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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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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, Intracellular Signaling Peptides and Proteins, 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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20 MeSH Terms
Fusion peptide of HIV-1 as a site of vulnerability to neutralizing antibody.
Kong R, Xu K, Zhou T, Acharya P, Lemmin T, Liu K, Ozorowski G, Soto C, Taft JD, Bailer RT, Cale EM, Chen L, Choi CW, Chuang GY, Doria-Rose NA, Druz A, Georgiev IS, Gorman J, Huang J, Joyce MG, Louder MK, Ma X, McKee K, O'Dell S, Pancera M, Yang Y, Blanchard SC, Mothes W, Burton DR, Koff WC, Connors M, Ward AB, Kwong PD, Mascola JR
(2016) Science 352: 828-33
MeSH Terms: AIDS Vaccines, Amino Acid Sequence, Antibodies, Neutralizing, Antibodies, Viral, B-Lymphocytes, Crystallography, X-Ray, HIV Envelope Protein gp120, HIV Envelope Protein gp41, HIV-1, Humans, Hydrophobic and Hydrophilic Interactions, Immunodominant Epitopes, Molecular Sequence Data, Peptides, Protein Conformation, Viral Fusion Proteins, Virus Internalization
Show Abstract · Added May 3, 2017
The HIV-1 fusion peptide, comprising 15 to 20 hydrophobic residues at the N terminus of the Env-gp41 subunit, is a critical component of the virus-cell entry machinery. Here, we report the identification of a neutralizing antibody, N123-VRC34.01, which targets the fusion peptide and blocks viral entry by inhibiting conformational changes in gp120 and gp41 subunits of Env required for entry. Crystal structures of N123-VRC34.01 liganded to the fusion peptide, and to the full Env trimer, revealed an epitope consisting of the N-terminal eight residues of the gp41 fusion peptide and glycan N88 of gp120, and molecular dynamics showed that the N-terminal portion of the fusion peptide can be solvent-exposed. These results reveal the fusion peptide to be a neutralizing antibody epitope and thus a target for vaccine design.
Copyright © 2016, American Association for the Advancement of Science.
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17 MeSH Terms
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, Intracellular Signaling Peptides and Proteins, 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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17 MeSH Terms
Human Metapneumovirus Is Capable of Entering Cells by Fusion with Endosomal Membranes.
Cox RG, Mainou BA, Johnson M, Hastings AK, Schuster JE, Dermody TS, Williams JV
(2015) PLoS Pathog 11: e1005303
MeSH Terms: Bronchi, Cell Line, Endosomes, Flow Cytometry, Humans, Metapneumovirus, Microscopy, Confocal, Paramyxoviridae Infections, RNA, Small Interfering, Respiratory Mucosa, Transfection, Viral Fusion Proteins, Virus Internalization
Show Abstract · Added February 4, 2016
Human metapneumovirus (HMPV), a member of the Paramyxoviridae family, is a leading cause of lower respiratory illness. Although receptor binding is thought to initiate fusion at the plasma membrane for paramyxoviruses, the entry mechanism for HMPV is largely uncharacterized. Here we sought to determine whether HMPV initiates fusion at the plasma membrane or following internalization. To study the HMPV entry process in human bronchial epithelial (BEAS-2B) cells, we used fluorescence microscopy, an R18-dequenching fusion assay, and developed a quantitative, fluorescence microscopy assay to follow virus binding, internalization, membrane fusion, and visualize the cellular site of HMPV fusion. We found that HMPV particles are internalized into human bronchial epithelial cells before fusing with endosomes. Using chemical inhibitors and RNA interference, we determined that HMPV particles are internalized via clathrin-mediated endocytosis in a dynamin-dependent manner. HMPV fusion and productive infection are promoted by RGD-binding integrin engagement, internalization, actin polymerization, and dynamin. Further, HMPV fusion is pH-independent, although infection with rare strains is modestly inhibited by RNA interference or chemical inhibition of endosomal acidification. Thus, HMPV can enter via endocytosis, but the viral fusion machinery is not triggered by low pH. Together, our results indicate that HMPV is capable of entering host cells by multiple pathways, including membrane fusion from endosomal compartments.
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13 MeSH Terms
Broadly Neutralizing Alphavirus Antibodies Bind an Epitope on E2 and Inhibit Entry and Egress.
Fox JM, Long F, Edeling MA, Lin H, van Duijl-Richter MKS, Fong RH, Kahle KM, Smit JM, Jin J, Simmons G, Doranz BJ, Crowe JE, Fremont DH, Rossmann MG, Diamond MS
(2015) Cell 163: 1095-1107
MeSH Terms: Alphavirus, Alphavirus Infections, Amino Acid Sequence, Animals, Antibodies, Monoclonal, Antibodies, Neutralizing, Antibodies, Viral, Chikungunya virus, Cryoelectron Microscopy, Epitopes, Glycoproteins, Humans, Immunoglobulin Fab Fragments, Mice, Models, Molecular, Molecular Sequence Data, Protein Structure, Tertiary, Sequence Alignment, Viral Envelope Proteins, Viral Vaccines, Virus Internalization
Show Abstract · Added January 26, 2016
We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O'nyong'nyong alphaviruses. Using alanine-scanning mutagenesis, loss-of-function recombinant proteins and viruses, and multiple functional assays, we determined that broadly neutralizing MAbs block multiple steps in the viral lifecycle, including entry and egress, and bind to a conserved epitope on the B domain of the E2 glycoprotein. A 16 Å resolution cryo-electron microscopy structure of a Fab fragment bound to CHIKV E2 B domain provided an explanation for its neutralizing activity. Binding to the B domain was associated with repositioning of the A domain of E2 that enabled cross-linking of neighboring spikes. Our results suggest that B domain antigenic determinants could be targeted for vaccine or antibody therapeutic development against multiple alphaviruses of global concern.
Copyright © 2015 Elsevier Inc. All rights reserved.
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21 MeSH Terms
Isolation and Characterization of Broad and Ultrapotent Human Monoclonal Antibodies with Therapeutic Activity against Chikungunya Virus.
Smith SA, Silva LA, Fox JM, Flyak AI, Kose N, Sapparapu G, Khomandiak S, Khomadiak S, Ashbrook AW, Kahle KM, Fong RH, Swayne S, Doranz BJ, McGee CE, Heise MT, Pal P, Brien JD, Austin SK, Diamond MS, Dermody TS, Crowe JE
(2015) Cell Host Microbe 18: 86-95
MeSH Terms: Animals, Antibodies, Monoclonal, Antibodies, Neutralizing, Antibodies, Viral, Chemoprevention, Chikungunya Fever, Chikungunya virus, Disease Models, Animal, Humans, Immunization, Passive, Inhibitory Concentration 50, Mice, Protein Binding, Survival Analysis, Treatment Outcome, Viral Envelope Proteins, Virus Internalization
Show Abstract · Added January 26, 2016
Chikungunya virus (CHIKV) is a mosquito-transmitted RNA virus that causes acute febrile infection associated with polyarthralgia in humans. Mechanisms of protective immunity against CHIKV are poorly understood, and no effective therapeutics or vaccines are available. We isolated and characterized human monoclonal antibodies (mAbs) that neutralize CHIKV infectivity. Among the 30 mAbs isolated, 13 had broad and ultrapotent neutralizing activity (IC50 < 10 ng/ml), and all of these mapped to domain A of the E2 envelope protein. Potent inhibitory mAbs blocked post-attachment steps required for CHIKV membrane fusion, and several were protective in a lethal challenge model in immunocompromised mice, even when administered at late time points after infection. These highly protective mAbs could be considered for prevention or treatment of CHIKV infection, and their epitope location in domain A of E2 could be targeted for rational structure-based vaccine development.
Copyright © 2015 Elsevier Inc. All rights reserved.
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17 MeSH Terms
Serotonin Receptor Agonist 5-Nonyloxytryptamine Alters the Kinetics of Reovirus Cell Entry.
Mainou BA, Ashbrook AW, Smith EC, Dorset DC, Denison MR, Dermody TS
(2015) J Virol 89: 8701-12
MeSH Terms: Animals, Antiviral Agents, Biological Transport, Cell Line, Cell Survival, Chikungunya virus, Chlorocebus aethiops, Cholera Toxin, Cricetinae, Cytoskeleton, Endosomes, HeLa Cells, Humans, Interferon-gamma, L Cells, Methiothepin, Mice, Murine hepatitis virus, Reoviridae, Reoviridae Infections, Serotonin Antagonists, Transferrin, Tryptamines, Vero Cells, Virus Assembly, Virus Attachment, Virus Internalization
Show Abstract · Added February 4, 2016
UNLABELLED - Mammalian orthoreoviruses (reoviruses) are nonenveloped double-stranded RNA viruses that infect most mammalian species, including humans. Reovirus binds to cell surface glycans, junctional adhesion molecule A (JAM-A), and the Nogo-1 receptor (depending on the cell type) and enters cells by receptor-mediated endocytosis. Within the endocytic compartment, reovirus undergoes stepwise disassembly, which is followed by release of the transcriptionally active viral core into the cytoplasm. In a small-molecule screen to identify host mediators of reovirus infection, we found that treatment of cells with 5-nonyloxytryptamine (5-NT), a prototype serotonin receptor agonist, diminished reovirus cytotoxicity. 5-NT also blocked reovirus infection. In contrast, treatment of cells with methiothepin mesylate, a serotonin antagonist, enhanced infection by reovirus. 5-NT did not alter cell surface expression of JAM-A or attachment of reovirus to cells. However, 5-NT altered the distribution of early endosomes with a concomitant impairment of reovirus transit to late endosomes and a delay in reovirus disassembly. Consistent with an inhibition of viral disassembly, 5-NT treatment did not alter infection by in vitro-generated infectious subvirion particles, which bind to JAM-A but bypass a requirement for proteolytic uncoating in endosomes to infect cells. We also found that treatment of cells with 5-NT decreased the infectivity of alphavirus chikungunya virus and coronavirus mouse hepatitis virus. These data suggest that serotonin receptor signaling influences cellular activities that regulate entry of diverse virus families and provides a new, potentially broad-spectrum target for antiviral drug development.
IMPORTANCE - Identification of well-characterized small molecules that modulate viral infection can accelerate development of antiviral therapeutics while also providing new tools to increase our understanding of the cellular processes that underlie virus-mediated cell injury. We conducted a small-molecule screen to identify compounds capable of inhibiting cytotoxicity caused by reovirus, a prototype double-stranded RNA virus. We found that 5-nonyloxytryptamine (5-NT) impairs reovirus infection by altering viral transport during cell entry. Remarkably, 5-NT also inhibits infection by an alphavirus and a coronavirus. The antiviral properties of 5-NT suggest that serotonin receptor signaling is an important regulator of infection by diverse virus families and illuminate a potential new drug target.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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27 MeSH Terms
HIV-1 Resistance to the Capsid-Targeting Inhibitor PF74 Results in Altered Dependence on Host Factors Required for Virus Nuclear Entry.
Zhou J, Price AJ, Halambage UD, James LC, Aiken C
(2015) J Virol 89: 9068-79
MeSH Terms: Amino Acid Substitution, Anti-HIV Agents, Binding Sites, CD4-Positive T-Lymphocytes, Capsid, Capsid Proteins, Cell Line, Drug Resistance, Viral, HIV Infections, HIV-1, Host-Pathogen Interactions, Humans, Indoles, Macrophages, Molecular Chaperones, Nuclear Pore Complex Proteins, Phenylalanine, Protein Binding, Protein Conformation, RNA Interference, RNA, Small Interfering, Virus Internalization, Virus Replication, beta Karyopherins, mRNA Cleavage and Polyadenylation Factors
Show Abstract · Added February 4, 2016
UNLABELLED - During HIV-1 infection of cells, the viral capsid plays critical roles in reverse transcription and nuclear entry of the virus. The capsid-targeting small molecule PF74 inhibits HIV-1 at early stages of infection. HIV-1 resistance to PF74 is complex, requiring multiple amino acid substitutions in the viral CA protein. Here we report the identification and analysis of a novel PF74-resistant mutant encoding amino acid changes in both domains of CA, three of which are near the pocket where PF74 binds. Interestingly, the mutant virus retained partial PF74 binding, and its replication was stimulated by the compound. The mutant capsid structure was not significantly perturbed by binding of PF74; rather, the mutations inhibited capsid interactions with CPSF6 and Nup153 and altered HIV-1 dependence on these host factors and on TNPO3. Moreover, the replication of the mutant virus was markedly impaired in activated primary CD4(+) T cells and macrophages. Our results suggest that HIV-1 escapes a capsid-targeting small molecule inhibitor by altering the virus's dependence on host factors normally required for entry into the nucleus. They further imply that clinical resistance to inhibitors targeting the PF74 binding pocket is likely to be strongly limited by functional constraints on HIV-1 evolution.
IMPORTANCE - The HIV-1 capsid plays critical roles in early steps of infection and is an attractive target for therapy. Here we show that selection for resistance to a capsid-targeting small molecule inhibitor can result in viral dependence on the compound. The mutant virus was debilitated in primary T cells and macrophages--cellular targets of infection in vivo. The mutations also altered the virus's dependence on cellular factors that are normally required for HIV-1 entry into the nucleus. This work provides new information regarding mechanisms of HIV-1 resistance that should be useful in efforts to develop clinically useful drugs targeting the HIV-1 capsid.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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25 MeSH Terms
Identification of residues in the human respiratory syncytial virus fusion protein that modulate fusion activity and pathogenesis.
Hotard AL, Lee S, Currier MG, Crowe JE, Sakamoto K, Newcomb DC, Peebles RS, Plemper RK, Moore ML
(2015) J Virol 89: 512-22
MeSH Terms: Animals, Cell Line, DNA Mutational Analysis, Disease Models, Animal, Female, Histocytochemistry, Humans, Lung, Mice, Inbred BALB C, Respiratory Syncytial Virus Infections, Respiratory Syncytial Virus, Human, Viral Fusion Proteins, Viral Load, Virus Internalization, Weight Loss
Show Abstract · Added January 20, 2015
UNLABELLED - Human respiratory syncytial virus (RSV) lower respiratory tract infection can result in inflammation and mucus plugging of airways. RSV strain A2-line19F induces relatively high viral load and mucus in mice. The line 19 fusion (F) protein harbors five unique residues compared to the non-mucus-inducing strains A2 and Long, at positions 79, 191, 357, 371, and 557. We hypothesized that differential fusion activity is a determinant of pathogenesis. In a cell-cell fusion assay, line 19 F was more fusogenic than Long F. We changed the residues unique to line 19 F to the corresponding residues in Long F and identified residues 79 and 191 together as responsible for high fusion activity. Surprisingly, mutation of residues 357 or 357 with 371 resulted in gain of fusion activity. Thus, we generated RSV F mutants with a range of defined fusion activity and engineered these into recombinant viruses. We found a clear, positive correlation between fusion activity and early viral load in mice; however, we did not detect a correlation between viral loads and levels of airway mucin expression. The F mutant with the highest fusion activity, A2-line19F-K357T/Y371N, induced high viral loads, severe lung histopathology, and weight loss but did not induce high levels of airway mucin expression. We defined residues 79/191 as critical for line 19 F fusion activity and 357/371 as playing a role in A2-line19F mucus induction. Defining the molecular basis of the role of RSV F in pathogenesis may aid vaccine and therapeutic strategies aimed at this protein.
IMPORTANCE - Human respiratory syncytial virus (RSV) is the most important lower respiratory tract pathogen of infants for which there is no vaccine. Elucidating mechanisms of RSV pathogenesis is important for rational vaccine and drug design. We defined specific amino acids in the fusion (F) protein of RSV strain line 19 critical for fusion activity and elucidated a correlation between fusion activity and viral load in mice. Further, we identified two distinct amino acids in F as contributing to the mucogenic phenotype of the A2-line19F virus. Taken together, these results illustrate a role for RSV F in virulence.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
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15 MeSH Terms