scholarly journals Insertion of the Two Cleavage Sites of the Respiratory Syncytial Virus Fusion Protein in Sendai Virus Fusion Protein Leads to Enhanced Cell-Cell Fusion and a Decreased Dependency on the HN Attachment Protein for Activity

2008 ◽  
Vol 82 (12) ◽  
pp. 5986-5998 ◽  
Author(s):  
Joanna Rawling ◽  
Blanca García-Barreno ◽  
José A. Melero
Keyword(s):  
Respiratory Syncytial Virus ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Sendai Virus ◽  
Cleavage Sites ◽  
Attachment Protein ◽  
F Protein ◽  
Syncytial Virus ◽  
Cell Cell

ABSTRACT Cell entry by paramyxoviruses requires fusion of the viral envelope with the target cell membrane. Fusion is mediated by the viral fusion (F) glycoprotein and usually requires the aid of the attachment glycoprotein (G, H or HN, depending on the virus). Human respiratory syncytial virus F protein (FRSV) is able to mediate membrane fusion in the absence of the attachment G protein and is unique in possessing two multibasic furin cleavage sites, separated by a region of 27 amino acids (pep27). Cleavage at both sites is required for cell-cell fusion. We have investigated the significance of the two cleavage sites and pep27 in the context of Sendai virus F protein (FSeV), which possesses a single monobasic cleavage site and requires both coexpression of the HN attachment protein and trypsin in order to fuse cells. Inclusion of both FRSV cleavage sites in FSeV resulted in a dramatic increase in cell-cell fusion activity in the presence of HN. Furthermore, chimeric FSeV mutants containing both FRSV cleavage sites demonstrated cell-cell fusion in the absence of HN. The presence of two multibasic cleavage sites may therefore represent a strategy to regulate activation of a paramyxovirus F protein for cell-cell fusion in the absence of an attachment protein.

scholarly journals A novel protein expression strategy using recombinant bovine respiratory syncytial virus (BRSV): modifications of the peptide sequence between the two furin cleavage sites of the BRSV fusion protein yield secreted proteins, but affect processing and function of the BRSV fusion protein

2004 ◽  
Vol 85 (7) ◽  
pp. 1815-1824 ◽  
Author(s):  
Patricia König ◽  
Katrin Giesow ◽  
Kathrin Schuldt ◽  
Ursula J. Buchholz ◽  
Günther M. Keil
Keyword(s):  
Respiratory Syncytial Virus ◽  
Amino Acid ◽  
Fusion Protein ◽  
Bovine Respiratory Syncytial Virus ◽  
Peptide Sequence ◽  
Target Cells ◽  
Cleavage Sites ◽  
Furin Cleavage ◽  
F Protein ◽  
Syncytial Virus

The bovine respiratory syncytial virus (BRSV) fusion (F) protein is cleaved at two furin cleavage sites, which results in generation of the disulfide-linked F1 and F2 subunits and release of an intervening peptide of 27 aa (pep27). A series of mutated open reading frames encoding F proteins that lacked the entire pep27, that contained an arbitrarily chosen 23 aa sequence instead of pep27 or in which pep27 was replaced by the amino acid sequences for the bovine cytokines interleukin 2 (boIL2), interleukin 4 (boIL4) or gamma interferon (boIFN-γ) was constructed. Transient expression experiments revealed that the sequence of the intervening peptide influenced intracellular transport, maturation of the F protein and F-mediated syncytium formation. Expression of boIL2, boIL4 or boIFN-γ in place of pep27 resulted in secretion of the cytokines into the culture medium. All mutated F proteins except the boIFN-γ-containing variant could be expressed by and were functional for recombinant BRSV. Characterization of the cell culture properties of the recombinants demonstrated that the amino acid sequence between the two furin cleavage sites affected entry into target cells, direct spreading of virions from cell to cell and virus growth. Secretion of boIL2 and boIL4 into the medium of cells infected with the respective recombinants demonstrated that the F protein can be used to express secreted heterologous bioactive peptides or (glyco)proteins, which might be of interest for the development of novel RSV vaccines.

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 A Conserved Region in the F2 Subunit of Paramyxovirus Fusion Proteins Is Involved In Fusion Regulation

2007 ◽  
Vol 81 (15) ◽  
pp. 8303-8314 ◽  
Author(s):  
Amanda E. Gardner ◽  
Rebecca E. Dutch
Keyword(s):  
Membrane Fusion ◽  
Cell Fusion ◽  
Critical Role ◽  
Simian Virus ◽  
Hendra Virus ◽  
Heptad Repeat ◽  
F Protein ◽  
Conserved Region ◽  
Heptad Repeats ◽  
Cell Cell

ABSTRACT Paramyxoviruses utilize both an attachment protein and a fusion (F) protein to drive virus-cell and cell-cell fusion. F exists functionally as a trimer of two disulfide-linked subunits: F1 and F2. Alignment and analysis of a set of paramyxovirus F protein sequences identified three conserved blocks (CB): one in the fusion peptide/heptad repeat A domain, known to play important roles in fusion promotion, one in the region between the heptad repeats of F1 (CBF1) (A. E. Gardner, K. L. Martin, and R. E. Dutch, Biochemistry 46:5094-5105, 2007), and one in the F2 subunit (CBF2). To analyze the functions of CBF2, alanine substitutions at conserved positions were created in both the simian virus 5 (SV5) and Hendra virus F proteins. A number of the CBF2 mutations resulted in folding and expression defects. However, the CBF2 mutants that were properly expressed and trafficked had altered fusion promotion activity. The Hendra virus CBF2 Y79A and P89A mutants showed significantly decreased levels of fusion, whereas the SV5 CBF2 I49A mutant exhibited greatly increased cell-cell fusion relative to that for wild-type F. Additional substitutions at SV5 F I49 suggest that both side chain volume and hydrophobicity at this position are important in the folding of the metastable, prefusion state and the subsequent triggering of membrane fusion. The recently published prefusogenic structure of parainfluenza virus 5/SV5 F (H. S. Yin et al., Nature 439:38-44, 2006) places CBF2 in direct contact with heptad repeat A. Our data therefore indicate that this conserved region plays a critical role in stabilizing the prefusion state, likely through interactions with heptad repeat A, and in triggering membrane fusion.

scholarly journals Thermostability of the human respiratory syncytial virus fusion protein before and after activation: implications for the membrane-fusion mechanism

2004 ◽  
Vol 85 (12) ◽  
pp. 3677-3687 ◽  
Author(s):  
M. Begoña Ruiz-Argüello ◽  
Diana Martín ◽  
Steve A. Wharton ◽  
Lesley J. Calder ◽  
Steve R. Martín ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Human Respiratory Syncytial Virus ◽  
Virus Fusion ◽  
Before And After ◽  
Syncytial Virus

scholarly journals The cytoplasmic domain of the F protein of Human respiratory syncytial virus is not required for cell fusion

2006 ◽  
Vol 87 (2) ◽  
pp. 395-398 ◽  
Author(s):  
Patrick J. Branigan ◽  
Nicole D. Day ◽  
Changbao Liu ◽  
Lester L. Gutshall ◽  
José A. Melero ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Cell Fusion ◽  
Fusion Proteins ◽  
Cytoplasmic Domain ◽  
Cysteine Residue ◽  
Human Respiratory Syncytial Virus ◽  
Cytoplasmic Domains ◽  
F Protein ◽  
Avian Pneumovirus ◽  
Syncytial Virus

The cytoplasmic domains of the fusion proteins encoded by several viruses play a role in cell fusion and contain sites for palmitoylation associated with viral protein trafficking and virus assembly. The fusion (F) protein of Human respiratory syncytial virus (HRSV) has a predicted cytoplasmic domain of 26 residues containing a single palmitoylated cysteine residue that is conserved in bovine RSV F protein, but not in the F proteins of other pneumoviruses such as pneumonia virus of mice, human metapneumovirus and avian pneumovirus. The cytoplasmic domains in other paramyxovirus fusion proteins such as Newcastle disease virus F protein play a role in fusion. In this study, it was shown that deletion of the entire cytoplasmic domain or mutation of the single cysteine residue (C550S) of the HRSV F protein had no effect on protein processing, cell-surface expression or fusion.

scholarly journals A Critical Phenylalanine Residue in the Respiratory Syncytial Virus Fusion Protein Cytoplasmic Tail Mediates Assembly of Internal Viral Proteins into Viral Filaments and Particles

mBio ◽  
2012 ◽  
Vol 3 (1) ◽  
Author(s):  
Fyza Y. Shaikh ◽  
Reagan G. Cox ◽  
Aaron W. Lifland ◽  
Anne L. Hotard ◽  
John V. Williams ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Cell Surface ◽  
Fusion Protein ◽  
Cytoplasmic Tail ◽  
Viral Proteins ◽  
General Strategy ◽  
Apical Surface ◽  
Filament Formation ◽  
F Protein ◽  
Syncytial Virus

ABSTRACTRespiratory syncytial virus (RSV) is a single-stranded RNA virus in theParamyxoviridaefamily that assembles into filamentous structures at the apical surface of polarized epithelial cells. These filaments contain viral genomic RNA and structural proteins, including the fusion (F) protein, matrix (M) protein, nucleoprotein (N), and phosphoprotein (P), while excluding F-actin. It is known that the F protein cytoplasmic tail (FCT) is necessary for filament formation, but the mechanism by which the FCT mediates assembly into filaments is not clear. We hypothesized that the FCT is necessary for interactions with other viral proteins in order to form filaments. In order to test this idea, we expressed the F protein with cytoplasmic tail (CT) truncations or specific point mutations and determined the abilities of these variant F proteins to form filaments independent of viral infection when coexpressed with M, N, and P. Deletion of the terminal three FCT residues (amino acids Phe-Ser-Asn) or mutation of the Phe residue resulted in a loss of filament formation but did not affect F-protein expression or trafficking to the cell surface. Filament formation could be restored by addition of residues Phe-Ser-Asn to an FCT deletion mutant and was unaffected by mutations to Ser or Asn residues. Second, deletion of residues Phe-Ser-Asn or mutation of the Phe residue resulted in a loss of M, N, and P incorporation into virus-like particles. These data suggest that a C-terminal Phe residue in the FCT mediates assembly through incorporation of internal virion proteins into virus filaments at the cell surface.IMPORTANCERespiratory syncytial virus (RSV) is a leading cause of bronchiolitis and pneumonia in infants and the elderly worldwide. There is no licensed RSV vaccine and only limited therapeutics for use in infected patients. Many aspects of the RSV life cycle have been studied, but the mechanisms that drive RSV assembly at the cell surface are not well understood. This study provides evidence that a specific residue in the RSV fusion protein cytoplasmic tail coordinates assembly into viral filaments by mediating the incorporation of internal virion proteins. Understanding the mechanisms that drive RSV assembly could lead to targeted development of novel antiviral drugs. Moreover, since RSV exits infected cells in an ESCRT (endosomal sorting complexes required for transport)-independent manner, these studies may contribute new knowledge about a general strategy by which ESCRT-independent viruses mediate outward bud formation using viral protein-mediated mechanisms during assembly and budding.

scholarly journals Respiratory Syncytial Virus Attachment Glycoprotein Contribution to Infection Depends on the Specific Fusion Protein

2015 ◽  
Vol 90 (1) ◽  
pp. 245-253 ◽  
Author(s):  
Jia Meng ◽  
Anne L. Hotard ◽  
Michael G. Currier ◽  
Sujin Lee ◽  
Christopher C. Stobart ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Fusion Protein ◽  
G Protein ◽  
Clinical Isolate ◽  
Protein Function ◽  
Virus Binding ◽  
Attachment Protein ◽  
Recombinant Viruses ◽  
Syncytial Virus

ABSTRACTHuman respiratory syncytial virus (RSV) is an important pathogen causing acute lower respiratory tract disease in children. The RSV attachment glycoprotein (G) is not required for infection, as G-null RSV replicates efficiently in several cell lines. Our laboratory previously reported that the viral fusion (F) protein is a determinant of strain-dependent pathogenesis. Here, we hypothesized that virus dependence on G is determined by the strain specificity of F. We generated recombinant viruses expressing G and F, or null for G, from the laboratory A2 strain (Katushka RSV-A2GA2F [kRSV-A2GA2F] and kRSV-GstopA2F) or the clinical isolate A2001/2-20 (kRSV-2-20G2-20F and kRSV-Gstop2-20F). We quantified the virus cell binding, entry kinetics, infectivity, and growth kinetics of these four recombinant virusesin vitro. RSV expressing the 2-20 G protein exhibited the greatest binding activity. Compared to the parental viruses expressing G and F, removal of 2-20 G had more deleterious effects on binding, entry, infectivity, and growth than removal of A2 G. Overall, RSV expressing 2-20 F had a high dependence on G for binding, entry, and infection.IMPORTANCERSV is the leading cause of childhood acute respiratory disease requiring hospitalization. As with other paramyxoviruses, two major RSV surface viral glycoproteins, the G attachment protein and the F fusion protein, mediate virus binding and subsequent membrane fusion, respectively. Previous work on the RSV A2 prototypical strain demonstrated that the G protein is functionally dispensable forin vitroreplication. This is in contrast to other paramyxoviruses that require attachment protein function as a prerequisite for fusion. We reevaluated this requirement for RSV using G and F proteins from clinical isolate 2-20. Compared to the laboratory A2 strain, the G protein from 2-20 had greater contributions to virus binding, entry, infectivity, andin vitrogrowth kinetics. Thus, the clinical isolate 2-20 F protein function depended more on its G protein, suggesting that RSV has a higher dependence on G than previously thought.

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.

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