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Structural and Functional Features of the Reovirus σ1 Tail.
Dietrich MH, Ogden KM, Long JM, Ebenhoch R, Thor A, Dermody TS, Stehle T
(2018) J Virol 92:
MeSH Terms: Amino Acid Sequence, Capsid Proteins, Cells, Cultured, Crystallography, X-Ray, Protein Binding, Protein Conformation, Receptors, Virus, Reoviridae, Reoviridae Infections, Sequence Homology, Virus Attachment, Virus Internalization, Virus Replication
Show Abstract · Added April 3, 2019
Mammalian orthoreovirus attachment to target cells is mediated by the outer capsid protein σ1, which projects from the virion surface. The σ1 protein is a homotrimer consisting of a filamentous tail, which is partly inserted into the virion; a body domain constructed from β-spiral repeats; and a globular head with receptor-binding properties. The σ1 tail is predicted to form an α-helical coiled coil. Although σ1 undergoes a conformational change during cell entry, the nature of this change and its contributions to viral replication are unknown. Electron micrographs of σ1 molecules released from virions identified three regions of flexibility, including one at the midpoint of the molecule, that may be involved in its structural rearrangement. To enable a detailed understanding of essential σ1 tail organization and properties, we determined high-resolution structures of the reovirus type 1 Lang (T1L) and type 3 Dearing (T3D) σ1 tail domains. Both molecules feature extended α-helical coiled coils, with T1L σ1 harboring central chloride ions. Each molecule displays a discontinuity (stutter) within the coiled coil and an unexpectedly seamless transition to the body domain. The transition region features conserved interdomain interactions and appears rigid rather than highly flexible. Functional analyses of reoviruses containing engineered σ1 mutations suggest that conserved residues predicted to stabilize the coiled-coil-to-body junction are essential for σ1 folding and encapsidation, whereas central chloride ion coordination and the stutter are dispensable for efficient replication. Together, these findings enable modeling of full-length reovirus σ1 and provide insight into the stabilization of a multidomain virus attachment protein. While it is established that different conformational states of attachment proteins of enveloped viruses mediate receptor binding and membrane fusion, less is understood about how such proteins mediate attachment and entry of nonenveloped viruses. The filamentous reovirus attachment protein σ1 binds cellular receptors; contains regions of predicted flexibility, including one at the fiber midpoint; and undergoes a conformational change during cell entry. Neither the nature of the structural change nor its contribution to viral infection is understood. We determined crystal structures of large σ1 fragments for two different reovirus serotypes. We observed an unexpectedly tight transition between two domains spanning the fiber midpoint, which allows for little flexibility. Studies of reoviruses with engineered changes near the σ1 midpoint suggest that the stabilization of this region is critical for function. Together with a previously determined structure, we now have a complete model of the full-length, elongated reovirus σ1 attachment protein.
Copyright © 2018 American Society for Microbiology.
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The Marburgvirus-Neutralizing Human Monoclonal Antibody MR191 Targets a Conserved Site to Block Virus Receptor Binding.
King LB, Fusco ML, Flyak AI, Ilinykh PA, Huang K, Gunn B, Kirchdoerfer RN, Hastie KM, Sangha AK, Meiler J, Alter G, Bukreyev A, Crowe JE, Saphire EO
(2018) Cell Host Microbe 23: 101-109.e4
MeSH Terms: Agrobacterium tumefaciens, Animals, Antibodies, Monoclonal, Antibodies, Neutralizing, Antibodies, Viral, Binding Sites, Carrier Proteins, Cell Line, Chlorocebus aethiops, Crystallography, X-Ray, Drosophila melanogaster, Humans, Marburgvirus, Membrane Glycoproteins, Receptors, Virus, Tobacco, Vero Cells, Viral Envelope Proteins, Viral Fusion Proteins, Virus Attachment
Show Abstract · Added March 17, 2018
Since their first identification 50 years ago, marburgviruses have emerged several times, with 83%-90% lethality in the largest outbreaks. Although no vaccines or therapeutics are available for human use, the human antibody MR191 provides complete protection in non-human primates when delivered several days after inoculation of a lethal marburgvirus dose. The detailed neutralization mechanism of MR191 remains outstanding. Here we present a 3.2 Å crystal structure of MR191 complexed with a trimeric marburgvirus surface glycoprotein (GP). MR191 neutralizes by occupying the conserved receptor-binding site and competing with the host receptor Niemann-Pick C1. The structure illuminates previously disordered regions of GP including the stalk, fusion loop, CXCC switch, and an N-terminal region of GP2 that wraps about the outside of GP1 to anchor a marburgvirus-specific "wing" antibody epitope. Virus escape mutations mapped far outside the MR191 receptor-binding site footprint suggest a role for these other regions in the GP quaternary structure.
Copyright © 2017 Elsevier Inc. All rights reserved.
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A "Trojan horse" bispecific-antibody strategy for broad protection against ebolaviruses.
Wec AZ, Nyakatura EK, Herbert AS, Howell KA, Holtsberg FW, Bakken RR, Mittler E, Christin JR, Shulenin S, Jangra RK, Bharrhan S, Kuehne AI, Bornholdt ZA, Flyak AI, Saphire EO, Crowe JE, Aman MJ, Dye JM, Lai JR, Chandran K
(2016) Science 354: 350-354
MeSH Terms: Animals, Antibodies, Bispecific, Antibodies, Monoclonal, Antibodies, Neutralizing, Antibodies, Viral, Binding Sites, Carrier Proteins, Cell Line, Tumor, Ebolavirus, Endosomes, Hemorrhagic Fever, Ebola, Humans, Immunotherapy, Membrane Glycoproteins, Mice, Mice, Inbred BALB C, Receptors, Virus, Viral Envelope Proteins, Virus Internalization
Show Abstract · Added April 13, 2017
There is an urgent need for monoclonal antibody (mAb) therapies that broadly protect against Ebola virus and other filoviruses. The conserved, essential interaction between the filovirus glycoprotein, GP, and its entry receptor Niemann-Pick C1 (NPC1) provides an attractive target for such mAbs but is shielded by multiple mechanisms, including physical sequestration in late endosomes. Here, we describe a bispecific-antibody strategy to target this interaction, in which mAbs specific for NPC1 or the GP receptor-binding site are coupled to a mAb against a conserved, surface-exposed GP epitope. Bispecific antibodies, but not parent mAbs, neutralized all known ebolaviruses by coopting viral particles themselves for endosomal delivery and conferred postexposure protection against multiple ebolaviruses in mice. Such "Trojan horse" bispecific antibodies have potential as broad antifilovirus immunotherapeutics.
Copyright © 2016, American Association for the Advancement of Science.
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Host-Primed Ebola Virus GP Exposes a Hydrophobic NPC1 Receptor-Binding Pocket, Revealing a Target for Broadly Neutralizing Antibodies.
Bornholdt ZA, Ndungo E, Fusco ML, Bale S, Flyak AI, Crowe JE, Chandran K, Saphire EO
(2016) mBio 7: e02154-15
MeSH Terms: Antibodies, Neutralizing, Antibodies, Viral, Carrier Proteins, Cell Line, Crystallography, X-Ray, Ebolavirus, Hemorrhagic Fever, Ebola, Humans, Hydrophobic and Hydrophilic Interactions, Membrane Glycoproteins, Mutagenesis, Mutation, Protein Binding, Receptors, Virus, Viral Envelope Proteins, Virus Internalization
Show Abstract · Added May 4, 2016
UNLABELLED - The filovirus surface glycoprotein (GP) mediates viral entry into host cells. Following viral internalization into endosomes, GP is cleaved by host cysteine proteases to expose a receptor-binding site (RBS) that is otherwise hidden from immune surveillance. Here, we present the crystal structure of proteolytically cleaved Ebola virus GP to a resolution of 3.3 Å. We use this structure in conjunction with functional analysis of a large panel of pseudotyped viruses bearing mutant GP proteins to map the Ebola virus GP endosomal RBS at molecular resolution. Our studies indicate that binding of GP to its endosomal receptor Niemann-Pick C1 occurs in two distinct stages: the initial electrostatic interactions are followed by specific interactions with a hydrophobic trough that is exposed on the endosomally cleaved GP1 subunit. Finally, we demonstrate that monoclonal antibodies targeting the filovirus RBS neutralize all known filovirus GPs, making this conserved pocket a promising target for the development of panfilovirus therapeutics.
IMPORTANCE - Ebola virus uses its glycoprotein (GP) to enter new host cells. During entry, GP must be cleaved by human enzymes in order for receptor binding to occur. Here, we provide the crystal structure of the cleaved form of Ebola virus GP. We demonstrate that cleavage exposes a site at the top of GP and that this site binds the critical domain C of the receptor, termed Niemann-Pick C1 (NPC1). We perform mutagenesis to find parts of the site essential for binding NPC1 and map distinct roles for an upper, charged crest and lower, hydrophobic trough in cleaved GP. We find that this 3-dimensional site is conserved across the filovirus family and that antibody directed against this site is able to bind cleaved GP from every filovirus tested and neutralize viruses bearing those GPs.
Copyright © 2016 Bornholdt et al.
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16 MeSH Terms
KIM-1-/TIM-1-mediated phagocytosis links ATG5-/ULK1-dependent clearance of apoptotic cells to antigen presentation.
Brooks CR, Yeung MY, Brooks YS, Chen H, Ichimura T, Henderson JM, Bonventre JV
(2015) EMBO J 34: 2441-64
MeSH Terms: Antigen Presentation, Apoptosis, Autophagy-Related Protein 5, Autophagy-Related Protein-1 Homolog, CD4-Positive T-Lymphocytes, Cell Proliferation, HEK293 Cells, Hepatitis A Virus Cellular Receptor 1, Humans, Intracellular Signaling Peptides and Proteins, Lipoylation, Membrane Glycoproteins, Microtubule-Associated Proteins, Phagocytosis, Protein-Serine-Threonine Kinases, Reactive Oxygen Species, Receptors, Virus
Show Abstract · Added September 12, 2016
Phagocytosis of apoptotic cells by both professional and semi-professional phagocytes is required for resolution of organ damage and maintenance of immune tolerance. KIM-1/TIM-1 is a phosphatidylserine receptor that is expressed on epithelial cells and can transform the cells into phagocytes. Here, we demonstrate that KIM-1 phosphorylation and association with p85 results in encapsulation of phagosomes by lipidated LC3 in multi-membrane organelles. KIM-1-mediated phagocytosis is not associated with increased ROS production, and NOX inhibition does not block LC3 lipidation. Autophagy gene expression is required for efficient clearance of apoptotic cells and phagosome maturation. KIM-1-mediated phagocytosis leads to pro-tolerogenic antigen presentation, which suppresses CD4 T-cell proliferation and increases the percentage of regulatory T cells in an autophagy gene-dependent manner. Taken together, these data reveal a novel mechanism of epithelial biology linking phagocytosis, autophagy and antigen presentation to regulation of the inflammatory response.
© 2015 The Authors.
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Structure of Serotype 1 Reovirus Attachment Protein σ1 in Complex with Junctional Adhesion Molecule A Reveals a Conserved Serotype-Independent Binding Epitope.
Stettner E, Dietrich MH, Reiss K, Dermody TS, Stehle T
(2015) J Virol 89: 6136-40
MeSH Terms: Amino Acid Sequence, Binding Sites, Capsid Proteins, Crystallography, X-Ray, Junctional Adhesion Molecule A, Models, Molecular, Molecular Sequence Data, Protein Binding, Protein Conformation, Receptors, Virus, Sequence Homology
Show Abstract · Added February 4, 2016
Mammalian orthoreoviruses use glycans and junctional adhesion molecule A (JAM-A) as attachment receptors. We determined the structure of serotype 1 reovirus attachment protein σ1 alone and in complex with JAM-A. Comparison with the structure of serotype 3 reovirus σ1 bound to JAM-A reveals that both σ1 proteins engage JAM-A with similar affinities and via conserved binding epitopes. Thus, σ1-JAM-A interactions are unlikely to explain the differences in pathogenesis displayed by these reovirus serotypes.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Glycan engagement dictates hydrocephalus induction by serotype 1 reovirus.
Stencel-Baerenwald J, Reiss K, Blaum BS, Colvin D, Li XN, Abel T, Boyd K, Stehle T, Dermody TS
(2015) mBio 6: e02356
MeSH Terms: Animals, Animals, Newborn, Cells, Cultured, Disease Models, Animal, G(M2) Ganglioside, Hydrocephalus, Mice, Receptors, Virus, Reoviridae, Reoviridae Infections, Serogroup, Virus Attachment
Show Abstract · Added February 4, 2016
UNLABELLED - Receptors expressed on the host cell surface adhere viruses to target cells and serve as determinants of viral tropism. Several viruses bind cell surface glycans to facilitate entry, but the contribution of specific glycan moieties to viral disease is incompletely understood. Reovirus provides a tractable experimental model for studies of viral neuropathogenesis. In newborn mice, serotype 1 (T1) reovirus causes hydrocephalus, whereas serotype 3 (T3) reovirus causes encephalitis. T1 and T3 reoviruses engage distinct glycans, suggesting that glycan-binding capacity contributes to these differences in pathogenesis. Using structure-guided mutagenesis, we engineered a mutant T1 reovirus incapable of binding the T1 reovirus-specific glycan receptor, GM2. The mutant virus induced substantially less hydrocephalus than wild-type virus, an effect phenocopied by wild-type virus infection of GM2-deficient mice. In comparison to wild-type virus, yields of mutant virus were diminished in cultured ependymal cells, the cell type that lines the brain ventricles. These findings suggest that GM2 engagement targets reovirus to ependymal cells in mice and illuminate the function of glycan engagement in reovirus serotype-dependent disease.
IMPORTANCE - Receptor utilization strongly influences viral disease, often dictating host range and target cell selection. Different reovirus serotypes bind to different glycans, but a precise function for these molecules in pathogenesis is unknown. We used type 1 (T1) reovirus deficient in binding the GM2 glycan and mice lacking GM2 to pinpoint a role for glycan engagement in hydrocephalus caused by T1 reovirus. This work indicates that engagement of a specific glycan can lead to infection of specific cells in the host and consequent disease at that site. Since reovirus is being developed as a vaccine vector and oncolytic agent, understanding reovirus-glycan interactions may allow manipulation of reovirus glycan-binding properties for therapeutic applications.
Copyright © 2015 Stencel-Baerenwald et al.
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12 MeSH Terms
Furosemide Stress Test and Biomarkers for the Prediction of AKI Severity.
Koyner JL, Davison DL, Brasha-Mitchell E, Chalikonda DM, Arthur JM, Shaw AD, Tumlin JA, Trevino SA, Bennett MR, Kimmel PL, Seneff MG, Chawla LS
(2015) J Am Soc Nephrol 26: 2023-31
MeSH Terms: Acute Kidney Injury, Acute-Phase Proteins, Aged, Albuminuria, Biomarkers, Creatinine, Disease Progression, Diuretics, Female, Furosemide, Hepatitis A Virus Cellular Receptor 1, Humans, Insulin-Like Growth Factor Binding Proteins, Interleukin-18, Lipocalin-2, Lipocalins, Male, Membrane Glycoproteins, Middle Aged, Proto-Oncogene Proteins, Receptors, Virus, Severity of Illness Index, Sodium, Tissue Inhibitor of Metalloproteinase-2, Uromodulin
Show Abstract · Added October 20, 2015
Clinicians have access to limited tools that predict which patients with early AKI will progress to more severe stages. In early AKI, urine output after a furosemide stress test (FST), which involves intravenous administration of furosemide (1.0 or 1.5 mg/kg), can predict the development of stage 3 AKI. We measured several AKI biomarkers in our previously published cohort of 77 patients with early AKI who received an FST and evaluated the ability of FST urine output and biomarkers to predict the development of stage 3 AKI (n=25 [32.5%]), receipt of RRT (n=11 [14.2%]), or inpatient mortality (n=16 [20.7%]). With an area under the curve (AUC)±SEM of 0.87±0.09 (P<0.0001), 2-hour urine output after FST was significantly better than each urinary biomarker tested in predicting progression to stage 3 (P<0.05). FST urine output was the only biomarker to significantly predict RRT (0.86±0.08; P=0.001). Regardless of the end point, combining FST urine output with individual biomarkers using logistic regression did not significantly improve risk stratification (ΔAUC, P>0.10 for all). When FST urine output was assessed in patients with increased biomarker levels, the AUC for progression to stage 3 improved to 0.90±0.06 and the AUC for receipt of RRT improved to 0.91±0.08. Overall, in the setting of early AKI, FST urine output outperformed biochemical biomarkers for prediction of progressive AKI, need for RRT, and inpatient mortality. Using a FST in patients with increased biomarker levels improves risk stratification, although further research is needed.
Copyright © 2015 by the American Society of Nephrology.
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Role of type I interferon signaling in human metapneumovirus pathogenesis and control of viral replication.
Hastings AK, Erickson JJ, Schuster JE, Boyd KL, Tollefson SJ, Johnson M, Gilchuk P, Joyce S, Williams JV
(2015) J Virol 89: 4405-20
MeSH Terms: Analysis of Variance, Animals, Antigen-Presenting Cells, CD8-Positive T-Lymphocytes, Flow Cytometry, Gene Expression Regulation, Hepatitis A Virus Cellular Receptor 2, Humans, Interferon Type I, Metapneumovirus, Mice, Mice, Inbred C57BL, Mice, Knockout, Oximetry, Paramyxoviridae Infections, Real-Time Polymerase Chain Reaction, Receptors, Virus, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction, Virus Replication
Show Abstract · Added October 2, 2015
UNLABELLED - Type I IFN signaling, which is initiated through activation of the alpha interferon receptor (IFNAR), regulates the expression of proteins that are crucial contributors to immune responses. Paramyxoviruses, including human metapneumovirus (HMPV), have evolved mechanisms to inhibit IFNAR signaling, but the specific contribution of IFNAR signaling to the control of HMPV replication, pathogenesis, and adaptive immunity is unknown. We used IFNAR-deficient (IFNAR(-/-)) mice to assess the effect of IFNAR signaling on HMPV replication and the CD8(+) T cell response. HMPV-infected IFNAR(-/-) mice had a higher peak of early viral replication but cleared the virus with kinetics similar to those of wild-type (WT) mice. However, IFNAR(-/-) mice infected with HMPV displayed less airway dysfunction and lung inflammation. CD8(+) T cells of IFNAR(-/-) mice after HMPV infection expressed levels of the inhibitory receptor programmed death 1 (PD-1) similar to those of WT mice. However, despite lower expression of inhibitory programmed death ligand 1 (PD-L1), HMPV-specific CD8(+) T cells of IFNAR(-/-) mice were more functionally impaired than those of WT mice and upregulated the inhibitory receptor Tim-3. Analysis of the antigen-presenting cell subsets in the lungs revealed that the expansion of PD-L1(low) dendritic cells (DCs), but not PD-L1(high) alveolar macrophages, was dependent on IFNAR signaling. Collectively, our results indicate a role for IFNAR signaling in the early control of HMPV replication, disease progression, and the development of an optimal adaptive immune response. Moreover, our findings suggest an IFNAR-independent mechanism of lung CD8(+) T cell impairment.
IMPORTANCE - Human metapneumovirus (HMPV) is a leading cause of acute respiratory illness. CD8(+) T cells are critical for clearing viral infection, yet recent evidence shows that HMPV and other respiratory viruses induce CD8(+) T cell impairment via PD-1-PD-L1 signaling. We sought to understand the role of type I interferon (IFN) in the innate and adaptive immune responses to HMPV by using a mouse model lacking IFN signaling. Although HMPV titers were higher in the absence of type I IFN, virus was nonetheless cleared and mice were less ill, indicating that type I IFN is not required to resolve HMPV infection but contributes to pathogenesis. Further, despite lower levels of the inhibitory ligand PD-L1 in mice lacking type I IFN, CD8(+) T cells were more impaired in these mice than in WT mice. Our data suggest that specific antigen-presenting cell subsets and the inhibitory receptor Tim-3 may contribute to CD8(+) T cell impairment.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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Influenza viruses with receptor-binding N1 neuraminidases occur sporadically in several lineages and show no attenuation in cell culture or mice.
Hooper KA, Crowe JE, Bloom JD
(2015) J Virol 89: 3737-45
MeSH Terms: Animals, Cell Line, Disease Models, Animal, Dogs, Female, Humans, Influenza A Virus, H1N1 Subtype, Influenza A Virus, H5N1 Subtype, Mice, Inbred BALB C, Mutant Proteins, Mutation, Missense, Neuraminidase, Orthomyxoviridae Infections, Protein Binding, Receptors, Virus, Reverse Genetics, Viral Proteins, Virus Attachment, Virus Cultivation
Show Abstract · Added February 2, 2015
UNLABELLED - In nearly all characterized influenza viruses, hemagglutinin (HA) is the receptor-binding protein while neuraminidase (NA) is a receptor-cleaving protein that aids in viral release. However, in recent years, several groups have described point mutations that confer receptor-binding activity on NA, albeit in laboratory rather than natural settings. One of these mutations, D151G, appears to arise in the NA of recent human H3N2 viruses upon passage in tissue culture. We inadvertently isolated the second of these mutations, G147R, in the NA of the lab-adapted A/WSN/33 (H1N1) strain while we were passaging a heavily engineered virus in the lab. G147R also occurs at low frequencies in the reported sequences of viruses from three different lineages: human 2009 pandemic H1N1 (pdmH1N1), human seasonal H1N1, and chicken H5N1. Here we reconstructed a representative G147R NA from each of these lineages and found that all of the proteins have acquired the ability to bind an unknown cellular receptor while retaining substantial sialidase activity. We then reconstructed a virus with the HA and NA of a reported G147R pdmH1N1 variant and found no attenuation of viral replication in cell culture or change in pathogenesis in mice. Furthermore, the G147R virus had modestly enhanced resistance to neutralization by the Fab of an antibody against the receptor-binding pocket of HA, although it remained completely sensitive to the full-length IgG. Overall, our results suggest that circulating N1 viruses occasionally may acquire the G147R NA receptor-binding mutation without impairment of replicative capacity.
IMPORTANCE - Influenza viruses have two main proteins on their surface: one (hemagglutinin) binds incoming viruses to cells, while the other (neuraminidase) helps release newly formed viruses from these same cells. Here we characterize unusual mutant neuraminidases that have acquired the ability to bind to cells. We show that the mutation that allows neuraminidase to bind cells has no apparent adverse effect on viral replication but does make the virus modestly more resistant to a fragment of an antibody that blocks the normal hemagglutinin-mediated mode of viral attachment. Our results suggest that viruses with receptor-binding neuraminidases may occur at low levels in circulating influenza virus lineages.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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19 MeSH Terms