scholarly journals Sendai virus recruits cellular villin to remodel actin cytoskeleton during fusion with hepatocytes

2017 ◽  
Vol 28 (26) ◽  
pp. 3801-3814 ◽  
Author(s):  
Sunandini Chandra ◽  
Raju Kalaivani ◽  
Manoj Kumar ◽  
Narayanaswamy Srinivasan ◽  
Debi P. Sarkar
Keyword(s):  
Membrane Fusion ◽  
Viral Entry ◽  
Sendai Virus ◽  
Viral Envelope ◽  
Bioactive Molecules ◽  
Host Cells ◽  
Actin Dynamics ◽  
Envelope Glycoproteins ◽  
Animal Cells ◽  
Viral Envelopes

Reconstituted Sendai viral envelopes (virosomes) are well recognized for their promising potential in membrane fusion–mediated delivery of bioactive molecules to liver cells. Despite the known function of viral envelope glycoproteins in catalyzing fusion with cellular membrane, the role of host cell proteins remains elusive. Here, we used two-dimensional differential in-gel electrophoresis to analyze hepatic cells in early response to virosome-induced membrane fusion. Quantitative mass spectrometry together with biochemical analysis revealed that villin, an actin-modifying protein, is differentially up-regulated and phosphorylated at threonine 206—an early molecular event during membrane fusion. We found that villin influences actin dynamics and that this influence, in turn, promotes membrane mixing through active participation of Sendai viral envelope glycoproteins. Modulation of villin in host cells also resulted in a discernible effect on the entry and egress of progeny Sendai virus. Taken together, these results suggest a novel mechanism of regulated viral entry in animal cells mediated by host factor villin.

Studies of membrane fusion. V. Fusion of erythrocytes with non-haemolytic Sendai virus

1979 ◽  
Vol 36 (1) ◽  
pp. 85-96
Author(s):  
S. Knutton
Keyword(s):  
Membrane Fusion ◽  
Cell Fusion ◽  
Erythrocyte Membrane ◽  
Sendai Virus ◽  
Lateral Diffusion ◽  
Viral Envelope ◽  
Cell Swelling ◽  
Viral Membrane ◽  
Cytoplasmic Bridges ◽  
Viral Envelopes

The fusion of human erythrocytes with non-haemolytic ‘1-day’ Sendai virus has been studied by electron microscopy. The mechanism of viral envelope-cell fusion is the same as that described previously for haemolytic ‘3-day’ Sendai virus except that fusion is frequently arrested at an initial stage when 2 segments of smooth linear viral membrane fuse and become incorporated into the erythrocyte membrane. After longer periods of incubation at 37 degrees C, in addition to many partly fused virus particles, long (up to 4 micrometer) lengths of smooth linear viral membrane are seen within the erythrocyte membrane which arise by linear aggregation of shorter (approximately 0.25 micrometer long) segments of smooth linear membrane derived from individual fused viral envelopes. Cell-Cell fusion, as a result of the fusion of a viral envelope with 2 adjacent erythrocytes also occurs but, in the absence of cell swelling, fusion is arrested at this stage with cells joined by one (or more) small cytoplasmic bridges. Typical fused cells are produced if such cells are swollen with hypotonic buffer. These observations provide further evidence that membrane fusion and cell swelling are distinct events in cell fusion and that cell swelling is the driving force both for completing the incorporation of the viral envelope into the cell membrane and for expanding cells connected by small cytoplasmic bridges to form spherical fused cells. Little lateral diffusion of viral envelope components occurs in the absence of cell swelling; in fact, some aggregation of components occurs. Comparison with previous studies using haemolytic ‘3-day’ Sendai virus suggests that virally induced cell swelling perturbs membrane structure so as to allow the rapid lateral diffusion of integrated viral envelope components.

scholarly journals Multiple Strategies Reveal a Bidentate Interaction between the Nipah Virus Attachment and Fusion Glycoproteins

2016 ◽  
Vol 90 (23) ◽  
pp. 10762-10773 ◽  
Author(s):  
Jacquelyn A. Stone ◽  
Bhadra M. Vemulapati ◽  
Birgit Bradel-Tretheway ◽  
Hector C. Aguilar
Keyword(s):  
Membrane Protein ◽  
Membrane Fusion ◽  
Conformational Changes ◽  
Protein Interactions ◽  
Viral Entry ◽  
Nipah Virus ◽  
Viral Envelope ◽  
Host Cells ◽  
Protein Protein Interactions ◽  
Flow Cytometric

ABSTRACTThe paramyxoviral family contains many medically important viruses, including measles virus, mumps virus, parainfluenza viruses, respiratory syncytial virus, human metapneumovirus, and the deadly zoonotic henipaviruses Hendra and Nipah virus (NiV). To both enter host cells and spread from cell to cell within infected hosts, the vast majority of paramyxoviruses utilize two viral envelope glycoproteins: the attachment glycoprotein (G, H, or hemagglutinin-neuraminidase [HN]) and the fusion glycoprotein (F). Binding of G/H/HN to a host cell receptor triggers structural changes in G/H/HN that in turn trigger F to undergo a series of conformational changes that result in virus-cell (viral entry) or cell-cell (syncytium formation) membrane fusion. The actual regions of G/H/HN and F that interact during the membrane fusion process remain relatively unknown though it is generally thought that the paramyxoviral G/H/HN stalk region interacts with the F head region. Studies to determine such interactive regions have relied heavily on coimmunoprecipitation approaches, whose limitations include the use of detergents and the micelle-mediated association of proteins. Here, we developed a flow-cytometric strategy capable of detecting membrane protein-protein interactions by interchangeably using the full-length form of G and a soluble form of F, or vice versa. Using both coimmunoprecipitation and flow-cytometric strategies, we found a bidentate interaction between NiV G and F, where both the stalk and head regions of NiV G interact with F. This is a new structural-biological finding for the paramyxoviruses. Additionally, our studies disclosed regions of the NiV G and F glycoproteins dispensable for the G and F interactions.IMPORTANCENipah virus (NiV) is a zoonotic paramyxovirus that causes high mortality rates in humans, with no approved treatment or vaccine available for human use. Viral entry into host cells relies on two viral envelope glycoproteins: the attachment (G) and fusion (F) glycoproteins. Binding of G to the ephrinB2 or ephrinB3 cell receptors triggers conformational changes in G that in turn cause F to undergo conformational changes that result in virus-host cell membrane fusion and viral entry. It is currently unknown, however, which specific regions of G and F interact during membrane fusion. Past efforts to determine the interacting regions have relied mainly on coimmunoprecipitation, a technique with some pitfalls. We developed a flow-cytometric assay to study membrane protein-protein interactions, and using this assay we report a bidentate interaction whereby both the head and stalk regions of NiV G interact with NiV F, a new finding for the paramyxovirus family.

Studies of membrane fusion. VI. Mechanism of the membrane fusion and cell swelling stages of Sendai virus-mediated cell fusion

1980 ◽  
Vol 43 (1) ◽  
pp. 103-118
Author(s):  
S. Knutton
Keyword(s):  
Membrane Fusion ◽  
Cell Fusion ◽  
Sendai Virus ◽  
Freeze Fracture ◽  
Viral Envelope ◽  
Cell Swelling ◽  
Fusion Event ◽  
Virus Envelope ◽  
Membrane Rupture ◽  
Membrane Tubules

The membrane fusion and cell swelling stages of Sendai virus-mediated cell-cell fusion have been studied by thin-section and freeze-fracture electron microscopy. Sites of membrane fusion have been detected in human erythrocytes arrested at the membrane fusion stage of cell fusion and in virtually all cases a fused viral envelope or envelope components has been identified thus providing further direct evidence that cell-viral envelope-cell bridge formation is the membrane fusion event in Sendai virus-induced cell fusion. Radial expansion of a single virus bridge connecting 2 cells is sufficient to produce a fused cell. Membrane redistribution which occurs during this cell swelling stage of the fusion process is often accompanied by the formation of a system of membrane tubules in the plane of expansion of the virus bridge. The tubules originate from points of fusion between the bridging virus envelope and the erythrocyte membrane and also expand radially as cells swell. Ultimately membrane rupture occurs and the tubules appear to break down as small vesicles. When previously observed in cross-sectioned cells these membrane tubules were interpreted as sites of direct membrane fusion. The present study indicates that this interpretation is incorrect and shows that the tubules are generated subsequent to membrane fusion when 2 cells connected by a virus bridge are induced to swell. A mechanism to explain the formation of this system of membrane tubules is proposed.

scholarly journals A Histidine Switch in Hemagglutinin-Neuraminidase Triggers Paramyxovirus-Cell Membrane Fusion

2008 ◽  
Vol 83 (4) ◽  
pp. 1727-1741 ◽  
Author(s):  
Anuja Krishnan ◽  
Santosh K. Verma ◽  
Prashant Mani ◽  
Rahul Gupta ◽  
Suman Kundu ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Fusion Proteins ◽  
Sendai Virus ◽  
Biological Activities ◽  
Viral Envelope ◽  
Attachment Protein ◽  
Histidine Residues ◽  
Human Parainfluenza Virus ◽  
Mimicking Peptides

ABSTRACT Most paramyxovirus fusion proteins require coexpression of and activation by a homotypic attachment protein, hemagglutinin-neuraminidase (HN), to promote membrane fusion. However, the molecular mechanism of the activation remains unknown. We previously showed that the incorporation of a monohistidylated lipid into F-virosome (Sendai viral envelope containing only fusion protein) enhanced its fusion to hepatocytes, suggesting that the histidine residue in the lipid accelerated membrane fusion. Therefore, we explored whether a histidine moiety in HN could similarly direct activation of the fusion protein. In membrane fusion assays, the histidine substitution mutants of HN (H247A of Sendai virus and H245A of human parainfluenza virus 3) had impaired membrane fusion promotion activity without significant changes in other biological activities. Synthetic 30-mer peptides corresponding to regions of the two HN proteins containing these histidine residues rescued the fusion promoting activity of the mutants, whereas peptides with histidine residues substituted by alanine did not. These histidine-containing peptides also activated F-virosome fusion with hepatocytes both in the presence and in the absence of mutant HN in the virosome. We provide evidence that the HN-mimicking peptides promote membrane fusion, revealing a specific histidine “switch” in HN that triggers fusion.

scholarly journals Scalable Analysis of Authentic Viral Envelopes on FRONTERA

2020 ◽  
Author(s):  
Fabio González-Arias ◽  
Tyler Reddy ◽  
John E. Stone ◽  
Jodi A. Hadden-Perilla ◽  
Juan R. Perilla
Keyword(s):  
Large Scale ◽  
Viral Envelope ◽  
Host Cells ◽  
Parallel Computer ◽  
Modeling And Analysis ◽  
Large Scale Analysis ◽  
Viral Envelopes ◽  
Novel Coronavirus ◽  
Dynamics Simulations ◽  
Scalable Analysis

AbstractEnveloped viruses infect host cells via fusion of their viral envelope with the plasma membrane. Upon cell entry, viruses gain access to all the macromolecular machinery necessary to replicate, assemble, and bud their progeny from the infected cell. By employing molecular dynamics simulations to characterize the dynamical and chemical-physical properties of viral envelopes, researchers can gain insights into key determinants of viral infection and propagation. Here, the Frontera supercomputer is leveraged for large-scale analysis of authentic viral envelopes, whose lipid compositions are complex and realistic. VMD with support for MPI is employed on the massive parallel computer to overcome previous computational limitations and enable investigation into virus biology at an unprecedented scale. The modeling and analysis techniques applied to authentic viral envelopes at two levels of particle resolution are broadly applicable to the study of other viruses, including the novel coronavirus that causes COVID-19. A framework for carrying out scalable analysis of multi-million particle MD simulation trajectories on Frontera is presented, expanding the the utility of the machine in humanity’s ongoing fight against infectious disease.

scholarly journals Displacement of the C Terminus of Herpes Simplex Virus gD Is Sufficient To Expose the Fusion-Activating Interfaces on gD

2013 ◽  
Vol 87 (23) ◽  
pp. 12656-12666 ◽  
Author(s):  
John R. Gallagher ◽  
Wan Ting Saw ◽  
Doina Atanasiu ◽  
Huan Lou ◽  
Roselyn J. Eisenberg ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Membrane Fusion ◽  
Receptor Binding ◽  
Viral Entry ◽  
Tertiary Structure ◽  
Host Cells ◽  
Binding Interface ◽  
C Terminus ◽  
Simplex Virus

Viral entry by herpes simplex virus (HSV) is executed and tightly regulated by four glycoproteins. While several viral glycoproteins can mediate viral adhesion to host cells, only binding of gD to cellular receptor can activate core fusion proteins gB and gH/gL to execute membrane fusion and viral entry. Atomic structures of gD bound to receptor indicate that the C terminus of the gD ectodomain must be displaced before receptor can bind to gD, but it is unclear which conformational changes in gD activate membrane fusion. We rationally designed mutations in gD to displace the C terminus and observe if fusion could be activated without receptor binding. Using a cell-based fusion assay, we found that gD V231W induced cell-cell fusion in the absence of receptor. Using recombinant gD V231W protein, we observed binding to conformationally sensitive antibodies or HSV receptor and concluded that there were changes proximal to the receptor binding interface, while the tertiary structure of gD V231W was similar to that of wild-type gD. We used a biosensor to analyze the kinetics of receptor binding and the extent to which the C terminus blocks binding to receptor. We found that the C terminus of gD V231W was enriched in the open or displaced conformation, indicating a mechanism for its function. We conclude that gD V231W triggers fusion through displacement of its C terminus and that this motion is indicative of how gD links receptor binding to exposure of interfaces on gD that activate fusion via gH/gL and gB.

scholarly journals Identification of Residues Controlling Restriction versus Enhancing Activities of IFITM Proteins on Entry of Human Coronaviruses

2017 ◽  
Vol 92 (6) ◽  
Author(s):  
Xuesen Zhao ◽  
Mohit Sehgal ◽  
Zhifei Hou ◽  
Junjun Cheng ◽  
Sainan Shu ◽  
...  
Keyword(s):  
Aspartic Acid ◽  
Viral Entry ◽  
Molecular Mechanisms ◽  
Biological Activities ◽  
Viral Envelope ◽  
Host Factors ◽  
Host Cells ◽  
Restriction Factors ◽  
Structural Motifs ◽  
Infectious Entry

ABSTRACTInterferon-induced transmembrane proteins (IFITMs) are restriction factors that inhibit the infectious entry of many enveloped RNA viruses. However, we demonstrated previously that human IFITM2 and IFITM3 are essential host factors facilitating the entry of human coronavirus (HCoV) OC43. In a continuing effort to decipher the molecular mechanism underlying IFITM differential modulation of HCoV entry, we investigated the roles of structural motifs important for IFITM protein posttranslational modifications, intracellular trafficking, and oligomerization in modulating the entry of five HCoVs. We found that three distinct mutations in IFITM1 or IFITM3 converted the host restriction factors to enhance entry driven by the spike proteins of severe acute respiratory syndrome coronavirus (SARS-CoV) and/or Middle East respiratory syndrome coronavirus (MERS-CoV). First, replacement of IFITM3 tyrosine 20 with either alanine or aspartic acid to mimic unphosphorylated or phosphorylated IFITM3 reduced its activity to inhibit the entry of HCoV-NL63 and -229E but enhanced the entry of SARS-CoV and MERS-CoV. Second, replacement of IFITM3 tyrosine 99 with either alanine or aspartic acid reduced its activity to inhibit the entry of HCoV-NL63 and SARS-CoV but promoted the entry of MERS-CoV. Third, deletion of the carboxyl-terminal 12 amino acid residues from IFITM1 enhanced the entry of MERS-CoV and HCoV-OC43. These findings suggest that these residues and structural motifs of IFITM proteins are key determinants for modulating the entry of HCoVs, most likely through interaction with viral and/or host cellular components at the site of viral entry to modulate the fusion of viral envelope and cellular membranes.IMPORTANCEThe differential effects of IFITM proteins on the entry of HCoVs that utilize divergent entry pathways and membrane fusion mechanisms even when using the same receptor make the HCoVs a valuable system for comparative investigation of the molecular mechanisms underlying IFITM restriction or promotion of virus entry into host cells. Identification of three distinct mutations that converted IFITM1 or IFITM3 from inhibitors to enhancers of MERS-CoV or SARS-CoV spike protein-mediated entry revealed key structural motifs or residues determining the biological activities of IFITM proteins. These findings have thus paved the way for further identification of viral and host factors that interact with those structural motifs of IFITM proteins to differentially modulate the infectious entry of HCoVs.

scholarly journals Conformational Dynamics Related to Membrane Fusion Observed in Single Viral Envelope Glycoproteins

2019 ◽  
Vol 116 (3) ◽  
pp. 40a
Author(s):  
Dibyendu Kumar Das ◽  
Natasha Durham ◽  
Angela Howard ◽  
James B. Munro
Keyword(s):  
Membrane Fusion ◽  
Conformational Dynamics ◽  
Viral Envelope ◽  
Envelope Glycoproteins

scholarly journals TMPRSS12 Is an Activating Protease for Subtype B Avian Metapneumovirus

2016 ◽  
Vol 90 (24) ◽  
pp. 11231-11246 ◽  
Author(s):  
Bingling Yun ◽  
Yao Zhang ◽  
Yongzhen Liu ◽  
Xiaolu Guan ◽  
Yongqiang Wang ◽  
...  
Keyword(s):  
Viral Replication ◽  
Membrane Fusion ◽  
Serine Protease ◽  
Viral Envelope ◽  
Host Cells ◽  
Protein Cleavage ◽  
Avian Metapneumovirus ◽  
F Protein ◽  
Subtype B ◽  
Transmembrane Serine Protease

ABSTRACTThe entry of avian metapneumovirus (aMPV) into host cells initially requires the fusion of viral and cell membranes, which is exclusively mediated by fusion (F) protein. Proteolysis of aMPV F protein by endogenous proteases of host cells allows F protein to induce membrane fusion; however, these proteases have not been identified. Here, we provide the first evidence that the transmembrane serine protease TMPRSS12 facilitates the cleavage of subtype B aMPV (aMPV/B) F protein. We found that overexpression of TMPRSS12 enhanced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. Subsequently, knockdown of TMPRSS12 with specific small interfering RNAs (siRNAs) reduced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. We also found a cleavage motif in the aMPV/B F protein (amino acids 100 and 101) that was recognized by TMPRSS12. The histidine, aspartic acid, and serine residue (HDS) triad of TMPRSS12 was shown to be essential for the proteolysis of aMPV/B F protein via mutation analysis. Notably, we observed TMPRSS12 mRNA expression in target organs of aMPV/B in chickens. Overall, our results indicate that TMPRSS12 is crucial for aMPV/B F protein proteolysis and aMPV/B infectivity and that TMPRSS12 may serve as a target for novel therapeutics and prophylactics for aMPV.IMPORTANCEProteolysis of the aMPV F protein is a prerequisite for F protein-mediated membrane fusion of virus and cell and for aMPV infection; however, the proteases usedin vitroandvivoare not clear. A combination of analyses, including overexpression, knockdown, and mutation methods, demonstrated that the transmembrane serine protease TMPRSS12 facilitated cleavage of subtype B aMPV (aMPV/B) F protein. Importantly, we located the motif in the aMPV/B F protein recognized by TMPRSS12 and the catalytic triad in TMPRSS12 that facilitated proteolysis of the aMPV/B F protein. This is the first report on TMPRSS12 as a protease for proteolysis of viral envelope glycoproteins. Our study will shed light on the mechanism of proteolysis of aMPV F protein and pathogenesis of aMPV.

scholarly journals Detection of Cell-Cell Fusion Mediated by Ebola Virus Glycoproteins

2006 ◽  
Vol 80 (6) ◽  
pp. 2815-2822 ◽  
Author(s):  
Séverine Bär ◽  
Ayato Takada ◽  
Yoshihiro Kawaoka ◽  
Marc Alizon
Keyword(s):  
Membrane Fusion ◽  
Viral Entry ◽  
Ebola Virus ◽  
Low Ph ◽  
Viral Envelope ◽  
Target Cells ◽  
Direct Role ◽  
Influenza Virus Hemagglutinin ◽  
Ph Dependent

ABSTRACT Ebola viruses (EboV) are enveloped RNA viruses infecting cells by a pH-dependent process mediated by viral glycoproteins (GP) involving endocytosis of virions and their routing into acidic endosomes. As with well-characterized pH-dependent viral entry proteins, in particular influenza virus hemagglutinin, it is thought that EboV GP require activation by low pH in order to mediate fusion of the viral envelope with the membrane of endosomes. However, it has not yet been possible to confirm the direct role of EboV GP in membrane fusion and the requirement for low-pH activation. It was in particular not possible to induce formation of syncytia by exposing cells expressing EboV GP to acidic medium. Here, we have used an assay based on the induction of a β-galactosidase (lacZ) reporter gene in target cells to detect cytoplasmic exchanges, indicating membrane fusion, with cells expressing EboV GP (Zaire species). Acidic activation of GP-expressing cells was required for efficient fusion with target cells. The direct role of EboV GP in this process is indicated by its inhibition by anti-GP antibodies and by the lack of activity of mutant GP normally expressed at the cell surface but defective for virus entry. Fusion was not observed when target cells underwent acidic treatment, for example, when they were placed in coculture with GP-expressing cells before the activation step. This unexpected feature, possibly related to the nature of the EboV receptor, could explain the impossibility of inducing formation of syncytia among GP-expressing cells.

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