Spin ballet for sweet encounters: saturation-transfer difference NMR and X-ray crystallography complement each other in the elucidation of protein–glycan interactions

Blaum, Bärbel S.; Neu, Ursula; Peters, Thomas; Stehle, Thilo

doi:10.1107/s2053230x18006581

Cited by 26 publications

(18 citation statements)

References 69 publications

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“…To verify the desired glycan binding abilities of the sialic acid mutants, we conducted saturation transfer difference (STD-) NMR experiments to probe for binding of sialylated oligosaccharidsses and GAG oligosaccharides to MCPyV wild-type and mutant virus-like particles (VLPs). STD-NMR makes use of energy transfer from proteins to their ligands upon binding and thus allows determination of binding specificities by comparing the changes in the resonance frequencies of the ligand molecules in association with wild-type or mutant viruses (30, 87). Here, 3’sialyllactose (3’SL) and a chemically well-defined heparan sulfate (HS)- pentasaccharide, Arixtra (Ax), were chosen as a minimal sialylated MCPyV ligand and a short GAG oligosaccharide, respectively (Fig.…”

Section: Resultsmentioning

confidence: 99%

Infectious Entry of Merkel Cell Polyomavirus

Becker

Dominguez

Greune

et al. 2018

Preprint

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27Merkel Cell Polyomavirus (MCPyV) is a small, non-enveloped tumor virus associated 28 with an aggressive form of skin cancer, the Merkel cell carcinoma (MCC). MCPyV 29 infections are highly prevalent in the human population with MCPyV virions being 30 continuously shed from human skin. However, the precise host cell tropism(s) of 31MCPyV remains unclear: MCPyV is able to replicate within a subset of dermal 32fibroblasts, but MCPyV DNA has also been detected in a variety of other tissues. 33However, MCPyV appears different from other polyomaviruses as it requires sulfated 34 polysaccharides such as heparan sulfates and/or chondroitin sulfates for initial 35 attachment. Like other polyomaviruses, MCPyV engages sialic acid as a (co-36 )receptor. To explore the infectious entry process of MCPyV, we analyzed the cell 37 biological determinants of MCPyV entry into A549 cells, a highly transducible lung 38 carcinoma cell line, in comparison to well-studied simian virus 40 and a number of 39 other viruses. Our results indicate that MCPyV enters cells via caveolar/lipid raft-40 mediated endocytosis but not macropinocytosis, clathrin-mediated endocytosis or 41 glycosphingolipid-enriched carriers. The viruses internalized in small endocytic pits 42 that led the virus to endosomes and from there to the endoplasmic reticulum (ER). 43Similar to other polyomaviruses, trafficking required microtubular transport, 44 acidification of endosomes, and a functional redox environment. To our surprise, the 45 virus was found to acquire a membrane envelope within endosomes, a phenomenon 46 not reported for other viruses. Only minor amounts of viruses reached the ER, while 47 the majority was retained in endosomal compartments suggesting that endosome-to-48 ER trafficking is a bottleneck during infectious entry. 49 50 51 52 3 Importance 53 MCPyV is the first polyomavirus directly implicated in the development of an 54 aggressive human cancer, the Merkel Cell Carcinoma (MCC). Although MCPyV is 55 constantly shed from healthy skin, MCC incidence increases among aging and 56 immunocompromised individuals. To date, the events connecting initial MCPyV 57 infection and subsequent transformation still remain elusive. MCPyV differs from 58 other known polyomaviruses concerning its cell tropism, entry receptor requirements, 59 and infection kinetics. In this study, we examined the cellular requirements for 60 endocytic entry as well as the subcellular localization of incoming virus particles. A 61 thorough understanding of the determinants of the infectious entry pathway and the 62 specific biological niche will benefit prevention of virus-derived cancers such as 63 MCC. 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 Polyomaviruses (PyV) are small, non-enveloped dsDNA viruses with a diameter of 80 45-50 nm. The icosahedral (T=7) capids consist of 72 homopentameric capsomers of 81 the major capid protein VP1 with minor capsid proteins VP2/VP3 located within a 82 cavity underneath the VP1 pentamers. The PyV capsid harbors a chromatinized, 83 circu...

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Section: Resultsmentioning

confidence: 99%

Infectious Entry of Merkel Cell Polyomavirus

Becker

Dominguez

Greune

et al. 2018

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…To verify the desired glycan binding abilities of the sialic acid mutants, we conducted saturation transfer difference-nuclear magnetic resonance (STD-NMR) experiments to probe for binding of sialylated oligosaccharides and GAG oligosaccharides to MCPyV wt and mutant virus-like particles (VLPs). STD-NMR makes use of energy transfer from proteins to their ligands upon binding and thus allows determination of binding specificities by comparing the changes in the resonance frequencies of the ligand molecules in association with wild-type or mutant viruses (30,90). Here, 3= sialyllactose (SL) and a chemically well-defined heparan sulfate (HS) pentasaccharide, Arixtra (Ax), were chosen as a minimal sialylated MCPyV ligand and a short GAG oligosaccharide, respectively ( Fig.…”

Section: Mcpyv Psv Cell Binding and Infection Depend On Sulfated Glycmentioning

confidence: 99%

Infectious Entry of Merkel Cell Polyomavirus

Becker

Dominguez

Greune

et al. 2019

J Virol

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Merkel cell polyomavirus (MCPyV) is a small, nonenveloped tumor virus associated with an aggressive form of skin cancer, Merkel cell carcinoma (MCC). MCPyV infections are highly prevalent in the human population, with MCPyV virions being continuously shed from human skin. However, the precise host cell tropism(s) of MCPyV remains unclear: MCPyV is able to replicate within a subset of dermal fibroblasts, but MCPyV DNA has also been detected in a variety of other tissues. However, MCPyV appears different from other polyomaviruses, as it requires sulfated polysaccharides, such as heparan sulfates and/or chondroitin sulfates, for initial attachment. Like other polyomaviruses, MCPyV engages sialic acid as a (co)receptor. To explore the infectious entry process of MCPyV, we analyzed the cell biological determinants of MCPyV entry into A549 cells, a highly transducible lung carcinoma cell line, in comparison to well-studied simian virus 40 and a number of other viruses. Our results indicate that MCPyV enters cells via caveolar/lipid raft-mediated endocytosis but not macropinocytosis, clathrin-mediated endocytosis, or glycosphingolipid-enriched carriers. The viruses were internalized in small endocytic pits that led the virus to endosomes and from there to the endoplasmic reticulum (ER). Similar to other polyomaviruses, trafficking required microtubular transport, acidification of endosomes, and a functional redox environment. To our surprise, the virus was found to acquire a membrane envelope within endosomes, a phenomenon not reported for other viruses. Only minor amounts of viruses reached the ER, while the majority was retained in endosomal compartments, suggesting that endosome-to-ER trafficking is a bottleneck during infectious entry. IMPORTANCE MCPyV is the first polyomavirus directly implicated in the development of an aggressive human cancer, Merkel cell carcinoma (MCC). Although MCPyV is constantly shed from healthy skin, the MCC incidence increases among aging and immunocompromised individuals. To date, the events connecting initial MCPyV infection and subsequent transformation still remain elusive. MCPyV differs from other known polyomaviruses concerning its cell tropism, entry receptor requirements, and infection kinetics. In this study, we examined the cellular requirements for endocytic entry as well as the subcellular localization of incoming virus particles. A thorough understanding of the determinants of the infectious entry pathway and the specific biological niche will benefit prevention of virus-derived cancers such as MCC.

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“…In order to confirm the observed interactions and to probe for specificity of binding, we examined the interactions of 3=SL and related glycans with NJPyV VP1 pentamers in solution using saturation transfer difference nuclear magnetic resonance (STD NMR) measurements (56,57). This technique has been used to study similar protein-glycan interactions that are typically characterized by weak binding affinities (57,58). It is based on the fast cross-relaxation of excited states in large molecules and the intermolecular nuclear Overhauser effect (NOE) between protons of a protein and protons of a ligand within a complex with a fast off-rate.…”

Section: Resultsmentioning

confidence: 99%

Structural Basis and Evolution of Glycan Receptor Specificities within the Polyomavirus Family

Stroh

Rustmeier

Blaum

et al. 2020

mBio

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Asymptomatic infections with polyomaviruses in humans are common, but these small viruses can cause severe diseases in immunocompromised hosts. New Jersey polyomavirus (NJPyV) was identified via a muscle biopsy in an organ transplant recipient with systemic vasculitis, myositis, and retinal blindness, and human polyomavirus 12 (HPyV12) was detected in human liver tissue. The evolutionary origins and potential diseases are not well understood for either virus. In order to define their receptor engagement strategies, we first used nuclear magnetic resonance (NMR) spectroscopy to establish that the major capsid proteins (VP1) of both viruses bind to sialic acid in solution. We then solved crystal structures of NJPyV and HPyV12 VP1 alone and in complex with sialylated glycans. NJPyV employs a novel binding site for a α2,3-linked sialic acid, whereas HPyV12 engages terminal α2,3- or α2,6-linked sialic acids in an exposed site similar to that found in Trichodysplasia spinulosa-associated polyomavirus (TSPyV). Gangliosides or glycoproteins, featuring in mammals usually terminal sialic acids, are therefore receptor candidates for both viruses. Structural analyses show that the sialic acid-binding site of NJPyV is conserved in chimpanzee polyomavirus (ChPyV) and that the sialic acid-binding site of HPyV12 is widely used across the entire polyomavirus family, including mammalian and avian polyomaviruses. A comparison with other polyomavirus-receptor complex structures shows that their capsids have evolved to generate several physically distinct virus-specific receptor-binding sites that can all specifically engage sialylated glycans through a limited number of contacts. Small changes in each site may have enabled host-switching events during the evolution of polyomaviruses. IMPORTANCE Virus attachment to cell surface receptors is critical for productive infection. In this study, we have used a structure-based approach to investigate the cell surface recognition event for New Jersey polyomavirus (NJPyV) and human polyomavirus 12 (HPyV12). These viruses belong to the polyomavirus family, whose members target different tissues and hosts, including mammals, birds, fish, and invertebrates. Polyomaviruses are nonenveloped viruses, and the receptor-binding site is located in their capsid protein VP1. The NJPyV capsid features a novel sialic acid-binding site that is shifted in comparison to other structurally characterized polyomaviruses but shared with a closely related simian virus. In contrast, HPyV12 VP1 engages terminal sialic acids in a manner similar to the human Trichodysplasia spinulosa-associated polyomavirus. Our structure-based phylogenetic analysis highlights that even distantly related avian polyomaviruses possess the same exposed sialic acid-binding site. These findings complement phylogenetic models of host-virus codivergence and may also reflect past host-switching events.

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Spin ballet for sweet encounters: saturation-transfer difference NMR and X-ray crystallography complement each other in the elucidation of protein–glycan interactions

Cited by 26 publications

References 69 publications

Infectious Entry of Merkel Cell Polyomavirus

Infectious Entry of Merkel Cell Polyomavirus

Infectious Entry of Merkel Cell Polyomavirus

Structural Basis and Evolution of Glycan Receptor Specificities within the Polyomavirus Family

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