scholarly journals Reovirus σ1 Conformational Flexibility Modulates the Efficiency of Host Cell Attachment

2020 ◽  
Vol 94 (23) ◽  
Author(s):  
Julia R. Diller ◽  
Sean R. Halloran ◽  
Melanie Koehler ◽  
Rita dos Santos Natividade ◽  
David Alsteens ◽  
...  
Keyword(s):  
Host Cell ◽  
Receptor Binding ◽  
Cell Attachment ◽  
Conformational Flexibility ◽  
Parental Virus ◽  
Receptor Binding Domain ◽  
Cross Link ◽  
Attachment Protein ◽  
Host Cell Attachment ◽  
Conformational Rearrangements

ABSTRACT Reovirus attachment protein σ1 is a trimeric molecule containing tail, body, and head domains. During infection, σ1 engages sialylated glycans and junctional adhesion molecule-A (JAM-A), triggering uptake into the endocytic compartment, where virions are proteolytically converted to infectious subvirion particles (ISVPs). Further disassembly allows σ1 release and escape of transcriptionally active reovirus cores into the cytosol. Electron microscopy has revealed a distinct conformational change in σ1 from a compact form on virions to an extended form on ISVPs. To determine the importance of σ1 conformational mobility, we used reverse genetics to introduce cysteine mutations that can cross-link σ1 by establishing disulfide bonds between structurally adjacent sites in the tail, body, and head domains. We detected phenotypic differences among the engineered viruses. A mutant with a cysteine pair in the head domain replicates with enhanced kinetics, forms large plaques, and displays increased avidity for JAM-A relative to the parental virus, mimicking properties of ISVPs. However, unlike ISVPs, particles containing cysteine mutations that cross-link the head domain uncoat and transcribe viral positive-sense RNA with kinetics similar to the parental virus and are sensitive to ammonium chloride, which blocks virion-to-ISVP conversion. Together, these data suggest that σ1 conformational flexibility modulates the efficiency of reovirus host cell attachment. IMPORTANCE Nonenveloped virus entry is an incompletely understood process. For reovirus, the functional significance of conformational rearrangements in the attachment protein, σ1, that occur during entry and particle uncoating are unknown. We engineered and characterized reoviruses containing cysteine mutations that cross-link σ1 monomers in nonreducing conditions. We found that the introduction of a cysteine pair in the receptor-binding domain of σ1 yielded a virus that replicates with faster kinetics than the parental virus and forms larger plaques. Using functional assays, we found that cross-linking the σ1 receptor-binding domain modulates reovirus attachment but not uncoating or transcription. These data suggest that σ1 conformational rearrangements mediate the efficiency of reovirus host cell binding.

scholarly journals Reovirus σ1 conformational flexibility modulates the efficiency of host cell attachment

2020 ◽  
Author(s):  
Julia R. Diller ◽  
Sean R. Halloran ◽  
Melanie Koehler ◽  
Rita dos Santos Natividade ◽  
David Alsteens ◽  
...  
Keyword(s):  
Host Cell ◽  
Receptor Binding ◽  
Cell Attachment ◽  
Conformational Flexibility ◽  
Binding Domain ◽  
Parental Virus ◽  
Receptor Binding Domain ◽  
Attachment Protein ◽  
Host Cell Attachment ◽  
Conformational Rearrangements

ABSTRACTReovirus attachment protein σ1 is a trimeric molecule containing tail, body, and head domains. During infection, σ1 engages sialylated glycans and junctional adhesion molecule-A (JAM-A), triggering uptake into the endocytic compartment, where virions are proteolytically converted to infectious subvirion particles (ISVPs). Further disassembly allows σ1 release and escape of transcriptionally active reovirus cores into the cytosol. Electron microscopy has revealed a distinct conformational change in σ1 from a compact form on virions to an extended form on ISVPs. To determine the importance of σ1 conformational mobility, we used reverse genetics to introduce cysteine mutations that can crosslink σ1 by establishing disulfide bonds between structurally adjacent sites in the tail, body, and head domains. We detected phenotypic differences among the engineered viruses. A mutant with a cysteine pair in the head domain replicates with enhanced kinetics, forms large plaques, and displays increased avidity for JAM-A relative to the parental virus, mimicking properties of ISVPs. However, unlike ISVPs, particles containing cysteine mutations that crosslink the head domain uncoat and transcribe viral positive-sense RNA with kinetics similar to the parental virus and are sensitive to ammonium chloride. Together, these data suggest that σ1 conformational flexibility modulates the efficiency of reovirus host cell attachment.IMPORTANCENonenveloped virus entry is an incompletely understood process. For reovirus, the functional significance of conformational rearrangements in the attachment protein, σ1, that occur during entry and particle uncoating are unknown. We engineered and characterized reoviruses containing cysteine mutations that crosslink σ1 monomers in non-reducing conditions. We found that the introduction of a cysteine pair in the receptor-binding domain of σ1 yielded a virus that replicates with faster kinetics than the parental virus and forms larger plaques. Using functional assays, we found that crosslinking the σ1 receptor-binding domain modulates reovirus attachment but not uncoating or transcription. These data suggest that σ1 conformational rearrangements mediate the efficiency of reovirus host cell attachment.

scholarly journals A structure-based rationale for sialic acid independent host-cell entry of Sosuga virus

2019 ◽  
Vol 116 (43) ◽  
pp. 21514-21520 ◽  
Author(s):  
Alice J. Stelfox ◽  
Thomas A. Bowden
Keyword(s):  
Sialic Acid ◽  
Host Cell ◽  
Receptor Binding ◽  
Cell Attachment ◽  
Cell Entry ◽  
Head Region ◽  
Close Proximity ◽  
Host Cell Attachment ◽  
Host Cell Entry

The bat-borne paramyxovirus, Sosuga virus (SosV), is one of many paramyxoviruses recently identified and classified within the newly established genus Pararubulavirus, family Paramyxoviridae. The envelope surface of SosV presents a receptor-binding protein (RBP), SosV-RBP, which facilitates host-cell attachment and entry. Unlike closely related hemagglutinin neuraminidase RBPs from other genera of the Paramyxoviridae, SosV-RBP and other pararubulavirus RBPs lack many of the stringently conserved residues required for sialic acid recognition and hydrolysis. We determined the crystal structure of the globular head region of SosV-RBP, revealing that while the glycoprotein presents a classical paramyxoviral six-bladed β-propeller fold and structurally classifies in close proximity to paramyxoviral RBPs with hemagglutinin-neuraminidase (HN) functionality, it presents a receptor-binding face incongruent with sialic acid recognition. Hemadsorption and neuraminidase activity analysis confirms the limited capacity of SosV-RBP to interact with sialic acid in vitro and indicates that SosV-RBP undergoes a nonclassical route of host-cell entry. The close overall structural conservation of SosV-RBP with other classical HN RBPs supports a model by which pararubulaviruses only recently diverged from sialic acid binding functionality.

scholarly journals Complementation of the fadA Mutation in Fusobacterium nucleatum Demonstrates that the Surface-Exposed Adhesin Promotes Cellular Invasion and Placental Colonization

2009 ◽  
Vol 77 (7) ◽  
pp. 3075-3079 ◽  
Author(s):  
Akihiko Ikegami ◽  
Peter Chung ◽  
Yiping W. Han
Keyword(s):  
Host Cell ◽  
Cell Attachment ◽  
Adverse Pregnancy Outcome ◽  
Fusobacterium Nucleatum ◽  
Host Cells ◽  
Mouse Placenta ◽  
Host Cell Attachment ◽  
Adverse Pregnancy

ABSTRACT Fusobacterium nucleatum is a gram-negative oral anaerobe implicated in periodontal disease and adverse pregnancy outcome. The organism colonizes the mouse placenta, causing localized infection and inflammation. The mechanism of placental colonization has not been elucidated. Previous studies identified a novel adhesin from F. nucleatum, FadA, as being involved in the attachment and invasion of host cells. The fadA deletion mutant F. nucleatum 12230 US1 was defective in host cell attachment and invasion in vitro, but it also exhibited pleiotropic effects with altered cell morphology and growth rate. In this study, a fadA-complementing clone, F. nucleatum 12230 USF81, was constructed. The expression of FadA on USF81 was confirmed by Western blotting and immunofluorescent labeling. USF81 restored host cell attachment and invasion activities. The ability of F. nucleatum 12230, US1, and USF81 to colonize the mouse placenta was examined. US1 was severely defective in placental colonization compared to the wild type and USF81. Thus, FadA plays an important role in F. nucleatum colonization in vivo. These results also represent the first complementation studies for F. nucleatum. FadA may be a therapeutic target for preventing F. nucleatum colonization of the host.

scholarly journals The autotransporter protein from Bordetella avium, Baa1, is involved in host cell attachment

2011 ◽  
Vol 167 (1) ◽  
pp. 55-60 ◽  
Author(s):  
S.B. Stockwell ◽  
H. Kuzmiak-Ngiam ◽  
N.M. Beach ◽  
D. Miyamoto ◽  
R. Fernandez ◽  
...  
Keyword(s):  
Host Cell ◽  
Cell Attachment ◽  
Bordetella Avium ◽  
Host Cell Attachment

scholarly journals Laser scanning cytometer-based assays for measuring host cell attachment and invasion by the human pathogenToxoplasma gondii

2005 ◽  
Vol 69A (1) ◽  
pp. 13-19 ◽  
Author(s):  
Jeffrey Mital ◽  
Janet Schwarz ◽  
Douglas J. Taatjes ◽  
Gary E. Ward
Keyword(s):  
Host Cell ◽  
Laser Scanning ◽  
Cell Attachment ◽  
Host Cell Attachment ◽  
Laser Scanning Cytometer

scholarly journals Monocyte-derived chicken macrophages exposed to Eimeria tenella sporozoites display reduced susceptibility to invasion by Toxoplasma gondii tachyzoites

2020 ◽  
Author(s):  
Runhui Zhang ◽  
Wanpeng Zheng ◽  
Arwid Daugschies ◽  
Berit Bangoura
Keyword(s):  
Host Cell ◽  
Cell Invasion ◽  
Cell Attachment ◽  
Gliding Motility ◽  
Phagocytic Cells ◽  
Apicomplexan Parasites ◽  
Cell Responses ◽  
Host Cell Attachment ◽  
Parasite Replication ◽  
Host Parasite

Abstract Background: Both Eimeria (E.) tenella and Toxoplasma (T.) gondii are common apicomplexan parasites in chickens. Host cell invasion by both protozoans includes gliding motility, host cell attachment and active penetration. Chicken macrophages as phagocytic cells participate in the innate host immune response against these two parasites. Methods: In this study, primary chicken monocyte-derived macrophages (MMs) were infected with both pathogens to investigate mutual and host-parasite interactions. MMs cultures were assigned to groups that were infected with E. tenella, T. gondii, or both. In co-infected cultures, MMs were first exposed to E. tenella sporozoites for 2 hours. Afterwards, T. gondii tachyzoite infection was performed. Live-cell imaging was carried out to observe cell invasion and survival of T. gondii by single parasite tracking over a period of 20 hours post infection (hpi). Quantitative analysis for parasite replication was performed at 2, 6, 12 and 24 hpi by real-time PCR (qPCR).Results: We found that during early co-infection T. gondii tachyzoites adhered for more than 4 hours to macrophages. Although they displayed high motility, ability to penetrate the cell membrane of the potential host cell was reduced. qPCR results confirmed that significantly less T. gondii entered E. tenella-activated MMs at 2 hpi, and a reduced proportion of intracellular T. gondii survived and replicated in these cells at 24 hpi. Conclusions: We conclude that E. tenella modulates host cell responses to another apicomplexan agent, T. gondii, reducing active invasion and multiplication in chicken primary macrophages.

scholarly journals A modular molecular framework for quickly estimating the binding affinity of the spike protein of SARS-CoV-2 variants for ACE2, in presence of mutations at the spike receptor binding domain.

2021 ◽  
Author(s):  
Vincenzo Tragni ◽  
Francesca Preziusi ◽  
Luna Laera ◽  
Angelo Onofrio ◽  
Simona Todisco ◽  
...  
Keyword(s):  
Host Cell ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Binding Domain ◽  
Host Cells ◽  
Spike Protein ◽  
Binding Affinities ◽  
Receptor Binding Domain ◽  
Rapid Spread ◽  
Related Proteins

The rapid spread of new SARS-CoV-2 variants needs the development of rapid tools for predicting the affinity of the mutated proteins responsible for the infection, i.e., the SARS-CoV-2 spike protein, for the human ACE2 receptor, aiming to understand if a variant can be more efficient in invading host cells. Here we show how our computational pipeline, previously used for studying SARS-CoV-2 spike receptor-binding domain (RBD)/ACE2 interactions and pre-/post-fusion conformational changes, can be used for predicting binding affinities of the human ACE2 receptor for the spike protein RBD of the characterized infectious variants of concern/interest B.1.1.7-UK (carrying the mutations N501Y, S494P, E484K at the RBD), P.1-Japan/Brazil (RBD mutations: K417N/T, E484K, N501Y), B.1.351-South Africa (RBD mutations: K417N, E484K, N501Y), B.1.427/B.1.429-California (RBD mutations: L452R), the B.1.141 variant (RBD mutations: N439K), and the recent B.1.617.1-India (RBD mutations: L452R; E484Q) and the B.1.620 (RBD mutations: S477N; E484K). Furthermore, we searched for ACE2 structurally related proteins that might be involved in interactions with the SARS-CoV-2 spike protein, in those tissues showing low ACE2 expression, revealing two new proteins, THOP1 and NLN, deserving to be investigated for their possible inclusion in the group of host-cell entry factors responsible for host-cell SARS-CoV-2 invasion and immunity response.

Antiviral Drug Design Based on the Opening Mechanism of Spike Glycoprotein in SARS-CoV-2

2021 ◽  
Author(s):  
Ruichao Mao ◽  
Lihua Bie ◽  
Maofeng Xu ◽  
Xiaocong Wang ◽  
Jun Gao
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Drug Design ◽  
Host Cell ◽  
Receptor Binding ◽  
Antiviral Drug ◽  
Binding Domain ◽  
The Novel ◽  
Receptor Binding Domain ◽  
Spike Glycoprotein

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the host cell after the receptor binding domain (RBD) of the virus spike (S) glycoprotein binding to the human angiotensin-converting...

scholarly journals Disruption of the Dimer-Dimer Interaction of the Mumps Virus Attachment Protein Head Domain, Aided by an Anion Located at the Interface, Compromises Membrane Fusion Triggering

2019 ◽  
Vol 94 (2) ◽  
Author(s):  
Marie Kubota ◽  
Iori Okabe ◽  
Shin-ichi Nakakita ◽  
Ayako Ueo ◽  
Yuta Shirogane ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Host Cell ◽  
Receptor Binding ◽  
Mumps Virus ◽  
Viral Envelope ◽  
Attachment Protein ◽  
F Protein ◽  
Virus Attachment ◽  
Head Domain

ABSTRACT Mumps virus (MuV), an enveloped negative-strand RNA virus belonging to the family Paramyxoviridae, enters the host cell through membrane fusion mediated by two viral envelope proteins, an attachment protein hemagglutinin-neuraminidase (MuV-HN) and a fusion (F) protein. However, how the binding of MuV-HN to glycan receptors triggers membrane fusion is not well understood. The crystal structure of the MuV-HN head domain forms a tetramer (dimer of dimers) like other paramyxovirus attachment proteins. In the structure, a sulfate ion (SO42−) was found at the interface between two dimers, which may be replaced by a hydrogen phosphate ion (HPO42−) under physiological conditions. The anion is captured by the side chain of a positively charged arginine residue at position 139 of one monomer each from both dimers. Substitution of alanine or lysine for arginine at this position compromised the fusion support activity of MuV-HN without affecting its cell surface expression, glycan-receptor binding, and interaction with the F protein. Furthermore, the substitution appeared to affect the tetramer formation of the head domain as revealed by blue native-PAGE analysis. These results, together with our previous similar findings with the measles virus attachment protein head domain, suggest that the dimer-dimer interaction within the tetramer may play an important role in triggering membrane fusion during paramyxovirus entry. IMPORTANCE Despite the use of effective live vaccines, mumps outbreaks still occur worldwide. Mumps virus (MuV) infection typically causes flu-like symptoms and parotid gland swelling but sometimes leads to orchitis, oophoritis, and neurological complications, such as meningitis, encephalitis, and deafness. MuV enters the host cell through membrane fusion mediated by two viral proteins, a receptor-binding attachment protein, and a fusion protein, but its detailed mechanism is not fully understood. In this study, we show that the tetramer (dimer of dimers) formation of the MuV attachment protein head domain is supported by an anion located at the interface between two dimers and that the dimer-dimer interaction plays an important role in triggering the activation of the fusion protein and causing membrane fusion. These results not only further our understanding of MuV entry but provide useful information about a possible target for antiviral drugs.

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