scholarly journals Analysis of Hendra Virus Fusion Protein N-Terminal Transmembrane Residues

Viruses ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2353
Author(s):  
Chelsea T. Barrett ◽  
Hadley E. Neal ◽  
Kearstin Edmonds ◽  
J. Lizbeth Reyes Zamora ◽  
Carole L. Moncman ◽  
...  
Keyword(s):  
Host Cell ◽  
Protein Function ◽  
Hendra Virus ◽  
Proteolytic Processing ◽  
Viral Envelope ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
F Protein ◽  
The Family ◽  
N Terminus

Hendra virus (HeV) is a zoonotic enveloped member of the family Paramyoxviridae. To successfully infect a host cell, HeV utilizes two surface glycoproteins: the attachment (G) protein to bind, and the trimeric fusion (F) protein to merge the viral envelope with the membrane of the host cell. The transmembrane (TM) region of HeV F has been shown to have roles in F protein stability and the overall trimeric association of F. Previously, alanine scanning mutagenesis has been performed on the C-terminal end of the protein, revealing the importance of β-branched residues in this region. Additionally, residues S490 and Y498 have been demonstrated to be important for F protein endocytosis, needed for the proteolytic processing of F required for fusion. To complete the analysis of the HeV F TM, we performed alanine scanning mutagenesis to explore the residues in the N-terminus of this region (residues 487–506). In addition to confirming the critical roles for S490 and Y498, we demonstrate that mutations at residues M491 and L492 alter F protein function, suggesting a role for these residues in the fusion process.

scholarly journals Pseudotyping Autographa californica Multicapsid Nucleopolyhedrovirus (AcMNPV): F Proteins from Group II NPVs Are Functionally Analogous to AcMNPV GP64

2002 ◽  
Vol 76 (11) ◽  
pp. 5729-5736 ◽  
Author(s):  
Oliver Lung ◽  
Marcel Westenberg ◽  
Just M. Vlak ◽  
Douwe Zuidema ◽  
Gary W. Blissard
Keyword(s):  
Membrane Fusion ◽  
Envelope Protein ◽  
Protein Gene ◽  
Autographa Californica ◽  
Viral Envelope ◽  
Viral Attachment ◽  
F Protein ◽  
Viral Propagation ◽  
The Family ◽  
Vesicular Stomatitis

ABSTRACT GP64, the major envelope glycoprotein of budded virions of the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), is involved in viral attachment, mediates membrane fusion during virus entry, and is required for efficient virion budding. Thus, GP64 is essential for viral propagation in cell culture and in animals. Recent genome sequences from a number of baculoviruses show that only a subset of closely related baculoviruses have gp64 genes, while other baculoviruses have a recently discovered unrelated envelope protein named F. F proteins from Lymantria dispar MNPV (LdMNPV) and Spodoptera exigua MNPV (SeMNPV) mediate membrane fusion and are therefore thought to serve roles similar to that of GP64. To determine whether F proteins are functionally analogous to GP64 proteins, we deleted the gp64 gene from an AcMNPV bacmid and inserted F protein genes from three different baculoviruses. In addition, we also inserted envelope protein genes from vesicular stomatitis virus (VSV) and Thogoto virus. Transfection of the gp64-null bacmid DNA into Sf9 cells does not generate infectious particles, but this defect was rescued by introducing either the F protein gene from LdMNPV or SeMNPV or the G protein gene from VSV. These results demonstrate that baculovirus F proteins are functionally analogous to GP64. Because baculovirus F proteins appear to be more widespread within the family and are much more divergent than GP64 proteins, gp64 may represent the acquisition of an envelope protein gene by an ancestral baculovirus. The AcMNPV pseudotyping system provides an efficient and powerful method for examining the functions and compatibilities of analogous or orthologous viral envelope proteins, and it could have important biotechnological applications.

scholarly journals Prediction of the binding interface between monoclonal antibody m102.4 and Nipah attachment glycoprotein using structure-guided alanine scanning and computational docking

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Phanthakarn Tit-oon ◽  
Kannan Tharakaraman ◽  
Charlermchai Artpradit ◽  
Abhinav Godavarthi ◽  
Pareenart Sungkeeree ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Structural Model ◽  
Affinity Maturation ◽  
Hendra Virus ◽  
World Health ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Computational Docking ◽  
Binding Interface ◽  
Starting Point

Abstract Nipah Virus (NiV) has been designated as a priority disease with an urgent need for therapeutic development by World Health Organization. The monoclonal antibody m102.4 binds to the immunodominant NiV receptor-binding glycoprotein (GP), and potently neutralizes NiV, indicating its potential as a therapeutic agent. Although the co-crystal structure of m102.3, an m102.4 derivative, in complex with the GP of the related Hendra Virus (HeV) has been solved, the structural interaction between m102.4 and NiV is uncharacterized. Herein, we used structure-guided alanine-scanning mutagenesis to map the functional epitope and paratope residues that govern the antigen–antibody interaction. Our results revealed that the binding of m102.4 is mediated predominantly by two residues in the HCDR3 region, which is unusually small for an antibody-antigen interaction. We performed computational docking to generate a structural model of m102.4-NiV interaction. Our model indicates that m102.4 targets the common hydrophobic central cavity and a hydrophilic rim on the GP, as observed for the m102.3-HeV co-crystal, albeit with Fv orientation differences. In summary, our study provides insight into the m102.4-NiV interaction, demonstrating that structure-guided alanine-scanning and computational modeling can serve as the starting point for additional antibody reengineering (e.g. affinity maturation) to generate potential therapeutic candidates.

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.

scholarly journals The Heptad Repeat C Domain of the Respiratory Syncytial Virus Fusion Protein Plays a Key Role in Membrane Fusion

2017 ◽  
pp. JVI.01323-17 ◽  
Author(s):  
Imogen M. Bermingham ◽  
Keith J. Chappell ◽  
Daniel Watterson ◽  
Paul R. Young
Keyword(s):  
Amino Acids ◽  
Respiratory Syncytial Virus ◽  
Membrane Fusion ◽  
Functional Characterization ◽  
Proteolytic Processing ◽  
Heptad Repeat ◽  
Alanine Scanning Mutagenesis ◽  
Viral Pathogen ◽  
F Protein ◽  
Syncytial Virus

Respiratory syncytial virus (RSV) mediates host cell entry through the fusion (F) protein, which undergoes a conformational change to facilitate the merger of viral and host lipid membrane envelopes. RSV F comprises a trimer of disulfide bonded F1and F2subunits that is present on the virion surface in a ‘metastable' pre-fusion state. This pre-fusion form is readily triggered to undergo refolding to bring two heptad repeats (HRA and HRB) into close proximity to form a six-helix bundle that stabilizes the post-fusion form and provides the free energy required for membrane fusion. This process can be triggered independently of other proteins. Here, we have performed a comprehensive analysis of a third heptad repeat region, HRC (amino acids 75-97), an amphipathic α-helix that lies at the interface of the pre-fusion F trimer and is a major structural feature of the F2subunit. We performed alanine scanning mutagenesis from Lys-75 to Met-97 and assessed all mutations in transient cell culture for expression, proteolytic processing, cell surface localization, protein conformation and membrane fusion. Functional characterization revealed a striking distribution of activity in which fusion-increasing mutations localized to one side of the helical face, while fusion-decreasing mutations clustered on the opposing face. Herein we propose a model in which HRC plays a stabilizing role within the globular head for the pre-fusion F trimer and is potentially involved in the early events of triggering, prompting fusion peptide release and transition into the post-fusion state.IMPORTANCERSV is recognized as the most important viral pathogen amongst pediatric populations worldwide, yet no vaccine or widely available therapeutic treatment is available. The F protein is critical for the viral replication process and is the major target for neutralizing antibodies. Recent years have seen the development of pre-fusion stabilized F protein based approaches to vaccine design. A detailed understanding of the specific domains and residues that contribute to protein stability and fusion function is fundamental to such efforts. Here we present a comprehensive mutagenesis based study of a region of the RSV F2subunit (amino acids 75 - 97), referred to as HRC, and propose a role for this helical region in maintaining the delicate stability of the pre-fusion form.

scholarly journals Subcellular Localization and Calcium and pH Requirements for Proteolytic Processing of the Hendra Virus Fusion Protein

2004 ◽  
Vol 78 (17) ◽  
pp. 9154-9163 ◽  
Author(s):  
Cara Theresia Pager ◽  
Mark Allen Wurth ◽  
Rebecca Ellis Dutch
Keyword(s):  
Subcellular Location ◽  
Vero Cells ◽  
Proteolytic Cleavage ◽  
Simian Virus ◽  
Hendra Virus ◽  
Proteolytic Processing ◽  
Viral Fusion ◽  
Proteolytic Activation ◽  
F Protein ◽  
Virus Fusion

ABSTRACT Proteolytic cleavage of the Hendra virus fusion (F) protein results in the formation of disulfide-linked F1 and F2 subunits, with cleavage occurring after residue K109 in the sequence GDVK↓L. This unusual cleavage site and efficient propagation of Hendra virus in a furin-deficient cell line indicate that the Hendra F protein is not cleaved by furin, the protease responsible for proteolytic activation of many viral fusion proteins. To identify the subcellular site of Hendra F processing, Vero cells transfected with pCAGGS-Hendra F or pCAGGS-SV5 F were metabolically labeled and chased in the absence and presence of inhibitors of exocytosis. The addition of carbonyl-cyanide-3-chlorophenylhydrazone, monensin, brefeldin A, or NaF-AlCl3 or incubation of cells at 20°C all inhibited processing of the Hendra F protein, suggesting that cleavage of Hendra F occurs either in secretory vesicles budding from the trans-Golgi network or at the cell surface. In contrast to proteolytic cleavage of the simian virus 5 (SV5) F protein by the Ca2+-dependent protease furin, proteolytic cleavage of the Hendra F protein was not significantly inhibited by decreases in Ca2+ levels following incubation with EGTA or A23187. However, in the presence of weak amines and H+ V-ATPase inhibitors, known to raise intracellular pH, cleavage of Hendra F protein was inhibited while processing of the SV5 F protein was not significantly affected. The subcellular location, sensitivity to pH changes, and decreased Ca2+ requirement suggest that the protease responsible for cleavage of Hendra F protein differs from proteases previously shown to be involved in the processing of other viral glycoproteins.

scholarly journals Separating Functions of the Phage-Encoded Quorum-Sensing-Activated Antirepressor Qtip

2019 ◽  
Author(s):  
Justin E. Silpe ◽  
Andrew A. Bridges ◽  
Xiuliang Huang ◽  
Daniela R. Coronado ◽  
Olivia P. Duddy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Binding ◽  
Quorum Sensing ◽  
Host Cell ◽  
Cell Density ◽  
Chemical Communication ◽  
Alanine Scanning Mutagenesis ◽  
Polar Localization ◽  
Coordinate Gene Expression ◽  
N Terminus

SummaryQuorum sensing is a process of chemical communication that bacteria use to track cell density and coordinate gene expression across a population. Bacteria-infecting viruses, called phages, can encode quorum-sensing components that enable them to integrate host cell density information into the lysis-lysogeny decision. Vibriophage VP882 is one such phage, and activation of its quorum-sensing pathway leads to the production of an antirepressor called Qtip. Qtip interferes with the prophage repressor (cIVP882), leading to host-cell lysis. Here, we show that Qtip interacts with the N-terminus of cIVP882, inhibiting both cIVP882 DNA-binding and cIVP882 autoproteolysis. Qtip also sequesters cIVP882, localizing it to the poles. Qtip can localize to the poles independently of cIVP882. Alanine-scanning mutagenesis of Qtip shows that its localization and interference with cIVP882 activities are separable. Comparison of Qtip to a canonical phage antirepressor reveals that, despite both proteins interacting with their partner repressors, only Qtip drives polar localization.

scholarly journals Identification of gp120 Binding Sites on CXCR4 by Using CD4-Independent Human Immunodeficiency Virus Type 2 Env Proteins

2003 ◽  
Vol 77 (2) ◽  
pp. 931-942 ◽  
Author(s):  
George Lin ◽  
Frédéric Baribaud ◽  
Josephine Romano ◽  
Robert W. Doms ◽  
James A. Hoxie
Keyword(s):  
Human Immunodeficiency Virus ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Chemokine Receptor ◽  
Binding Sites ◽  
Viral Envelope ◽  
Alanine Scanning Mutagenesis ◽  
Immunodeficiency Virus ◽  
N Terminus ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus (HIV) and simian (SIV) immunodeficiency virus entry is mediated by binding of the viral envelope glycoprotein (Env) to CD4 and chemokine receptors, CCR5 and/or CXCR4. CD4 induces extensive conformational changes that expose and/or induce formation of a chemokine receptor binding site on gp120. CD4-independent Env's of HIV type 1 (HIV-1), HIV-2, and SIV have been identified that exhibit exposed chemokine receptor binding sites and can bind directly to CCR5 or CXCR4 in the absence of CD4. While many studies have examined determinants for gp120-CCR5 binding, analysis of gp120-CXCR4 binding has been hindered by the apparently lower affinity of this interaction for X4-tropic HIV-1 isolates. We show here that gp120 proteins from two CD4-independent HIV-2 Env's, VCP and ROD/B, bind directly to CXCR4 with an apparently high affinity. By use of CXCR4 N-terminal deletion constructs, CXCR4-CXCR2 chimeras, and human-rat CXCR4 chimeras, binding determinants were shown to reside in the amino (N) terminus, extracellular loop 2 (ECL2), and ECL3. Alanine-scanning mutagenesis of charged residues, tyrosines, and phenylalanines in extracellular CXCR4 domains implicated multiple amino acids in the N terminus (E14/E15, D20, Y21, and D22), ECL2 (D187, R188, F189, Y190, and D193), and ECL3 (D262, E268, E277, and E282) in binding, although minor differences were noted between VCP and ROD/B. However, mutations in CXCR4 that markedly reduced binding did not necessarily hinder cell-cell fusion by VCP or ROD/B, especially in the presence of CD4. These gp120 proteins will be useful in dissecting determinants for CXCR4 binding and Env triggering and in evaluating pharmacologic inhibitors of the gp120-CXCR4 interaction.

scholarly journals Cathepsin L Is Involved in Proteolytic Processing of the Hendra Virus Fusion Protein

2005 ◽  
Vol 79 (20) ◽  
pp. 12714-12720 ◽  
Author(s):  
Cara Theresia Pager ◽  
Rebecca Ellis Dutch
Keyword(s):  
Cathepsin L ◽  
Vero Cells ◽  
Simian Virus ◽  
Hendra Virus ◽  
Proteolytic Processing ◽  
Protein Cleavage ◽  
F Protein ◽  
Metalloprotease Inhibitors ◽  
Specific Protease ◽  
Cellular Protease

ABSTRACT Proteolytic processing of paramyxovirus fusion (F) proteins is essential for the generation of a mature and fusogenic form of the F protein. Although many paramyxovirus F proteins are proteolytically processed by the cellular protease furin at a multibasic cleavage motif, cleavage of the newly emerged Hendra virus F protein occurs by a previously unidentified cellular protease following a single lysine at residue 109. We demonstrate here that the cellular protease cathepsin L is involved in converting the Hendra virus precursor F protein (F0) to the active F1 + F2 disulfide-linked heterodimer. To initially identify the class of protease involved in Hendra virus F protein cleavage, Vero cells transfected with pCAGGS-Hendra F or pCAGGS-SV5 F (known to be proteolytically processed by furin) were metabolically labeled and chased in the absence or presence of serine, cysteine, aspartyl, and metalloprotease inhibitors. Nonspecific and specific protease inhibitors known to decrease cathepsin activity inhibited proteolytic processing of Hendra virus F but had no effect on simian virus 5 F processing. We next designed shRNA oligonucleotides to cathepsin L which dramatically reduced cathepsin L protein expression and enzyme activity. Cathepsin L shRNA-expressing Vero cells transfected with pCAGGS-Hendra F demonstrated a nondetectable amount of cleavage of the Hendra virus F protein and significantly decreased membrane fusion activity. Additionally, we found that purified human cathepsin L processed immunopurified Hendra virus F0 into F1 and F2 fragments. These studies introduce a novel mechanism for primary proteolytic processing of viral glycoproteins and also suggest a previously unreported biological role for cathepsin L.

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