LitCovid-sample-sentences  

PMC:7253482 / 5511-27036 JSONTXT 27 Projects

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Id Subject Object Predicate Lexical cue
T30 0-22 Sentence denotes Results and discussion
T31 24-82 Sentence denotes Glycan processing of trimeric SARS and MERS spike proteins
T32 83-295 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 296-399 Sentence denotes SARS, MERS and HKU1 S genes encode many N-linked glycan sequons; 23, 23 and 29, respectively (Fig. 1a).
T34 400-525 Sentence denotes We initially sought to quantitatively assess the composition of the carbohydrate structures displayed on the S glycoproteins.
T35 526-705 Sentence denotes N-linked glycans were enzymatically released, fluorescently labelled, and subjected to hydrophilic interaction chromatography-ultra-performance liquid chromatography (HILIC-UPLC).
T36 706-861 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 862-1066 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 1067-1316 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 1317-1506 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 1507-1668 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 1669-1991 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 1992-2053 Sentence denotes Fig. 1 Compositional analysis of SARS, MERS and HKU1 glycans.
T43 2054-2239 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 2240-2568 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 2569-2779 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 2780-2909 Sentence denotes Oligomannose-type glycans are schematically annotated with mannose residues as green circles and GlcNAc residues as blue squares.
T47 2910-3130 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 3131-3301 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 3302-3448 Sentence denotes The glycan compositions characterised in the spectra were largely invariant among the coronaviruses with no major structural differences observed.
T50 3450-3496 Sentence denotes Clustering of underprocessed glycans on MERS S
T51 3497-3646 Sentence denotes We subsequently performed glycopeptide analysis to ascertain the compositions of glycans at all of the potential N-linked glycosylation sites (PNGs).
T52 3647-3863 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 3864-3972 Sentence denotes This revealed differential levels of oligomannose, hybrid, and complex-type glycan populations (Fig. 2a, b).
T54 3973-4039 Sentence denotes Using structures of the trimeric MERS and SARS S proteins (PDB ID:
T55 4040-4196 Sentence denotes 5X59 and 5X58, respectively), we generated models of fully glycosylated coronavirus spikes using experimentally determined glycan compositions (Fig. 3a, b).
T56 4197-4377 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 4378-4564 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 4565-4671 Sentence denotes As such, we performed mutagenesis to knock out glycosylation sites with N155A, N166A, and N236A mutations.
T59 4672-4844 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 4845-5006 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 5007-5115 Sentence denotes Fig. 2 Quantitative site-specific N-linked glycan analysis of SARS and MERS coronavirus spike glycoproteins.
T62 5116-5260 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 5261-5410 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 5411-5531 Sentence denotes Glycan compositions are based on the glycan library generated from negative-ion mass spectrometry of released N-glycans.
T65 5532-5883 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 5884-5933 Sentence denotes Left to right; least processed to most processed.
T67 5934-5995 Sentence denotes The pie charts summarise the quantification of these glycans.
T68 5996-6118 Sentence denotes Additional compositional information regarding the distribution of fucosylation and sialylation can be found in SI Fig. 3.
T69 6119-6198 Sentence denotes Fig. 3 Structure-based mapping of N-linked glycans on MERS and SARS S proteins.
T70 6199-6458 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 6459-6636 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 6637-6718 Sentence denotes The glycans are colored according to oligomannose content, as defined by the key.
T73 6719-6840 Sentence denotes DPP4 receptor-binding sites and ACE2 receptor-binding sites for MERS and SARS, respectively, are indicated in light blue.
T74 6841-6915 Sentence denotes The S1 and S2 subunits are colored light grey and dark grey, respectively.
T75 6916-6987 Sentence denotes Fig. 4 Amino-acid sequence diversification across SARS and MERS spikes.
T76 6988-7045 Sentence denotes a Amino-acid diversity in SARS and MERS S gene sequences.
T77 7046-7237 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 7238-7496 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 7497-7597 Sentence denotes S proteins are presented as backbone traces with residues colored according to amino-acid diversity.
T80 7598-7704 Sentence denotes Residues with elevated diversity are colored in red, and N-linked glycans are presented as white surfaces.
T81 7705-7895 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 7896-8074 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 8075-8287 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 8288-8627 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 8628-8932 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 8934-8972 Sentence denotes Sequence diversification of CoV spikes
T87 8973-9223 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 9224-9503 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 9504-9872 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 9873-10066 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 10067-10160 Sentence denotes Buried residues on SARS had mean dN/dS ratios of 0.31 compared with 2.82 for exposed resides.
T92 10161-10288 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 10289-10619 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 10620-10766 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 10767-10872 Sentence denotes We would suggest that this may result from the intrinsic receptor-binding functionality of these domains.
T96 10873-11186 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 11187-11360 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 11361-11470 Sentence denotes MERS was characterized by repeated spillover events from camels into humans, where it circulated transiently.
T99 11471-11592 Sentence denotes In contrast, the SARS outbreak corresponded to a single zoonotic event followed by extensive human-to-human transmission.
T100 11593-11710 Sentence denotes Consequently, inferring the degree of selection acting upon MERS and SARS from dN/dS analysis is extremely difficult.
T101 11711-12015 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 12017-12073 Sentence denotes Visualising the HIV-1 and SARS glycan shields by cryo-EM
T103 12074-12275 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 12276-12436 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 12437-12591 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 12592-12788 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 12789-12964 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 12965-13118 Sentence denotes This structure defines diffuse boundaries between buried and exposed surface protein surface, which can serve to define potential sites of vulnerability.
T109 13119-13299 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 13300-13553 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 13554-13872 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 13873-14324 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 14325-14400 Sentence denotes Fig. 5 Comparative cryo-EM analysis of SARS S and HIV-1 Env glycan shields.
T114 14401-14414 Sentence denotes a Left panel:
T115 14415-14623 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 14624-14637 Sentence denotes Middle panel:
T117 14638-14777 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 14778-14940 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 14942-14995 Sentence denotes Disparate shielding efficacies of viral glycosylation
T120 14996-15185 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 15186-15282 Sentence denotes For example, both LASV GPC and coronavirus S proteins consist of 25% glycan by molecular weight.
T122 15283-15590 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 15591-15851 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 15852-16197 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 16198-16449 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 16450-16523 Sentence denotes Fig. 6 Comparison of the glycan shields of viral class I fusion proteins.
T127 16524-16852 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 16853-17049 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 17050-17252 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 17253-17434 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 17435-17555 Sentence denotes Whether the restricted glycan shielding observed on coronaviruses is linked to the zoonosis of the pathogens is unknown.
T132 17556-17810 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 17811-18015 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 18016-18303 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 18304-18634 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 18635-18847 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 18848-18994 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 18995-19117 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 19118-19255 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 19256-19434 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 19435-19598 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 19599-19825 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 19826-20043 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 20044-20257 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 20258-20421 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 20422-20747 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 20748-21103 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 21104-21323 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 21324-21525 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.