LitCovid-sample-sentences  

PMC:7253482 JSONTXT 37 Projects

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Id Subject Object Predicate Lexical cue
T1 0-77 Sentence denotes Vulnerabilities in coronavirus glycan shields despite extensive glycosylation
T2 79-87 Sentence denotes Abstract
T3 88-265 Sentence denotes Severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) coronaviruses (CoVs) are zoonotic pathogens with high fatality rates and pandemic potential.
T4 266-390 Sentence denotes Vaccine development focuses on the principal target of the neutralizing humoral immune response, the spike (S) glycoprotein.
T5 391-514 Sentence denotes Coronavirus S proteins are extensively glycosylated, encoding around 66–87 N-linked glycosylation sites per trimeric spike.
T6 515-688 Sentence denotes Here, we reveal a specific area of high glycan density on MERS S that results in the formation of oligomannose-type glycan clusters, which were absent on SARS and HKU1 CoVs.
T7 689-952 Sentence denotes We provide a comparison of the global glycan density of coronavirus spikes with other viral proteins including HIV-1 envelope, Lassa virus glycoprotein complex, and influenza hemagglutinin, where glycosylation plays a known role in shielding immunogenic epitopes.
T8 953-1162 Sentence denotes Overall, our data reveal how organisation of glycosylation across class I viral fusion proteins influence not only individual glycan compositions but also the immunological pressure across the protein surface.
T9 1164-1260 Sentence denotes Glycosylation plays a key role in shielding of immunogenic epitopes on viral spike (S) proteins.
T10 1261-1476 Sentence denotes Here Watanabe et al. report that glycans of coronavirus SARS and MERS S proteins are heterogeneously distributed and do not form an efficacious high-density global shield which would ensure efficient immune evasion.
T11 1478-1490 Sentence denotes Introduction
T12 1491-1614 Sentence denotes Coronaviruses (CoVs) are enveloped pathogens responsible for multiple respiratory disorders of varying severity in humans1.
T13 1615-1880 Sentence denotes Certain CoVs represent a significant threat to global human health, as illustrated by outbreaks of severe acute respiratory syndrome coronavirus (SARS-CoV) in 20032, Middle East respiratory syndrome coronavirus (MERS-CoV) in 20123, and most recently of SARS-CoV-24.
T14 1881-2131 Sentence denotes Given their mortality rates, the current lack of targeted treatments and licensed vaccines, and their capacity to transmit between humans and across species barriers5,6, there is an urgent need for effective countermeasures to combat these pathogens.
T15 2132-2299 Sentence denotes Ongoing vaccine development efforts focus on the spike (S) proteins that protrude from the viral envelope and constitute the main target of neutralizing antibodies7,8.
T16 2300-2479 Sentence denotes These trimeric S proteins mediate host-cell entry with the S1 and S2 subunits responsible for binding to the host-cell receptor and facilitating membrane fusion, respectively9–11.
T17 2480-2641 Sentence denotes MERS S binds to dipeptidyl-peptidase 4 (DPP4)12, whereas SARS S13 and SARS-CoV-214,15 utilize angiotensin-converting enzyme 2 (ACE2) as a host cellular receptor.
T18 2642-2876 Sentence denotes CoV S proteins are the largest class I viral fusion proteins known9, and are extensively glycosylated, with SARS and MERS S glycoproteins both encoding 69 N-linked glycan sequons per trimeric spike with SARS-CoV-2 containing 66 sites.
T19 2877-3022 Sentence denotes These modifications often mask immunogenic protein epitopes from the host humoral immune system by occluding them with host-derived glycans16–18.
T20 3023-3291 Sentence denotes This phenomenon of immune evasion by molecular mimicry and glycan shielding has been well characterised across other viral glycoproteins, such as HIV-1 envelope protein (Env)19–21, influenza hemagglutinin (HA)22,23 and Lassa virus glycoprotein complex (LASV GPC)24–26.
T21 3292-3675 Sentence denotes Previous analyses of viral glycan shields have revealed the presence of underprocessed oligomannose-type glycans that seemingly arise due to steric constraints that prevent access of glycan processing enzymes to substrate glycans24,27,28, especially when the viral glycoprotein has evolved to mask immunogenic epitopes with a particularly dense array of host-derived glycans26,29–34.
T22 3676-3874 Sentence denotes Restricted access to these glycan sites or interference with surrounding protein surface or neighbouring glycan residues can render glycan processing enzymes ineffective in specific regions27,28,35.
T23 3875-4197 Sentence denotes Glycan processing on soluble glycoproteins has also been shown to be a strong reporter of native-like protein architecture and thus immunogen integrity36–38; and glycan processing on a successful immunogen candidate should therefore mimic, as closely as possible, the structural features observed on the native virus39,40.
T24 4198-4448 Sentence denotes Here, we provide global and site-specific analyses of N-linked glycosylation on soluble SARS, MERS and HKU1 CoV S glycoproteins and reveal extensive heterogeneity, ranging from oligomannose-type glycans to highly-processed complex-type glycosylation.
T25 4449-4669 Sentence denotes The structural mapping of glycans of trimeric S proteins revealed that some of these glycans contribute to the formation of a cluster of oligomannose-type glycans at specific regions of high glycan density on MERS-CoV S.
T26 4670-4864 Sentence denotes Molecular evolution analysis of SARS and MERS S genes also reveals a higher incidence of amino-acid diversity on the exposed surfaces of the S proteins that are not occluded by N-linked glycans.
T27 4865-5140 Sentence denotes In addition, we compare the structures of the respective glycan coats of SARS and HIV-1 envelope proteins using cryo-electron microscopy (cryo-EM) and computational modelling, which delineate a sparse glycan shield exhibited on SARS S compared with other viral glycoproteins.
T28 5141-5420 Sentence denotes We therefore undertook a comparative analysis of viral glycan shields from characterized class I fusion proteins to highlight how glycosylation density influences oligomannose-type glycan abundance, and the relationship between effective glycan shields and viral evasion ability.
T29 5421-5509 Sentence denotes Together, these data underscore the importance of glycosylation in viral immune evasion.
T30 5511-5533 Sentence denotes Results and discussion
T31 5535-5593 Sentence denotes Glycan processing of trimeric SARS and MERS spike proteins
T32 5594-5806 Sentence denotes To generate a soluble mimic of the viral S proteins, we used the 2P-stabilised native-like SARS and MERS S protein antigens, the design and structures of which have been described previously by Pallesen et al.41.
T33 5807-5910 Sentence denotes SARS, MERS and HKU1 S genes encode many N-linked glycan sequons; 23, 23 and 29, respectively (Fig. 1a).
T34 5911-6036 Sentence denotes We initially sought to quantitatively assess the composition of the carbohydrate structures displayed on the S glycoproteins.
T35 6037-6216 Sentence denotes N-linked glycans were enzymatically released, fluorescently labelled, and subjected to hydrophilic interaction chromatography-ultra-performance liquid chromatography (HILIC-UPLC).
T36 6217-6372 Sentence denotes Treatment with endoglycosidase H (Endo H) revealed a population (SARS 32.2%; MERS 33.8%, HKU1 25.0%) of underprocessed oligomannose-type glycans (Fig. 1b).
T37 6373-6577 Sentence denotes This observation of both complex and oligomannose-type glycans reveals that the majority of N-linked glycans can be processed, although there is limited processing at specific sites across the S proteins.
T38 6578-6827 Sentence denotes It is also interesting to note that the distribution of oligomannose-type glycans was broad, with Man5GlcNAc2 to Man9GlcNAc2 glycans all present, without one particular dominant peak, as is the case for some viral glycoproteins, such as HIV-1 Env36.
T39 6828-7017 Sentence denotes The proportion of oligomannose-type glycans on recombinant coronavirus S proteins is consistent with previous studies performed on virally derived MERS and SARS coronavirus S proteins17,42.
T40 7018-7179 Sentence denotes Coronaviruses have been previously been reported to form virions by budding into the lumen of endoplasmic reticulum-Golgi intermediate compartments (ERGIC)43,44.
T41 7180-7502 Sentence denotes Observations of hybrid- and complex-type glycans on virally derived material17,42 would, however, suggest that it is likely that coronavirus virions travel through the Golgi apparatus after virion formation in the ERGIC en route to the cell surface, thus supporting recombinant immunogens as models of viral glycoproteins.
T42 7503-7564 Sentence denotes Fig. 1 Compositional analysis of SARS, MERS and HKU1 glycans.
T43 7565-7750 Sentence denotes a Schematic representation of SARS, MERS and HKU1 coronavirus S glycoproteins, showing the positions of N-linked glycosylation amino-acid sequons (NXS/T, where X ≠ P) shown as branches.
T44 7751-8079 Sentence denotes The domains of the S glycoproteins are illustrated: N-terminal domain (NTD), receptor-binding domain (RBD), sub-domain 1/2 (SD1/2), fusion peptide (FP), heptad repeat 1/2 (HR1/2), central helix (CH), and transmembrane domain (TM). b HILIC-UPLC chromatograms of fluorescently labelled N-linked glycans from SARS, MERS and HKU1 S.
T45 8080-8290 Sentence denotes Oligomannose-type glycans (M5 to M9; Man5GlcNAc2–Man9GlcNAc2) (green) and complex-type glycans (magenta) were identified by Endo H digestion, with quantification of major glycan types summarised as a pie chart.
T46 8291-8420 Sentence denotes Oligomannose-type glycans are schematically annotated with mannose residues as green circles and GlcNAc residues as blue squares.
T47 8421-8641 Sentence denotes To ascertain the precise structures of N-linked glycans, glycan pools of each coronavirus S protein were analysed by negative-ion ion-mobility-electrospray ionisation mass spectrometry (IM-ESI MS) (Supplementary Fig. 1).
T48 8642-8812 Sentence denotes Consistent with the UPLC data, IM-ESI MS confirmed an array of complex-type glycans ranging from mono- to tetra-antennary, but also oligomannose- and hybrid-type glycans.
T49 8813-8959 Sentence denotes The glycan compositions characterised in the spectra were largely invariant among the coronaviruses with no major structural differences observed.
T50 8961-9007 Sentence denotes Clustering of underprocessed glycans on MERS S
T51 9008-9157 Sentence denotes We subsequently performed glycopeptide analysis to ascertain the compositions of glycans at all of the potential N-linked glycosylation sites (PNGs).
T52 9158-9374 Sentence denotes MERS, SARS and HKU1 recombinant S proteins were reduced, alkylated and digested with an assortment of proteases to yield glycopeptides, which were subjected to in-line liquid chromatography-mass spectrometry (LC-MS).
T53 9375-9483 Sentence denotes This revealed differential levels of oligomannose, hybrid, and complex-type glycan populations (Fig. 2a, b).
T54 9484-9550 Sentence denotes Using structures of the trimeric MERS and SARS S proteins (PDB ID:
T55 9551-9707 Sentence denotes 5X59 and 5X58, respectively), we generated models of fully glycosylated coronavirus spikes using experimentally determined glycan compositions (Fig. 3a, b).
T56 9708-9888 Sentence denotes This revealed that oligomannose-type glycans on MERS S co-localize to specific clusters on the head of the S protein, consisting of glycans at Asn155, Asn166, and Asn236 (Fig. 3a).
T57 9889-10075 Sentence denotes We hypothesized that the fully oligomannose-type glycan population in this cluster arises due to the hindered accessibility of glycan processing enzymes to access the substrate glycan28.
T58 10076-10182 Sentence denotes As such, we performed mutagenesis to knock out glycosylation sites with N155A, N166A, and N236A mutations.
T59 10183-10355 Sentence denotes Site-specific analysis of these glycan-KO mutants revealed enhanced trimming of mannose residues, i.e. increased processing, when glycan clustering was reduced (SI Fig. 4).
T60 10356-10517 Sentence denotes The presence of clustered oligomannose-type glycans is reminiscent of that found on other viral glycoproteins, including HIV-1 Env and LASV GPC24,31,34,36,45,46.
T61 10518-10626 Sentence denotes Fig. 2 Quantitative site-specific N-linked glycan analysis of SARS and MERS coronavirus spike glycoproteins.
T62 10627-10771 Sentence denotes Purified (a) MERS and b SARS S proteins were digested. Quantitative site-specific N-linked glycan analysis of a MERS and b SARS S glycoproteins.
T63 10772-10921 Sentence denotes Purified S proteins were digested with trypsin, chymotrypsin, alpha-lytic protease, Glu-C, and trypsin plus chymotrypsin, then analysed by LC-ESI MS.
T64 10922-11042 Sentence denotes Glycan compositions are based on the glycan library generated from negative-ion mass spectrometry of released N-glycans.
T65 11043-11394 Sentence denotes The bar graphs represent the relative quantities of each glycan group with oligomannose-type glycan series (M9 to M5; Man9GlcNAc2 to Man5GlcNAc2) (green), afucosylated and fucosylated hybrid glycans (Hybrid & F Hybrid) (dashed pink), and complex glycans grouped according to the number of antennae and presence of core fucosylation (A1 to FA4) (pink).
T66 11395-11444 Sentence denotes Left to right; least processed to most processed.
T67 11445-11506 Sentence denotes The pie charts summarise the quantification of these glycans.
T68 11507-11629 Sentence denotes Additional compositional information regarding the distribution of fucosylation and sialylation can be found in SI Fig. 3.
T69 11630-11709 Sentence denotes Fig. 3 Structure-based mapping of N-linked glycans on MERS and SARS S proteins.
T70 11710-11969 Sentence denotes The modelling of the experimentally observed glycosylation is illustrated on the prefusion structure of trimeric a MERS S (PDB ID 5X59)11 and b SARS S (PDB ID 5X58)11 glycoproteins. Structural-based mapping of N-linked glycans on a MERS and b SARS S proteins.
T71 11970-12147 Sentence denotes The modelling of the experimentally observed glycosylation is illustrated on the prefusion structure of trimeric MERS S (PDB ID 5X59)11 and SARS S (PDB ID 5X58)11 glycoproteins.
T72 12148-12229 Sentence denotes The glycans are colored according to oligomannose content, as defined by the key.
T73 12230-12351 Sentence denotes DPP4 receptor-binding sites and ACE2 receptor-binding sites for MERS and SARS, respectively, are indicated in light blue.
T74 12352-12426 Sentence denotes The S1 and S2 subunits are colored light grey and dark grey, respectively.
T75 12427-12498 Sentence denotes Fig. 4 Amino-acid sequence diversification across SARS and MERS spikes.
T76 12499-12556 Sentence denotes a Amino-acid diversity in SARS and MERS S gene sequences.
T77 12557-12748 Sentence denotes Averaged values for each domain are also shown below. b Comparison of dN/dS values between buried and exposed residues across SARS and MERS S (n = 70 and 100 for SARS and MERS, respectively).
T78 12749-13007 Sentence denotes The error bars correspond to the 95% highest posterior density intervals while the circles indicate mean dN/dS values. c Mapping of the per residue amino-acid diversity shown in A onto the structures of SARS and MERS S (PDB ID 5X58 and 5X59, respectively)11.
T79 13008-13108 Sentence denotes S proteins are presented as backbone traces with residues colored according to amino-acid diversity.
T80 13109-13215 Sentence denotes Residues with elevated diversity are colored in red, and N-linked glycans are presented as white surfaces.
T81 13216-13406 Sentence denotes Interestingly, SARS and HKU1 (SI Fig. 2) S proteins did not exhibit specific mannose clusters that contribute to the overall mannose abundance, but only isolated glycans were underprocessed.
T82 13407-13585 Sentence denotes We speculate that the oligomannose-type glycans here arise from protein-directed inhibition of glycan processing, as opposed to the glycan-influenced processing observed on MERS.
T83 13586-13798 Sentence denotes Importantly, oligomannose-type glycans has also been implicated in innate immune recognition of coronaviruses by lectins47,48 that recognise these underprocessed glycans as pathogen-associated molecular patterns.
T84 13799-14138 Sentence denotes Given that the receptor-binding domain is the main target of neutralising antibodies8, it is surprising that the DPP4 receptor-binding site of MERS S was not occluded by glycans (Fig. 3a), as observed for other receptor-binding sites of class I viral fusion proteins, including SARS S (Fig. 3b), HIV-1 Env49, LASV GPC24 and influenza HA50.
T85 14139-14443 Sentence denotes We suggest that this is likely due to the intrinsic functionality of the receptor-binding domain of MERS S, that would be sterically hindered by the presence of N-linked glycans, whereas other viruses are able to accommodate the post-translational modifications, without greatly perturbing functionality.
T86 14445-14483 Sentence denotes Sequence diversification of CoV spikes
T87 14484-14734 Sentence denotes We hypothesized that solvent-accessible, amino-acid residues on S proteins would be undergoing higher rates of mutations compared with buried residues and regions that are occluded by glycans, which are unable to be targeted by host immune responses.
T88 14735-15014 Sentence denotes To that end, we performed an evaluation of amino-acid diversification on a residue-specific level, using publicly available gene sequences of SARS and MERS S, which was calculated as the number of observed pairwise differences divided by the total number of pairwise comparisons.
T89 15015-15383 Sentence denotes Firstly, we found that amino-acid diversity was elevated at known epitopes targeted by neutralizing antibodies, such as the N-terminal domain and the receptor-binding domains, and reduced in the regions in the S2 domain, such as the fusion peptide, heptad repeat one, and the central helix domains, which are likely subject to greater functional constraints (Fig. 4a).
T90 15384-15577 Sentence denotes Analysis of the relative ratio of non-synonymous to synonymous nucleotide substitutions (i.e. dN/dS ratios) revealed that exposed residues exhibited significantly higher dN/dS values (Fig. 4b).
T91 15578-15671 Sentence denotes Buried residues on SARS had mean dN/dS ratios of 0.31 compared with 2.82 for exposed resides.
T92 15672-15799 Sentence denotes Likewise, the buried residues on MERS had a calculated dN/dS ratio of 0.10 compared with exposed residues with a value of 0.45.
T93 15800-16130 Sentence denotes Furthermore, when per-site amino-acid diversities were mapped onto the fully glycosylated structural model of the respective CoV S proteins (Fig. 4c), hotspots of mutations were highlighted on the protein surface throughout the trimer revealing extensive vulnerabilities permeating through the glycan shield of SARS and MERS CoVs.
T94 16131-16277 Sentence denotes It is interesting to note the lack of amino-acid diversity on the receptor-binding domains of MERS S proteins that protrude away from the glycans.
T95 16278-16383 Sentence denotes We would suggest that this may result from the intrinsic receptor-binding functionality of these domains.
T96 16384-16697 Sentence denotes Although dN/dS estimates are comparable within each viral outbreak, they are not directly comparable between viral families as they can only be considered in the environment in which they are measured (i.e. multiple differences in transmission ecology and host-virus interactions disallow meaningful comparisons).
T97 16698-16871 Sentence denotes For example, differences in the epidemic behaviour and host immune environment of MERS and SARS outbreaks likely contribute to the observed genetic diversity and thus dN/dS.
T98 16872-16981 Sentence denotes MERS was characterized by repeated spillover events from camels into humans, where it circulated transiently.
T99 16982-17103 Sentence denotes In contrast, the SARS outbreak corresponded to a single zoonotic event followed by extensive human-to-human transmission.
T100 17104-17221 Sentence denotes Consequently, inferring the degree of selection acting upon MERS and SARS from dN/dS analysis is extremely difficult.
T101 17222-17526 Sentence denotes Importantly, while similar analyses of SARS-CoV-2 is desirable, due to the low genetic variation among the current SARS-CoV-2 sequences (as of 17 March 2020), which likely include deleterious mutations that will be removed by selection over time, the resulting bioinformatic analyses would be unreliable.
T102 17528-17584 Sentence denotes Visualising the HIV-1 and SARS glycan shields by cryo-EM
T103 17585-17786 Sentence denotes HIV-1 Env is a prototypic viral class I fusion protein that exhibits extensive surface glycosylation, resulting in an effective glycan shield to aid evasion from the host adaptive immune response21,31.
T104 17787-17947 Sentence denotes In order to visualize the structure of the respective glycan “shields” of HIV-1 and SARS coronavirus we used single-particle cryo-electron microscopy (cryo-EM).
T105 17948-18102 Sentence denotes The results for HIV-1 Env were reproduced directly from Berndsen et al.51 while the previously published SARS 2P dataset52 was reprocessed for this study.
T106 18103-18299 Sentence denotes Although cryo-EM datasets of fully glycosylated MERS S41 and chimpanzee simian immunodeficiency virus (SIVcpz)53 are also available, only the HIV and SARS data were of sufficient quality (Fig. 5).
T107 18300-18475 Sentence denotes We recently showed51 that dynamics in surface exposed glycans HIV-1 Env leads to an extensive network of interactions that drive higher-order structuring in the glycan shield.
T108 18476-18629 Sentence denotes This structure defines diffuse boundaries between buried and exposed surface protein surface, which can serve to define potential sites of vulnerability.
T109 18630-18810 Sentence denotes Cryo-EM captures the ensemble-average structure of biomolecules and therefore glycan dynamics results in blurred density at the resolutions necessary for building atomic structure.
T110 18811-19064 Sentence denotes However, we showed how a simple combination of low-pass filtering and auto-thresholding, as well as 3D variability analysis, can reveal the previously hidden structure of the SARS glycan shield and compare it with the HIV-1 Env glycan shield51 (Fig. 5).
T111 19065-19383 Sentence denotes We observe the nearly all-encompassing glycan density on HIV-1 Env and evidence for extensive glycan–glycan interactions, especially in the oligomannose patch regions, whereas the glycans on SARS S appear more isolated and lack the wide-ranging glycan networks that are the hallmark of an effective glycan shield54,55.
T112 19384-19835 Sentence denotes The 3D variability maps are more sensitive to low intensity signal and reveal additional glycan–glycan interactions in both maps, however the S1 receptor-binding domains in the SARS dataset were shown to exist in both up and down conformations52, leading to poor resolution and significant 2D-variability which is convolved with the variability coming from glycans and limits the interpretability of glycan shielding effects in this region of the map.
T113 19836-19911 Sentence denotes Fig. 5 Comparative cryo-EM analysis of SARS S and HIV-1 Env glycan shields.
T114 19912-19925 Sentence denotes a Left panel:
T115 19926-20134 Sentence denotes Sharpened 3.2-Å-resolution C3-symmetric cryo-EM map of SARS S 2P ectodomain52 visualized at a high contour level with disordered S1 receptor-binding and N-terminal domains extending out from the central core.
T116 20135-20148 Sentence denotes Middle panel:
T117 20149-20288 Sentence denotes Low-pass filtered (lpf) cryo-EM map of the glycoprotein visualised at a low contour level along with a simulated peptide-only map overlaid.
T118 20289-20451 Sentence denotes Right panel: SPARX 3D variability map51. b Same as in (a) but for HIV-1 Env BG505 SOSIP.664 construct in complex with three copies of RM20A3 base-specific Fabs51.
T119 20453-20506 Sentence denotes Disparate shielding efficacies of viral glycosylation
T120 20507-20696 Sentence denotes Viral envelope proteins are glycosylated to varying degrees, but depending on their overall mass, surface area, and volume, the overall density of glycan shielding may differ significantly.
T121 20697-20793 Sentence denotes For example, both LASV GPC and coronavirus S proteins consist of 25% glycan by molecular weight.
T122 20794-21101 Sentence denotes However, given the significantly larger protein surface area and volume of coronavirus S proteins, coverage of the glycan “shield” over the proteinaceous surface is considerably sparser in comparison to the smaller LASV GPC, which occludes a far greater proportion of the protein surface with fewer glycans.
T123 21102-21362 Sentence denotes To demonstrate that the presence of glycosylation plays a major role in the immune response to these different glycoproteins, we studied the glycome of several biomedically important coronaviruses and compared their glycan compositions in a structural context.
T124 21363-21708 Sentence denotes We then investigated the glycan shield densities of seven viral class I fusion proteins using a global structural approach which was calculated by dividing the number of amino-acids that interact with glycans by the number of solvent-accessible amino-acid residues of each respective glycoprotein and plotted this against oligomannose abundance.
T125 21709-21960 Sentence denotes A strong correlation was observed (Fig. 6) and viruses historically classified as “evasion strong”56 had significantly elevated glycan shield densities and oligomannose abundance, which underscores the importance of glycan shielding in immune evasion.
T126 21961-22034 Sentence denotes Fig. 6 Comparison of the glycan shields of viral class I fusion proteins.
T127 22035-22363 Sentence denotes Glycan shield densities were calculated using Proteins, Interfaces, Structures and Assemblies (PISA)83 analyses of fully glycosylated models of SARS S, MERS S, HKU1 S, LASV GPC, HIV-1 Env (BG505), Influenza H3N2 hemagglutinin (Victoria 2011), SIV Env (PDB ID 5X58, 5X59, 5I08, 5VK2, 4ZMJ, 4O5N, 6OHY, respectively)9,11,53,84–86.
T128 22364-22560 Sentence denotes Oligomannose abundances of viral glycoproteins were ascertained by HILIC-UPLC analysis of PNGase F released N-linked glycans that were fluorescently labelled with procainamide24,45,53 (SI Fig. 5).
T129 22561-22763 Sentence denotes The number of amino-acid residues interacting with N-linked glycans was divided by the number of solvent-accessible amino-acid residues of the glycoprotein as a measure for global glycan shield density.
T130 22764-22945 Sentence denotes All viral glycoproteins analysed were expressed as trimers in HEK293F cells apart from LASV GPC, which was derived from virus-like particles from Madin-Darby canine kidney II cells.
T131 22946-23066 Sentence denotes Whether the restricted glycan shielding observed on coronaviruses is linked to the zoonosis of the pathogens is unknown.
T132 23067-23321 Sentence denotes However, it is tempting to speculate, for example, that MERS has not evolved a dense shield since it would not offer as much of a protective advantage against camel nanobodies (also known as single-domain antibodies) which could more easily penetrate it.
T133 23322-23526 Sentence denotes Investigation of the host immune response to viruses in their natural reservoirs may offer a route to understanding why coronavirus glycosylation does not reach the density of other viruses such as HIV-1.
T134 23527-23814 Sentence denotes In addition, it may be that functional constraints, such as maintaining flexibility of the receptor-binding domains, limit the accretion of glycans on coronavirus spikes, which would render it incapable of performing its primary functions, including receptor-binding and membrane fusion.
T135 23815-24145 Sentence denotes This phenomenon has been observed on other viral glycoproteins, including influenza HAs, where there is a limit to the accumulation of glycosylation sites that can be incorporated in vivo57,58, compared with in vitro59, with H3N2 and H1N1 HAs replacing existing PNGs rather than continually adding them upon the glycoprotein22,58.
T136 24146-24358 Sentence denotes The importance of glycosylation in modulation of viral infectivity and immune responses have also been investigated during influenza vaccine research22,60 and should be considered in coronavirus vaccine research.
T137 24359-24505 Sentence denotes More topically, it is interesting to note the conservation of N-linked glycosylation sites on S proteins from the SARS-CoV-2 and SARS (SI Fig. 6).
T138 24506-24628 Sentence denotes SARS-CoV-2 possesses a total of 22 N-linked glycan sites compared with 23 on SARS, with 18 of these sites being in common.
T139 24629-24766 Sentence denotes As such, it is likely that these glycans on this novel coronavirus would shield similar immunogenic epitopes that are observed on SARS S.
T140 24767-24945 Sentence denotes As expected, most of the differences between the two viruses are observed on the S1 subunit, due to its amenability to substitutions while still remaining functionally competent.
T141 24946-25109 Sentence denotes Furthermore, likely targets for the majority of antibodies targeting the spike are located on S1, resulting in greater levels of immune pressure upon this subunit.
T142 25110-25336 Sentence denotes This notion is further reflected in terms of glycosylation, with all of the glycan sites conserved on the S2 subunit between SARS and SARS-CoV-2, whereas the S1 subunit exhibits glycan site additions and deletions (SI Fig. 7).
T143 25337-25554 Sentence denotes Bioinformatic analysis of current SARS-CoV-2 spike genes (n = 566 as of 17 March 2020) from nextstrain61 (https://nextstrain.org/ncov) revealed low sequence diversity and no changes in glycosylation sites (SI Fig. 8).
T144 25555-25768 Sentence denotes Although it is difficult to directly compare viruses in terms of immunogenic responses, on the one hand, SARS and MERS coronaviruses readily elicit neutralizing antibodies following infection or immunization62–65.
T145 25769-25932 Sentence denotes Indeed, many potential MERS-CoV vaccine candidates are able to elicit high titres of serum IgG upon immunization but fail to produce sufficient mucosal immunity65.
T146 25933-26258 Sentence denotes In contrast, the high mutation rate66 and the evolving glycan shield of HIV-120,39, which firmly exemplifies it as “evasion strong” virus, hinders the development of broadly neutralizing antibodies67.Viruses classified as “evasion strong”26,56 may then differ due to varied efficacies of protein surface shielding by glycans.
T147 26259-26614 Sentence denotes Overall, this study adds further evidence suggesting that extensive N-linked glycan modifications of SARS and MERS CoV S proteins do not constitute an effective shield, in comparison to glycan shields of certain other viruses, which is reflected by the overall structure, density and oligomannose abundance across the corresponding trimeric glycoproteins.
T148 26615-26834 Sentence denotes We also demonstrate that amino-acid diversification indeed occurs at antibody accessible regions on the trimer, which confirms that glycans play a role in occluding specific regions if vulnerability on the glycoprotein.
T149 26835-27036 Sentence denotes Furthermore, comparisons between glycan shields from a number of viruses highlight the importance of a glycan shield in immune evasion and reveal structural principles that govern glycosylation status.
T150 27038-27045 Sentence denotes Methods
T151 27047-27109 Sentence denotes Expression and purification of coronavirus spike glycoproteins
T152 27110-27388 Sentence denotes Human embryonic kidney 293 Freestyle (HEK293F) cells were transfected with mammalian-codon-optimised genes encoding 2P-stabilised SARS MERS and HKU1 S proteins containing a C-terminal T4 fibritin trimerization domain, an HRV3C cleavage site, an 8xHis-tag and a Twin-Strep-tag41.
T153 27389-27462 Sentence denotes H3N2 Victoria 2011 hemagglutinin was also expressed in the HEK293F cells.
T154 27463-27670 Sentence denotes The 200 ml cultures were harvested 6 days after transfection, filtered and purified by nickel-affinity chromatography and size exclusion chromatography using a SuperdexTM 16/600 75 pg column (GE Healthcare).
T155 27672-27713 Sentence denotes Release and labelling of N-linked glycans
T156 27714-27835 Sentence denotes Excised coronavirus S gel bands were washed alternately with acetonitrile and water before drying in a vacuum centrifuge.
T157 27836-27930 Sentence denotes The bands were rehydrated with 100 μL of water and incubated with PNGase F at 37 °C overnight.
T158 27931-28193 Sentence denotes Aliquots of released N-linked glycans were also fluorescently labelled with procainamide, by adding 100 μL of labelling mixture (110 mg/mL procainamide and 60 mg/mL sodium cyanoborohydrate in 70% DMSO and 30% glacial acetic acid) and incubating for 4 h at 65 °C.
T159 28194-28289 Sentence denotes Procainamide labelled glycans were purified using Spe-ed Amide 2 columns (Applied Separations).
T160 28291-28320 Sentence denotes Glycan analysis by HILIC-UPLC
T161 28321-28533 Sentence denotes Labelled glycans were analysed using a 2.1 mm × 150 mm Acquity BEH Glycan column (Waters) on an Acquity H-Class UPLC instrument (Waters), with fluorescence measurements occurring at λex = 310 nm and λem = 370 nm.
T162 28534-28580 Sentence denotes The following gradient was used: time (t) = 0:
T163 28581-28629 Sentence denotes 22% A, 78% B (flow rate = 0.5 mL/min); t = 38.5:
T164 28630-28670 Sentence denotes 44.1% A, 55.9% B (0.5 mL/min); t = 39.5:
T165 28671-28708 Sentence denotes 100% A, 0% B (0.25 mL/min); t = 44.5:
T166 28709-28746 Sentence denotes 100% A, 0% B (0.25 mL/min); t = 46.5:
T167 28747-28849 Sentence denotes 22% A, 78% B (0.5 mL/min), where solvent A was 50 mM ammonium formate (pH 4.4) and B was acetonitrile.
T168 28850-29030 Sentence denotes Quantification of oligomannose-type glycans was achieved by digestion of fluorescently labelled glycans with Endo H, and clean-up using a PVDF protein-binding membrane (Millipore).
T169 29031-29088 Sentence denotes Empower 3 software (Waters) was used for data processing.
T170 29090-29118 Sentence denotes Mass spectrometry of glycans
T171 29119-29372 Sentence denotes Prior to ion-mobility electrospray ionisation MS and tandem MS analysis, PNGase F released N-linked glycans were purified on a Nafion® 117 membrane (Sigma-Aldrich) and a trace amount of ammonium phosphate was added to promote phosphate adduct formation.
T172 29373-29789 Sentence denotes Glycans were analyzed by direct infusion using a Synapt G2Si instrument (Waters) with the following settings: capillary voltage, 0.8–1.0 kV; sample cone, 150 V; extraction cone, 150 V; cone gas, 40 l/h; source temperature, 80 °C; trap collision voltage, 4–160 V; transfer collision voltage, 4 V; trap DC bias, 60 V; IMS wave velocity, 450 m/s; IMS wave height, 40 V; trap gas flow, 2 ml/min; IMS gas flow, 80 ml/min.
T173 29790-29887 Sentence denotes Data were acquired and processed with MassLynx v4.1 and Driftscope version 2.8 software (Waters).
T174 29889-29923 Sentence denotes Mass spectrometry of glycopeptides
T175 29924-30031 Sentence denotes Aliquots of 30–50 μg of coronavirus spikes were denatured, reduced and alkylated as described previously36.
T176 30032-30176 Sentence denotes Proteins were proteolytically digested with trypsin (Promega), chymotrypsin (Promega), alpha-lytic protease (Sigma-Aldrich) and Glu-C (Promega).
T177 30177-30320 Sentence denotes Reaction mixtures were dried and peptides/glycopeptides were extracted using C18 Zip-tip (MerckMilipore) following the manufacturer’s protocol.
T178 30321-30532 Sentence denotes Samples were resuspended in 0.1% formic acid prior to analysis by liquid chromatography-mass spectrometry using an Easy-nLC 1200 system coupled to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific).
T179 30533-30761 Sentence denotes Glycopeptides were separated using an EasySpray PepMap RSLC C18 column (75 μm × 75 cm) with a 240-min linear solvent gradient of 0–32% acetonitrile in 0.1% formic acid, followed by 35 min of 80% acetonitrile in 0.1% formic acid.
T180 30762-30909 Sentence denotes Other settings include an LC flow rate of 200 nL/min, spray voltage of 2.8 kV, capillary temperature of 275 °C, and an HCD collision energy of 50%.
T181 30910-31041 Sentence denotes Precursor and fragmentation detection were performed using an Orbitrap at the following resolution: MS1 = 100,000 and MS2 = 30,000.
T182 31042-31160 Sentence denotes The automatic gain control (AGC) targets were MS1 = 4e5 and MS2 = 5e4, and injection times were MS1 = 50 and MS2 = 54.
T183 31161-31303 Sentence denotes The following cleavage sites were used for the respective proteases; trypsin=R/K, chymotrypsin=F/Y/W, alpha lytic protease=T/A/S/V, Glu C=E/D.
T184 31304-31345 Sentence denotes Number of missed cleavages were set at 3.
T185 31346-31393 Sentence denotes The following modifications were also included:
T186 31394-31691 Sentence denotes Carbamidomethyl (+57.021464, target=C, fine control=fixed), Oxidation (+15.994915, target=M, fine control=variable rare 1), Glu to pyro-Glu (−18.010565, target=peptide N-term E, fine control=variable rare 1), and Gln to pyro-Glu (−17.026549, target peptide N-term Q, fine control=variable rare 1).
T187 31692-31839 Sentence denotes Glycopeptide fragmentation data were extracted form raw files using ByonicTM (Version 3.5.0) and ByologicTM (Version 3.5-15; Protein Metrics Inc.).
T188 31840-32051 Sentence denotes Glycopeptide fragmentation data were manually evaluated with true-positive assignments given when correct b- and y-fragments and oxonium ions corresponding to the peptide and glycan, respectively, were observed.
T189 32052-32146 Sentence denotes The precursor mass tolerance was set at 4 ppm for precursor ions and 10 ppm for fragment ions.
T190 32147-32240 Sentence denotes MS data were searched using a glycan library (SI Fig. 9) with the identical peptide sequence.
T191 32241-32285 Sentence denotes A 1% false discovery rate (FDR) was applied.
T192 32286-32503 Sentence denotes The extracted ion chromatographic areas for each true-positive glycopeptide, with the same amino-acid sequence, were compared to determine the relative quantitation of glycoforms at each specific N-linked glycan site.
T193 32505-32523 Sentence denotes Model construction
T194 32524-32797 Sentence denotes Structural models of N-linked glycan presentation on SARS, MERS and HKU1 S were created using electron microscopy structures (PDB ID 5X58, 5X59, and 5I08, respectively)9,11, along with complex-, hybrid-, and oligomannose-type N-linked glycans (PDB ID 4BYH, 4B7I, and 2WAH).
T195 32798-32921 Sentence denotes The most dominant glycoform presented at each site was modelled on to the N-linked carbohydrate attachment sites in Coot68.
T196 32923-32951 Sentence denotes Molecular evolution analysis
T197 32952-33090 Sentence denotes Publicly available sequences encoding full-length GPC spike gene for SARS-CoV (3765 bp) were downloaded from GenBank and manually aligned.
T198 33091-33172 Sentence denotes For MERS-CoV, we leveraged the whole genome alignment collated by Dudas et al.69.
T199 33173-33299 Sentence denotes Specifically, the alignment corresponding to the spike gene was extracted (4059 bp), excluding sequences isolated from humans.
T200 33300-33391 Sentence denotes Final alignments for SARS- and MERS-CoV corresponded to 70 and 100 sequences, respectively.
T201 33392-33531 Sentence denotes For the dN/dS analysis, we first estimated Bayesian molecular clock phylogenies for SARS- and MERS-CoV independently using BEAST v 1.8.470.
T202 33532-33699 Sentence denotes For both viruses, we assumed an uncorrelated log-normal distributed molecular clock71, Bayesian Skyline coalescent prior72 and a codon-structured substitution model73.
T203 33700-33830 Sentence denotes Multiple independent MCMC runs of 10–20 million steps were executed to ensure that stationarity and convergence had been achieved.
T204 33831-34124 Sentence denotes Empirical distributions of time-scaled phylogenies were obtained by combining (after the removal of burnin) the posterior tree distributions from the separate runs, which were subsequently used to estimate dN/dS ratios using the renaissance counting approach74,75 implemented in BEAST v 1.8.4.
T205 34125-34338 Sentence denotes We also estimated per-site amino-acid diversity, which was calculated as the average number of amino-acid difference between two sequences at an amino-acid position in all possible pairs in the sequence alignment.
T206 34340-34379 Sentence denotes Cryo-EM data analysis and visualization
T207 34380-34511 Sentence denotes Single-particle cryo-EM data analysis of BG505 SOSIP.664 in complex with RM20A3 Fab was reproduced directly from Berndsen et al.51.
T208 34512-34738 Sentence denotes Data for the SARS-CoV S 2P ectodomain was previously published52 and the final particle stack and alignment parameters from the published reconstruction were used for 3D variability analysis in the SPARX software package76,77.
T209 34739-34873 Sentence denotes All metadata for these reconstructions along with raw data images and FSC resolution curves can be found in the original publications.
T210 34874-35031 Sentence denotes In summary, both datasets were acquired on a FEI Titan Krios (Thermo Fisher) operating at 300 KeV equipped with a K2 Summit Direct Electron Detector (Gatan).
T211 35032-35144 Sentence denotes Movie micrographs were aligned and dose weighted with MotionCor278 and CTF estimation was performed with Gctf79.
T212 35145-35230 Sentence denotes Single-particle data processing was performed using CryoSparc v.280 and Relion v.381.
T213 35231-35316 Sentence denotes Maps were low-pass filtered using a Gaussian kernel and visualized in UCSF chimera82.
T214 35317-35483 Sentence denotes A detailed description of the auto-thresholding method used to set the isosurface value for visualisation of low-pass filtered maps can be found in Berndsen et al.51.
T215 35485-35528 Sentence denotes Clustering analysis of viral glycan shields
T216 35529-35742 Sentence denotes Solvent-accessible residues and interactions between N-linked glycans and amino-acid residues were calculated using Proteins, Interfaces, Structures and Assemblies (PISA) European Bioinformatics Institute (EBI)83.
T217 35743-35912 Sentence denotes Glycan shield density was calculated by the number of amino-acid residues interacting with glycans divided by the total number of solvent-accessible amino-acid residues.
T218 35914-35931 Sentence denotes Reporting summary
T219 35932-36048 Sentence denotes Further information on research design is available in the Nature Research Reporting Summary linked to this article.
T220 36050-36075 Sentence denotes Supplementary information
T221 36077-36102 Sentence denotes Supplementary Information
T222 36103-36120 Sentence denotes Reporting Summary
T223 36122-36185 Sentence denotes Peer review information Nature Communications thanks Matthew B.
T224 36186-36282 Sentence denotes Renfrow and other, anonymous, reviewers for their contributions to the peer review of this work.
T225 36283-36418 Sentence denotes Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
T226 36420-36445 Sentence denotes Supplementary information
T227 36446-36530 Sentence denotes Supplementary information is available for this paper at 10.1038/s41467-020-16567-0.
T228 36532-36548 Sentence denotes Acknowledgements
T229 36549-36624 Sentence denotes The Wellcome Centre for Human Genetics is supported by grant 203141/Z/16/Z.
T230 36625-36712 Sentence denotes We thank the Medical Research Council (MR/S007555/1 to T.A.B.), NIH (R56 AI127371 to I.
T231 36713-37156 Sentence denotes A.W., R01 AI127521 to J.S.M. and A.B.W.), Bill and Melinda Gates Foundation (grants OPP1115782 to A.B.W. and M.C., and OPP1170236 to I.A.W. and A.B.W.), the International AIDS Vaccine Initiative, Bill and Melinda Gates Foundation through the Collaboration for AIDS Discovery (grants OPP1084519 and OPP1196345 to I.A.W., A.B.W. and M.C.), and the Scripps Consortium for HIV Vaccine Development (CHAVD) (UM1 AI144462 to M.C., A.B.W. and I.A.W.).
T232 37158-37175 Sentence denotes Data availability
T233 37176-37558 Sentence denotes In addition to the data reported in this paper and accompanying supplementary materials, the raw mass spectrometric data that support the findings of this study presented have been deposited on the MassIVE server (https://massive.ucsd.edu) with the accession codes (MSV000084993 for glycopeptides analysis [10.25345/C58T21]; and MSV000085152 for N-linked glycans [10.25345/C54705]).
T234 37560-37577 Sentence denotes Code availability
T235 37578-37613 Sentence denotes All code is available upon request.
T236 37615-37634 Sentence denotes Competing interests
T237 37635-37697 Sentence denotes J.S.M. and A.B.W. are inventors on U.S. patent application no.
T238 37698-37764 Sentence denotes 62/412,703 (“Prefusion Coronavirus Spike Proteins and Their Use”).
T239 37765-37817 Sentence denotes J.S.M. is an inventor on U.S. patent application no.
T240 37818-37851 Sentence denotes 62/972,886 (“2019-nCoV Vaccine”).