semliki 
 forest virus ( sfv ) is a prototypic alphavirus within the togaviridae family and was first isolated in aedes albopictus mosquitoes in uganda in 1944 . although 
 sfv infection only causes mild febrile 
 illness in humans , it is highly pathogenic in rodents and has become 
 a model system for investigating viral encephalitis caused by other neurotropic alphaviruses such as sindbis 
 virus , chikungunya virus ( chikv ) , and venezuelan equine encephalitis 
 virus . the positive - sense , single - stranded rna alphavirus genome 
 encodes 
 nine proteins , three of which are structural and encoded in the second 
 of two open reading frames . in the mature particle , the capsid protein 
 c forms an icosahedral shell ( triangulation t = 4 ) 
 that encapsulates the viral rna and is surrounded by a lipid bilayer . two membrane - bound glycoproteins , e1 and e2 , form an outer icosahedral 
 shell ( triangulation t = 4 ) and 
 are responsible for host cell fusion and attachment , respectively . e1 and e2 are generated by proteolytic cleavage 
 of polyprotein precursor via the e1p62 intermediate . alphaviruses assemble in the plasma membrane , 
 where the cytoplasmic 
 tail of the e2 glycoprotein interacts with the rna - containing capsid , 
 leading to budding of mature virions . co - translational 
 formation of the e1p62 heterodimer is necessary for proper 
 folding . the e1p62 heterodimer 
 forms concomitantly to protein folding in the er , and the glycans 
 are initially processed in the context of this structure . the initial formation of single trimeric viral 
 spikes , ( e1p62)3 , is thought to occur in this stage . proteolytic cleavage by furin in the trans - golgi 
 network is required to cleave p62 into e2 and a third glycoprotein , 
 e3 . this cleavage primes e1 for fusion 
 and enables the formation of trimers of e1/e2 heterodimers on infectious 
 virus particle . the e1 glycoprotein 
 forms a class - ii fusion glycoprotein fold with a sequence - internal hydrophobic fusion 
 peptide that is buried within the e1e2 interface . upon endosomal acidification during clathrin - mediated 
 host cell entry , e1 undergoes conformational rearrangements into fusogenic 
 homotrimers , which merge the virion and endosomal membranes . the structure of the e2 glycoprotein was revealed through crystallographic 
 analysis of the e1e2 complex from chikv . e2 is composed of three immunoglobulin - like domains and 
 forms a 2500  protein infectious particles are 
 targeted by the host antibody response , 
 which is directed predominantly to e2 . this is consistent with e2 being more membrane distal and solvent - exposed 
 than e1 . the capacity of alphaviruses 
 to infect a wide range of species is suggested to be , in large part , 
 due to the ability of the e2 glycoprotein to attach to and interact 
 with multiple cellular receptors . alphaviral glycosylation is an important determinant of cellular 
 tropism and virulence , and the composition of alphavirion glycans derived from mammalian 
 cell lines has been the subject of extensive study . the alphaviral e1 and e2 glycoproteins are post - translationally 
 modified with n - linked glycosylation . for example , sfv has one n - linked 
 sequon on e1 and two on e2 . across alphaviruses , 
 it has been generally observed that glycans from the e1 fusion glycoprotein 
 derived from mammalian cell lines are highly processed and composed 
 of complex - type glycosylation . conversely , glycans from the e2 glycoprotein 
 exhibit endoglycosidase h ( endo h ) sensitivity and are , therefore , 
 composed of hybrid- or oligomannose - type structures . for example , 
 across the eastern equine encephalitis alphaviruses , e1 exhibits complex - type 
 glycosylation , while e2 has a high level of endo h - sensitive structures . similarly , barmah forest virus exhibits predominantly 
 complex - type glycosylation on e1 but has no glycans on e2 . in sindbis virus , e1 primarily exhibited complex - type glycosylation , 
 while e2 exhibited a greater degree of oligomannose - type glycosylation . importantly , the location of the n - linked glycan sequons is not 
 well - conserved across the alphaviruses , and the precise environment 
 of a glycan can heavily influence processing . the identification of less - processed structures has implicated 
 c - type lectins , dc - sign and l - sign , as putative mammalian attachment 
 receptors for alphaviruses . host species 
 have a significant impact on glycosylation processing , however , which 
 may introduce or eliminate these lectin - binding motifs . for example , sfv derived from mosquito c6/36 
 cells exhibited endo d - sensitive paucimannose structures on e1 , while 
 e2 was almost entirely sensitive to endo h. the formation of paucimannose structures necessitates extensive 
 golgi - mediated processing for biosynthesis . these structures are therefore biosynthetically analogous to the 
 processed complex - type glycosylation of mammals . it has been 
 long appreciated that both cellular ( e.g. , cell type ) 
 and viral factors ( e.g. , protein - directed glycosylation ) play an important 
 role in determining the chemical composition of glycans . we selected the prototypic alphavirus , 
 sfv , to investigate the compositional variation of glycosylation through 
 a side - by - side comparison of virus derived from both arthropod and 
 rodent cell lines . we performed mass spectrometric analysis of the 
 glycans from sfv purified from both types of cell lines using ion - mobility 
 mass spectrometry and collision - induced dissociation analysis . we 
 observe that in contrast with the fully processed e1 glycoprotein , 
 e2 preserves under - processed oligomannose - type structures regardless 
 of host species . by integrating this analysis with known crystallographic 
 and electron microscopy data , we rationalize these observations and 
 propose that glycan processing on e2 is restrained by local steric - hindrance , 
 whereas the e1 glycan is accessible to enzymes despite being located 
 at the base of the multimeric viral spike . our mass spectrometric 
 study comprises the first structural and compositional analysis of 
 alphavirus n - linked glycosylation . these data reveal the consequence 
 of steric protection and cellular processing enzymes on the composition 
 of the virion glycome during interspecies transmission of sfv . bhk-21 ( baby hamster kidney-21 ) cells 
 were maintained in glasgow minimal essential medium supplemented with 
 10% tryptose phosphate broth and 5% fetal bovine serum ( fbs ) at 37 
 c in a humidified atmosphere containing 5% co2 . c6/36 
 cells were maintained in schneider s drosophila medium supplemented 
 with 10% fbs and 2% glutamine and grown at 28 c in a humidified 
 atmosphere containing 5% co2 . wild - type sfv ( strain 
 psp6-sfv4 ) was kindly provided by prof . m. kielian ( albert einstein college of medicine , ny ) and propagated 
 in bhk-21 cells to determine virus titer . c6/36 cells and bhk-21 were 
 infected at a multiplicity of infection of 1 and 0.1 , respectively . media containing secreted sfv were collected 136 h following 
 infection in c6/36 cells and 22 h after infection in bhk-21 
 cells , similar to previous methods . supernatants were cleared from cell debris by 
 low - speed centrifugation , and viral particles were pelleted by ultracentrifugation . 
 for sfv isolation from bhk-21 cells , an additional sucrose density 
 gradient centrifugation step was added . virus purity and endoglycosidase 
 f1 ( endo f1 ) susceptibility was confirmed by sds - page analysis . endo f1 was expressed as a fusion protein with glutathione 
 s - transferase ( gst ) and has been previously described by grueninger - leitch 
 et al . to confirm sample integrity , we 
 vitrified purified sfv from bhk cells by rapid plunge - freezing on 
  c - flat  electron microscopy grids ( protochips , raleigh , 
 nc ) into a mixture of liquid ethane ( 37% ) and propane ( 63% ) . polara  transmission electron microscope ( fei , 
 eindhoven , netherlands ) operated at a temperature of 80 k. 
 images of sfv were taken at 4 m defocus using a charge - coupled 
 device camera ( ultrascan 4000sp ; gatan , pleasanton , ca ) at a nominal 
 magnification of 93  000 , corresponding to a calibrated 
 pixel size of 0.24 nm with a dose of 12 e /  . oligosaccharides 
 were released from 
 sfv e1 and e2 glycoproteins with peptide - n - glycosidase 
 f ( new england biolabs , ipswich , ma ) from coomassie blue - stained page 
 gels , as previously described . gel bands 
 were excised , washed five times alternatively with acetonitrile and 
 deionized water , and rehydrated with 30 units of aqueous pngase f 
 solution . after incubation for 12 h at 37 c , enzymatically released 
 glycans were eluted with water . a 1 l aliquot was cleaned 
 with a nafion membrane and diluted with 
 water ( 4 l ) , methanol ( 5 l ) , and 0.1 m ammonium phosphate 
 ( 0.5 l , to maximize formation of [ m+h2po4 ] ions ) and centrifuged at 9500 g for 1 min . electrospray ( esi ) and ion mobility experiments were 
 carried out in negative ion mode with a waters synapt g2 traveling 
 wave ion mobility mass spectrometer ( waters ms - technologies , manchester , 
 u.k . ) fitted with an esi ion source . the esi capillary voltage was 1.2 kv , the cone voltage was 80 v , and 
 the ion source temperature was maintained at 80 c . the t - wave 
 velocity and peak height voltages were 450 m / sec and 40 v , respectively . the t - wave mobility cell ( nitrogen ) was operated 
 at a pressure of 0.55 mbar . fragmentation was performed after mobility 
 separation in the transfer cell with argon as the collision gas . data 
 acquisition and processing were carried out using the waters driftscope 
 ( version 2.1 ) software and masslynx ( version 4.1 ) . analysis of ms 
 data was performed using the previously described methods by comparison with spectra of well - characterized n - linked glycans 
 from ribonuclease b , bovine fetuin , and human transferrin . a model of n - linked glycan presentation 
 on the alphavirus surface was created using the crystal structure 
 of the chikv e1e2 envelope glycoprotein complex fit into the 
 sindbis virus cryo - em map ( pdb accession number 2xfb(12 ) ) . n - linked glycans , representative of those observed on 
 sfv when produced in bhk and c6/36 cells ( see results 
 and discussion ) , were modeled onto the n - linked carbohydrate 
 attachment sites asn141 on e1 as well as asn200 and asn262 on e2 . the symbolic representation of glycans 
 follows that of harvey et al . with residues 
 in both the schematic diagrams and molecular graphics following the 
 color scheme of the centre for functional glycomics , as previously 
 implemented . thus , ions retaining charge on the nonreducing 
 terminus are named  a  ( cross - ring ) ,  b  , 
 and  c  ( glycosidic ) with corresponding ions from the 
 reducing terminus named x , y , and z. subscript numbers define the 
 bonds cleaved in the glycan sequence . for fragmentation analysis , 
 we applied an extension of the domon and costello nomenclature by 
 harvey et al . this nomenclature describes fragmentations of different glycan cores 
 to avoid confusion by the subscript number changing in response to 
 different antenna lengths . bonds cleaved in the cross - ring cleavage 
 reactions are indicated by superscript numbers . the glycosylation of alphavirus has been extensively studied , and 
 the sfv e1 and e2 glycoproteins have been shown to exhibit differing 
 degrees of biosynthetic processing of n - linked glycans . however , the 
 composition of these glycans , including that arising from different 
 host cells , has not been determined . we have applied ion - mobility 
 mass spectrometry to define the virion glycome in two principal host 
 species , rodent and mosquito . for 
 virus propagation and purification , bhk and mosquito c6/36 cells were 
 infected with sfv . these cell lines were chosen because the natural 
 hosts of sfv include mosquitoes and rodents . to purify and isolate virions , we subjected 
 media from infected cells to ultracentrifugation . we verified that 
 our production and purification system was generating mature intact 
 particles by electron microscopy and sds - page analysis ( figure 1 ) . the electron micrographs revealed uniform virions 
 consistent with their t = 4 icosahedral symmetry ( figure 1a ) . sds - page 
 analysis revealed three discrete bands consistent with e1 , e2 , and 
 the capsid protein ( figure 1b ) . ( a ) electron 
 cryomicroscopy image of sfv virions , purified from bhk cells , taken 
 at 4 m defocus . ( b ) sds - page analysis 
 of sfv purified from bhk ( lanes 1 and 2 ) and c6/36 cells ( lanes 3 
 and 4 ) . the gel was stained with coomassie blue , and the protein bands 
 correspond to the sfv structural proteins : capsid ( labeled  c  ; 
 white triangle ) , e1 ( gray triangle ) , and e2 ( black triangle ) proteins . bhk- and c6/36-derived sfv were treated with endoglycosidase f1 ( endo f1 ; star ) in lanes 2 and 4 , respectively . endo f1 was expressed as a fusion protein with gst , as 
 previously reported . following previous investigations of alphaviral glycosylation , we studied the sensitivity of the virion - associated e1 and e2 glycans 
 to endo f1 digestion ( figure 1b ) . the endo f1 used in this study has been previously described 
 by grueninger - leitch et al . endo f1 hydrolyzes the di - n - acetylchitobiose core 
 ( glcnac14glcnac ) of oligomannose - type and hybrid - type 
 glycans . e1 , regardless 
 of being derived from bhk or c6/36 cells , was largely insensitive 
 to endo f1 ( figure 1b ) and is therefore 
 largely devoid of oligomannose- and hybrid - type glycosylation . in 
 contrast , e2 exhibited a reduction in apparent molecular mass by several 
 kilodaltons ( figure 1b ) , indicating the presence 
 of oligomannose- or hybrid - type glycan structures . these results are 
 consistent with the role of e2 in c - type lectin binding as well as with previously described radiolabeling 
 experiments , which revealed differential processing of e1 and e2 in 
 c6/36 cells . while e2 from both cell 
 lines migrated similarly , e1 from bhk cells was measurably larger 
 in apparent molecular mass than e1 from c6/36 cells ( figure 1b ) . these results suggest that n - linked post - translational 
 modifications are more substantial in bhk cells than in c6/36 cells . our findings support the conclusion that sfv exhibits glycosylation 
 directed by viral and cellular factors on e2 and e1 glycoproteins , 
 respectively . to identify the chemical composition of the n - linked glycans 
 from e1 and e2 of sfv , we performed mass spectrometric analysis of 
 enzymatically released glycans . because low - abundance biological samples 
 often pose challenges to detailed characterization , we chose to employ ion mobility ms . this technique enables 
 the separation of glycan ions that are otherwise obscured by contamination 
 arising during sample preparation . we 
 hypothesized that this method would enable analysis of glycans from 
 intact virions at sufficient signal - to - noise ratio , which hitherto 
 has been limited . for ion mobility ms , e1 and e2 glycans from 
 both c6/36 and bhk cells were released by pngase f exoglycosidase 
 treatment . this treatment releases all n - linked glycans except for 
 those with core 13-fucosylation . the resulting spectra of the extracted glycans from e1 
 and e2 are shown in figures 2 and 3 , respectively , and reveal the 
 utility of ion mobility ms for studying low abundance samples.figure 2mobility - extracted 
 singly negatively charged n - glycan ions from 
 the e1 glycoproteins from the mosquito ( spectrum a ) and rodent cell 
 lines ( spectrum b ) . a key to the symbols used for the glycan structures 
 is displayed in the upper right - hand corner of panel a. the linkages 
 are shown by the angle of the lines connecting the symbols ( | = 2-link , 
 / = 3-link ,  = 4-link , and -bonds are 
 shown with broken lines and -bonds are shown with full lines . oligomannose - type glycans are highlighted in green , and 
 fragment ions are shown in gray . mobility - extracted 
 singly negatively charged n - glycan ions from 
 the e1 glycoproteins from the mosquito ( spectrum a ) and rodent cell 
 lines ( spectrum b ) . a key to the symbols used for the glycan structures 
 is displayed in the upper right - hand corner of panel a. the linkages 
 are shown by the angle of the lines connecting the symbols ( | = 2-link , 
 / = 3-link ,  = 4-link , and \ = 6-link ) . -bonds are 
 shown with broken lines and -bonds are shown with full lines . oligomannose - type glycans are highlighted in green , and 
 fragment ions are shown in gray . mobility - extracted singly negatively charged n - glycan ions from 
 the e2 glycoproteins from the mosquito ( spectrum a ) and rodent cell 
 lines ( spectrum b ) . oligomannose - type glycans 
 are highlighted in green , and fragment ions are shown in gray . consistent with 
 the protein migration shift observed in figure 1b , ion mobility ms revealed that the molecular mass of e1 glycans 
 produced in the rodent cells ( bhk ) was considerably larger than e1 
 glycans produced in the mosquito cells ( c6/36 ) ( figure 2 ) . the spectrum of mosquito cell - derived e1 glycans was dominated 
 by paucimannose structures and minor fucosylated derivates , such as 
 man3glcnac2 and man3glcnac2fuc , respectively ( figure 2a ) . this is consistent 
 with extensive glycan processing in the mosquito golgi apparatus . in this insect system , glycans are trapped in 
 the paucimannose state by hexosaminidase activity , which removes the 
 gnt i - mediated glcnac necessary for the action of other transferases . in contrast , the spectrum of e1 glycans derived 
 from the rodent cell line exhibited considerable complexity , dominated 
 by biantennary glycans exhibiting variable occupancy of terminal galactose 
 and sialic acid ( figure 2b ) . this observation 
 supports a model whereby the e1 glycan is fully accessible to enzymatic 
 processing . hybrid - type glycans are also present together with a minor 
 population of smaller oligomannose - type glycans . importantly , the 
 relative population of oligomannose - type glycans matched that observed 
 in the spectrum of mosquito - derived e1 with man5glcnac2 > man6glcnac2 > man7glcnac2 . we suggest this is likely to arise from 
 similar glycan presentation and accessibility to biosynthetic enzymes 
 in the er . comparison of the 
 spectra of e2 glycans from mosquito and rodent cell lines revealed 
 substantial convergence of composition despite the considerable variation 
 in the processing machinery of the two systems ( figure 3 ) . both spectra were dominated by the full oligomannose series , 
 man59glcnac2 , with only a minor population 
 of glycans exhibiting golgi processing , characteristic of that primarily 
 observed on e1 glycans . in contrast with the conserved distribution 
 of oligomannose - type structures in e1 ( figure 3 ) , the distribution of oligomannose - type structures differed between 
 rodent and mosquito - derived e2 . in mosquito - derived e2 , the man9glcnac2 glycan was most abundant with decreasing 
 levels down the series to man5glcnac2 . this reversal indicates 
 that while e2 glycans are largely protected from processing following 
 the calnexin / calreticulin folding checkpoint , the efficacy of the 
 mosquito -mannosidases differs from that of the rodent system . we would not expect the physical presentation of these conserved , 
 early glycans to differ between systems , and these differences are 
 therefore likely driven by dissimilarities in specific activity of 
 cell resident -mannosidases or in transit time . the variance 
 in e2 oligomannose processing between cell lines may also indicate 
 that the conservation of the processing of the minor oligomannose 
 series observed on e1 is somewhat coincidental . given the functional 
 importance of oligomannose - type glycans , it would be valuable to investigate the stability of -mannosidase 
 processing in related alphaviruses . it should also be noted that the 
 temperature difference between insect and mammalian viral production 
 can influence viral architecture , and this may influence glycan processing . finally , e3 is shed in various stages during alphavirus biogenesis , and the influence of e3 on glycan ion 
 mobility combined with 
 ms / ms analysis involves a highly sensitive ion - extraction technique , 
 able to separate low abundance glycan ions from contaminants , with 
 the generation of fragmentation fingerprints characteristic of particular 
 substructures within glycans . this also enables accurate assignments 
 of isomers , for example , the effective discrimination between isobaric 
 complex and hybrid - type structures on the virion surface . the 
 glycan assignments previously discussed were based on detailed ms / ms 
 fragmentation analysis following mobility separation ( figures 4 and 5 ) . the mass of each 
 glycan gave the constituent monosaccharide composition in terms of 
 isobaric residues such as hexose and n - acetylhexose 
 ( hexnac ) . structures of the di - n - acetylchitobiose 
 core , the 6- and 3-antennae , isomers ( when present ) , and the complex 
 biantennary glycans were assigned based on combination of the parent 
 molecular ion mass and the pattern of fragmentation ions produced 
 by collision - induced dissociation . this assignment process is described 
 later . examples of mobility - extracted , negative ion cid ( transfer region ) 
 spectra of n - glycans from sfv derived from a mosquito cell line . ( a ) 
 man8glcnac2 , ( b ) man7glcnac2 , ( c ) man3glcnac2 , and ( d ) fucosylated man3glcnac2 . ion nomenclature 
 follows that proposed by domon and costello with spectral interpretation as described by harvey et al . examples of mobility - extracted , negative ion 
 cid ( transfer region ) 
 spectra of n - glycans from sfv derived from rodent cell lines . ( a ) 
 man5glcnac2 , ( b ) hybrid - type glycan ( man5glcnac3gal1 ) , ( c ) biantennary glycan 
 ( man3glcnac4gal2 ) , and ( d ) sialylated 
 biantennary glycan ( man3glcnac4gal2fuc1neu5ac1 ) . the di - n - acetylchitobiose core region of 
 all glycans 
 was characterized by the production of abundant ar , br-1 , and ar-1 ions . ( see the nomenclature section . ) 
 for example , these ions correspond to peaks at m / z 1072 , 1012 , and 869 in the spectrum of man5glcnac2 ( figure 5a ) . the phosphate 
 adducts of the glycans resulted in the ar ion appearing at 259 mass units below that of the molecular ( [ m+h2po4 ] ) ion . a loss of 405 mass 
 units , as observed in the spectrum of the fucose - containing man3glcnac2fuc1 ( figure 4d ) , reveals that the fucose is located at the six - position 
 of the reducing - terminal glcnac . ( note that the proton from the 3-oh 
 group is lost when forming this ion , leaving the six - position as the 
 only remaining position for substitution . ) the composition of the 6-antenna , and by difference the 3-antenna , 
 was defined by analysis of the d and d-18 ions ( these ions are formed 
 by the formal loss of the di - n - acetylchitobiose core 
 3-antenna ) and ar-2 and ar-2 ions from the core branching mannose residue . these ions appear at m / z 647 , 629 , 
 575 , and 545 , respectively , in man5glcnac2 ( figure 5a ) and at m / z 809 , 
 791 , 737 , and 707 in that of man8glcnac2 ( figure 4a ) , where there is an additional mannose residue 
 in the 6-antenna . localization of this mannose to the 6-branch of 
 this antenna was facilitated by identification of the abundant ion 
 at m / z 485 , termed d. where 
 isomers were present , as in the case of man7glcnac2 ( figure 4b ) , two groups of ions appeared 
 depending on the number of mannose residues in the 6-antenna . in this 
 spectrum , ions at m / z 647 , 629 , 
 and 545 and at m / z 809 , 791 , 737 , 
 and 707 , respectively , revealed the presence of three and four mannose 
 residues in the 6-antenna . the presence of these ions also informed 
 on the composition of hybrid - type glycans in the bhk - derived samples 
 ( figure 5b , c ) . for example , ions at m / z 647 , 629 , 575 , and 545 in the spectrum 
 of man5gal1glcnac3 ( figure 5b ) revealed glycans at the 6-antenna identical to 
 those observed in the high - mannose glycan , man5glcnac2 ( figure 5a ) . an additional feature 
 of hybrid - type glycans ( figure 5b , c ) is the 
 presence of the a3 ion at m / z 424 , which defines a gal - glcnac composition at 
 the 3-antenna . the single c1 ion at m / z 179 in the glycoforms derived from both c6/36 and bhk 
 cells ( figures 4 and 5 ) confirms hexose as the terminating residue for all antennae . structures of the complex biantennary glycans , such as those depicted 
 in figure 5c , d , were similarly determined by 
 characteristic fragmentation ions . d and d-18 ions were located at m / z 688 and 670 , respectively , while the 
 single a3 ion at m / z 424 confirmed the structure of the gal - glcnac antennae . termination of these fragments with galactose is supported by identification 
 of the c1 ion at m / z 179 . substitution of this glycan with sialic acid ( figure 5d ) gave a spectrum similar to that of the neutral glycan . the ar , br-1 , and ar-1 ions were formed with concomitant 
 loss of the sialic acid moiety , and their mass at m / z 1478 , 1418 , and 1275 confirmed the presence of 
 the core fucose . confirmation of the sialic acid ( neu5ac ) residue 
 was made by the b1 ion at m / z 290 , and the absence of the ion at m / z 306 showed that this residue was predominantly 23 
 linked . our analysis supports the conclusion 
 that the glycans of the e2 glycoprotein 
 are largely resistant to enzymatic processing . there are many examples 
 where the local environment of glycans limits processing in the er 
 and golgi apparatus in both mammalian and viral glycoproteins . one extreme example is the processing of the 
 envelope attachment glycoprotein gp120 from the human immunodeficiency 
 virus ( hiv-1 ) . the density of n - linked 
 glycans is sufficient to induce a minimally processed more commonly , protein  carbohydrate packing can also disrupt 
 the processing of n - linked glycosylation . the presence of oligomannose - type 
 glycans on sfv can influence cellular tropism and virulence and suggests that there may have been evolutionary 
 pressure on the virus to control the processing of host glycosylation 
 by indirectly altering the proteinous environment around the glycans . 
 when considering the structural influences on glycan processing , it 
 is important to evaluate the presentation of glycans to -mannosidases 
 both from initial folding in the er and golgi transit . in sfv biosynthesis , 
 initially isolated e1p62 heterodimers are formed in the er 
 and are then thought to trimerize . the 
 trimers are transported from the er to the golgi apparatus and further 
 toward the plasma membrane . during later stages of transport , ( e1p62)3 complexes are thought to form higher order assemblies similar 
 to those observed in the virion . to rationalize the structure of the glycans on the mature virion 
 surface , we constructed a model of the mature ( e1e2)3 viral spike from the structure of the related alphaviral chikv glycoproteins . glycans were modeled onto the e1 and e2 glycoprotein using the crystallographic 
 coordinates reported for a sialylated complex - type glycan and an oligomannose - type glycan , respectively . a model for the virion surface was further constructed by using the glycosylated chikv e1e2 
 structure fitted into the electron microscopy map of sindbis virus for sfv produced in both rodent ( figure 6a ) and mosquito ( figure 6b ) cells . models of the glycosylated alphavirus surface as derived from rodent 
 ( a ) and mosquito ( b ) cells . the crystal structure of the chkv e1e2 
 envelope glycoprotein complex was fitted into the sindbis virus cryo - em 
 map ( pdb accession number 2xfb ) . e1 and e2 are shown 
 in a surface representation in dark gray and light gray , respectively . oligomannose - type glycans , presented by e2 at asn200 and asn262 , are 
 shown as light- and dark - green spheres , respectively ( man9glcnac2 , from pdb accession code 2wah(75 ) ) . at asn141 , glycan structures are 
 modeled as complex - type glycans for rodent - derived alphavirus ( panel 
 a ; pink spheres , from pdb accession code 4byh(74 ) ) and paucimannose - type 
 for mosquito - derived alphavirus ( panel b ; orange spheres , from pdb 
 accession code 2wah ) . despite being located in the crevices 
 created by the virion spike , the e1 glycan is fully accessible both in isolated ( e1e2)3 complexes , which are likely to be observed in the golgi apparatus 
 and er , and in the mature virion through the hexagonal space formed 
 between protomers ( figure 6 ) . this presentation 
 is entirely consistent with host - dependent glycosylation in rodent 
 ( figure 6a ) and mosquito ( figure 6b ) cellular secretion pathways , whereby glycosylhydrolases 
 and glycosyltransferases help to determine glycan composition . in 
 contrast with e1 , the e2 glycans are more membrane - distal and are 
 located on the side of the viral spike , which is presumably sufficient 
 to limit -mannosidase processing in the er and early golgi 
 ( figure 6 ) . in the mature virion , the glycans 
 are driven into close proximity to the protein surface of opposing 
 protomers of the trimer and yet are accessible for cell attachment 
 by dc - sign recognition . our study provides a glycomic analysis of the n - linked glycans 
 displayed on the major glycoproteins from sfv , a prototypic alphavirus . consistent with early electrophoretic analyses of alphaviral glycans , we show that e1 and e2 display predominantly complex- and oligomannose - type 
 glycosylation , respectively . these results indicate that in contrast 
 with e1 , which presents glycans characteristic of the host species , 
 the glycan processing of e2 is particularly restricted . such compositional 
 differences may be attributed to localized steric - controlled hindrance 
 of enzymatic processing imposed by the virus . the presence of 
 both complex- and oligomannose - type glycosylation 
 on the envelope glycoproteins of membranous viruses is an emerging 
 theme in viral glycomics . as an extreme , the oligomannose - type glycans 
 on gp120 of hiv-1 are thought to arise 
 by local steric constraints imposed by both glycan  glycan clustering 
 and glycan  protein interactions . in addition , broadly neutralizing antibodies 
 that recognize mixed glycan / protein epitopes have emerged as guides 
 to hiv vaccine design and even as potential antivirals . to this end , our analysis of sfv virion glycosylation assists in 
 the definition of the alphaviral antigenic surface . defining how the 
 host modulates alphavirion glycosylation will assist in understanding 
 how the mammalian immune system differs in the recognition of insect- 
 and mammalian - derived particles . we anticipate that the sensitive 
 ion mobility methodologies adopted 
 in this study will enable the precise analysis of glycans from membrane 
 viruses where low abundance has impeded traditional mass spectrometric 
 approaches . the resolution of our method further allows us to explore 
 the range of structures displayed by each of the alphaviral glycoproteins . we show that specific glycan types are not exclusive to either glycoprotein 
 and observe that both proteins display a full range of glycan structures , 
 ranging from oligomannose series with little biosynthetic processing 
 to fully processed complex - type glycans . such compositional variation , which is either imposed by the virus or the host , is likely to influence 
 host - cell tropism and immunological responses to alphavirus infection .
cross - species 
 viral transmission subjects parent and progeny alphaviruses 
 to differential post - translational processing of viral envelope glycoproteins . 
 alphavirus biogenesis has been extensively studied , and the semliki 
 forest virus e1 and e2 glycoproteins have been shown to exhibit differing 
 degrees of processing of n - linked glycans . however 
 the composition 
 of these glycans , including that arising from different host cells , 
 has not been determined . 
 here we determined the chemical composition 
 of the glycans from the prototypic alphavirus , semliki forest virus , 
 propagated in both arthropod and rodent cell lines , by using ion - mobility 
 mass spectrometry and collision - induced dissociation analysis . 
 we 
 observe that both the membrane - proximal e1 fusion glycoprotein and 
 the protruding e2 attachment glycoprotein display heterogeneous glycosylation 
 that contains n - linked glycans exhibiting both limited and extensive 
 processing . 
 however , e1 contained predominantly highly processed glycans 
 dependent on the host cell , with rodent and mosquito - derived e1 exhibiting 
 complex - type and paucimannose - type glycosylation , respectively . 
 in 
 contrast , the protruding e2 attachment glycoprotein primarily contained 
 conserved under - processed oligomannose - type structures when produced 
 in both rodent and mosquito cell lines . 
 it is likely that glycan processing 
 of e2 is structurally restricted by steric - hindrance imposed by local 
 viral protein structure . 
 this contrasts e1 , which presents glycans 
 characteristic of the host cell and is accessible to enzymes . 
 we integrated 
 our findings with previous cryo - electron microscopy and crystallographic 
 analyses to produce a detailed model of the glycosylated mature virion 
 surface . 
 taken together , these data reveal the degree to which virally 
 encoded protein structure and cellular processing enzymes shape the 
 virion glycome during interspecies transmission of semliki forest 
 virus .