acquired in the presence of vlp or p particle using 
 the procedure outlined in experimental section yields gm3 affinities of 2600  200 and 5500  600 m for the p particle and vlp , respectively .
the slight 
 differences in the magnitude of the affinities measured for the binding 
 of a common carbohydrate ligand to the p dimer , p particle , and vlp 
 of a nov ( the first such data set to be reported ) , likely reflect 
 subtle differences in the structure of the carbohydrate binding site 
 presented by these related protein complexes .
these differences notwhithstanding , the present results suggest 
 that the p dimer can serve as a surrogate of the vlp for carbohydrate 
 binding studies .
representative esi mass spectra measured in positive ion 
 mode for 
 aqueous ammonium acetate solutions ( 160 mm , ph 7 and 25 c ) of 
 pproxy ( gal-3c , 3.0 m ) , pref ( ubq , 1.0 
 m ) , and gm3 trisaccharide ( 40 m ) without ( a ) or with 
 ( b ) nov va387 vlp ( 570 nm , 180-mer ) .
insets show the fraction of free 
 and gm3-bound pproxy , after correction for nonspecific 
 ligand binding .
( c ) plot of the abundance ratio of gm3-bound pproxy to free pproxy ( rproxy ) versus vlp concentration .
the solution conditions for each measurement 
 were the same as in ( a ) , but with the addition of vlp .
it has been proposed that nov 
 va387 has a binding interface that 
 recognizes hbgas through the -l - fuc epitope as the major binding 
 interaction and either the -d - galnac or -d - gal epitope 
 as a minor binding interaction .
however , these core recognition elements are missing in the ganglioside 
 ligands identified in the present study .
therefore , it is of interest 
 to establish whether the ganglioside ligands interact with the nov 
 through the hbga binding site or through a distinct ganglioside binding 
 site .
it is not possible to answer this question through competitive 
 binding measurements carried out using a ganglioside oligosaccharide 
 ( e.g. , gm3 trisaccharide ) and va387 p dimer in the presence of varying 
 concentrations of a hbga oligosaccharide ligand due to the low affinities 
 of these ligands . instead
, future efforts will rely on x - ray crystallography 
 to establish whether va387 nov has distinct binding sites for hbga 
 and ganglioside ligands .
the aforementioned 
 binding data reveal that nov va387 binds to mono- and disialylated 
 gangliosides , with affinities comparable to those of the highest affinity 
 hbga oligosaccharide ligands . to demonstrate that this is not an isolated 
 example of a human nov that recognizes gangliosides , the affinities 
 of the 13 ganglioside oligosaccharides for the p dimer of nov va115 
 ( gi.3 genotype ) , which does not bind to human hbgas , were also measured ( table 1 ) .
a 
 representative esi mass spectrum acquired for an aqueous ammonium 
 acetate solution ( 200 mm , ph 7 , 25 c ) of nov va115 p dimer ( 12 
 m ) and gm3 trisaccharide ( 80 m ) is shown in figure 2b as well as the distribution of ligand - bound p 
 dimer after correction for nonspecific binding .
notably , the va115 
 p dimer binds to all 13 oligosaccharides tested , and overall , the 
 affinities are slightly higher than those for va387 .
additional evidence for the recognition of sialic 
 acid by human novs comes from elisa measurements carried out on the 
 va387 vlp , p particle , and gst - p fusion protein as well as va115 gst - p fusion protein , with paa - conjugated neu5ac , 
 6-sialylacnac , and gm3 trisaccharide . as shown in figure 4 ,
it is curious that the va387 vlp 
 exhibited weaker binding than that of the p particle to the three 
 glycoconjugates and the cause of the weaker binding is , at this time , 
 unknown .
nevertheless , the fact that all three assemblies of nov capsid 
 protein exhibit a similar binding pattern to the three glycoconjugates 
 ( gm3 > 6-sialylacnac > neu5ac ) validate their applications 
 as models for nov  ligand interaction . moreover , comparing the 
 binding of gst - p fusion protein of va387 to that of va115 indicates 
 that the sialic acid - containing glycoconjugates have slightly higher 
 affinities for va115 , consistent with the esi - ms data .
these results , 
 together with those from esi - ms , suggest both -(2,3)- and -(2,6)- 
 linked sialic acids as critical motifs in va387 and va115 binding , 
 similar to what has been reported for mnv1 and psav .  
it is important to point out that , although 
 sialic acid - containing oligosaccharides have been identified as receptors 
 for an animal nov ( mnv1 ) and two other animal 
 cvs ( fcv and psav ) , human novs generally recognize 
 gangliosides in addition to hbgas . furthermore , human novs differ 
 greatly from mnvs in many other important aspects , including host 
 tropism ( human vs mouse ) , clinical manifestation ( with vs without 
 diarrhea / vomiting ) , and pathogenesis . binding 
 of nov vlp , p particles , and gst - p fusion protein of va387 
 as well as gst - p fusion protein of va115 to paa - conjugated gm3 trisaccharide , 
 6-sialylacnac , and neu5ac in 1x pbs ( ph 7.4 ) .
gst , which does 
 not show binding to any of the three glycoconjugates , served as a 
 negative control .
taken 
 together , the results of esi - ms and elisa measurements performed 
 on two human novs representing two different genogroups ( gi and gii ) 
 provide the first experimental evidence of interactions between human 
 novs and gangliosides and sialic acid - containing glycoconjugates . 

notably , the affinities measured for the oligosaccharides of the ganglioside 
 ligands by esi - ms are comparable in magnitude to those reported for 
 the oligosaccharides of known hbga receptors .
these experimental data 
 demonstrate sialic acid - containing oligosaccharides as alternative 
 ( to hbgas ) ligands for human novs and suggest a new mechanism of human 
 nov 
host interaction , one that involves hbga and sialic acid - containing 
 oligosaccharide receptors and co - receptors for attachment and penetration 
 into host cells and opens a new direction in human nov research .
further 
 studies to characterize the role of cell surface sialic acids / gangliosides 
 in the early stage of viral infection and its potential coordination 
 with hbgas for viral attachment and/or entry are needed .
 human 
 noroviruses ( novs ) are known to recognize histo - blood group 
 antigens ( hbgas ) as attachment factors . 
 we report the first experimental 
 evidence that sialic acid - containing glycosphingolipids ( gangliosides ) 
 are also ligands for human novs . 
 electrospray ionization mass spectrometry - based 
 carbohydrate binding measurements performed on assemblies ( p dimer , 
 p particle , and virus - like particle ) of recombinant viral capsid proteins 
 of two nov strains , va387 ( gii.4 ) and va115 ( gi.3 ) , identified binding 
 to the oligosaccharides of mono- , di- , and trisialylated gangliosides . 
 
 the intrinsic ( per binding site ) affinities measured for these ligands 
 are similar in magnitude ( 102103 m1 ) to those of human hbgas . binding of nov vlps , p 
 particles , and glutathione s - transferase ( gst)-p domain fusion proteins 
 to sialic acid - containing glycoconjugates , observed in enzyme - linked 
 immunosorbent assays , provided additional confirmation of the nov  ganglioside 
 interactions . 