Identification of Respiratory Syncytial Virus Fusion Protein Inhibitor: In silico Screening and Molecular Docking Approach

Fusion Protein ◽

Therapeutic Potential ◽

Viral Envelope ◽

Host Cells ◽

Inhibition Constant ◽

Molecular Docking Study ◽

F Protein ◽

In young children, immunocompromised individuals, and elderly people, the respiratory syncytial virus (RSV) is the primary source of acute lower respiratory tract infection. Intervention with RSV-specific small-molecule antivirals may provide significant therapeutic potential. For virus entry, the RSV fusion protein (F) is crucial as it facilitates viral and hosts membrane fusion. To date, no approved vaccine or drug molecule is available to treat RSV. With this purpose, in the present study, virtual screening of a library of natural compounds against the active site of F protein was performed, followed by an in-depth molecular docking study of top-scored compounds. Selected hits ZINC8740013, ZINC4029781 and ZINC898642were found to strongly bind with RSV F protein relative to the other compounds as well as the control. The binding energy (BE) and inhibition constant for ‘ZINC8740013-RSV F’, ‘ZINC4029781-RSV F’, and ‘ZINC898642-RSVF’ complexes were found as ‘-7.8 kcal/mol and 63.27 µM’, ‘-7.7 kcal/mol and 19.04 µM’, and ‘-7.5 kcal/mol and 3.31 µM’, respectively. However, BE and inhibition constant of control (JNJ-53718678) with RSV F protein was found as -6.1 kcal/mol and 563.26 µM, respectively. Phe140 and Phe488 are the main interacting residues of RSV F protein with JNJ-53718678 and selected hit compounds. The finding of this study suggests that these hits can be utilized as the RSV Fprotein inhibitor to prevent the fusion of the viral envelope with the host cells. Further, bench work experiments are required to optimize these hit compounds as RSV Fprotein inhibitors.

RAGE inhibits human respiratory syncytial virus syncytium formation by interfering with F-protein function

Journal of General Virology ◽

10.1099/vir.0.049254-0 ◽

2013 ◽

Vol 94 (8) ◽

pp. 1691-1700 ◽

Cited By ~ 7

Author(s):

Jane Tian ◽

Kelly Huang ◽

Subramaniam Krishnan ◽

Catherine Svabek ◽

Daniel C. Rowe ◽

...

Keyword(s):

Epithelial Cells ◽

Syncytium Formation ◽

Human Respiratory Syncytial Virus ◽

Host Cells ◽

Type I ◽

F Protein ◽

Viral Spread ◽

Infected Cells ◽

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection. Infection is critically dependent on the RSV fusion (F) protein, which mediates fusion between the viral envelope and airway epithelial cells. The F protein is also expressed on infected cells and is responsible for fusion of infected cells with adjacent cells, resulting in the formation of multinucleate syncytia. The receptor for advanced glycation end products (RAGE) is a pattern-recognition receptor that is constitutively highly expressed by type I alveolar epithelial cells. Here, we report that RAGE protected HEK cells from RSV-induced cell death and reduced viral titres in vitro. RAGE appeared to interact directly with the F protein, but, rather than inhibiting RSV entry into host cells, virus replication and budding, membrane-expressed RAGE or soluble RAGE blocked F-protein-mediated syncytium formation and sloughing. These data indicate that RAGE may contribute to protecting the lower airways from RSV by inhibiting the formation of syncytia, viral spread, epithelial damage and airway obstruction.

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 ◽

10.1128/mbio.00270-11 ◽

2012 ◽

Vol 3 (1) ◽

Cited By ~ 31

Author(s):

Fyza Y. Shaikh ◽

Reagan G. Cox ◽

Aaron W. Lifland ◽

Anne L. Hotard ◽

John V. Williams ◽

...

Keyword(s):

Cell Surface ◽

Fusion Protein ◽

Cytoplasmic Tail ◽

Viral Proteins ◽

General Strategy ◽

Apical Surface ◽

Filament Formation ◽

F Protein ◽

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.

Multiple glycosylated forms of the respiratory syncytial virus fusion protein are expressed in virus-infected cells

Journal of General Virology ◽

10.1099/0022-1317-83-1-61 ◽

2002 ◽

Vol 83 (1) ◽

pp. 61-66 ◽

Cited By ~ 12

Author(s):

Helen W. McL. Rixon ◽

Craig Brown ◽

Gaie Brown ◽

Richard J. Sugrue

Keyword(s):

Fusion Protein ◽

Vero Cells ◽

Cross Linking ◽

Protein Species ◽

Glycosylation Pattern ◽

F Protein ◽

Virus Fusion ◽

Infected Cells ◽

Analysis of the respiratory syncytial virus (RSV) fusion (F) protein in RSV-infected Vero cells showed the presence of a single F1 subunit and at least two different forms of the F2 subunit, designated F2a (21 kDa) and F2b (16 kDa), which were collectively referred to as [F2]a/b. Enzymatic deglycosylation of [F2]a/b produced a single 10 kDa product suggesting that [F2]a/b arises from differences in the glycosylation pattern of F2a and F2b. The detection of [F2]a/b was dependent upon the post-translational cleavage of the F protein by furin, since its appearance was prevented in RSV-infected Vero cells treated with the furin inhibitor dec-RVKR-cmk. Analysis by protein cross-linking revealed that the F1 subunit interacted with [F2]a/b, via disulphide bonding, to produce equivalent F protein trimers, which were expressed on the surface of infected cells. Collectively, these data show that multiple F protein species are expressed in RSV-infected cells.

N-Glycans of F Protein Differentially Affect Fusion Activity of Human Respiratory Syncytial Virus

Journal of Virology ◽

10.1128/jvi.75.10.4744-4751.2001 ◽

2001 ◽

Vol 75 (10) ◽

pp. 4744-4751 ◽

Cited By ~ 51

Author(s):

Gert Zimmer ◽

Ina Trotz ◽

Georg Herrler

Keyword(s):

Cell Surface ◽

Fusion Protein ◽

Syncytium Formation ◽

Human Respiratory Syncytial Virus ◽

Fusion Activity ◽

Triple Mutant ◽

Activity Increase ◽

F Protein ◽

ABSTRACT The human respiratory syncytial virus (Long strain) fusion protein contains six potential N-glycosylation sites: N27, N70, N116, N120, N126, and N500. Site-directed mutagenesis of these positions revealed that the mature fusion protein contains three N-linked oligosaccharides, attached to N27, N70, and N500. By introducing these mutations into the F gene in different combinations, four more mutants were generated. All mutants, including a triple mutant devoid of any N-linked oligosaccharide, were efficiently transported to the plasma membrane, as determined by flow cytometry and cell surface biotinylation. None of the glycosylation mutations interfered with proteolytic activation of the fusion protein. Despite similar levels of cell surface expression, the glycosylation mutants affected fusion activity in different ways. While the N27Q mutation did not have an effect on syncytium formation, loss of the N70-glycan caused a fusion activity increase of 40%. Elimination of both N-glycans (N27/70Q mutant) reduced the fusion activity by about 50%. A more pronounced reduction of the fusion activity of about 90% was observed with the mutants N500Q, N27/500Q, and N70/500Q. Almost no fusion activity was detected with the triple mutant N27/70/500Q. These data indicate that N-glycosylation of the F2 subunit at N27 and N70 is of minor importance for the fusion activity of the F protein. The single N-glycan of the F1 subunit attached to N500, however, is required for efficient syncytium formation.

The Optimal Concentration of Formaldehyde is Key to Stabilizing the Pre-Fusion Conformation of Respiratory Syncytial Virus Fusion Protein

Viruses ◽

10.3390/v11070628 ◽

2019 ◽

Vol 11 (7) ◽

pp. 628 ◽

Cited By ~ 1

Author(s):

Wei Zhang ◽

Lu-Jing Zhang ◽

Lu-Ting Zhan ◽

Min Zhao ◽

Guang-Hua Wu ◽

...

Keyword(s):

Fusion Protein ◽

Neutralizing Antibody ◽

Neutralization Assay ◽

Mean Value ◽

Optimal Concentration ◽

F Protein ◽

Rsv Infection ◽

Antibody Titers ◽

Background: To date, there is no licensed vaccine available to prevent respiratory syncytial virus (RSV) infection. The valuable pre-fusion conformation of the fusion protein (pre-F) is prone to lose high neutralizing antigenic sites. The goals of this study were to stabilize pre-F protein by fixatives and try to find the possibility of developing an inactivated RSV vaccine. Methods: The screen of the optimal fixative condition was performed with flow cytometry. BALB/c mice were immunized intramuscularly with different immunogens. The serum neutralizing antibody titers of immunized mice were determined by neutralization assay. The protection and safety of these immunogens were assessed. Results: Fixation in an optimal concentration of formaldehyde (0.0244%–0.0977%) or paraformaldehyde (0.0625%–1%) was able to stabilize pre-F. Additionally, BALB/c mice inoculated with optimally stabilized pre-F protein (opti-fixed) induced a higher anti-RSV neutralization (9.7 log2, mean value of dilution rate) than those inoculated with unstable (unfixed, 8.91 log2, p < 0.01) or excessively fixed (exce-fixed, 7.28 log2, p < 0.01) pre-F protein. Furthermore, the opti-fixed immunogen did not induce enhanced RSV disease. Conclusions: Only the proper concentration of fixatives could stabilize pre-F and the optimal formaldehyde condition provides a potential reference for development of an inactivated RSV vaccine.

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

Journal of General Virology ◽

10.1099/vir.0.80010-0 ◽

2004 ◽

Vol 85 (7) ◽

pp. 1815-1824 ◽

Cited By ~ 11

Author(s):

Patricia König ◽

Katrin Giesow ◽

Kathrin Schuldt ◽

Ursula J. Buchholz ◽

Günther M. Keil

Keyword(s):

Amino Acid ◽

Fusion Protein ◽

Bovine Respiratory Syncytial Virus ◽

Peptide Sequence ◽

Target Cells ◽

Cleavage Sites ◽

Furin Cleavage ◽

F Protein ◽

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.

Cleavage at the Furin Consensus Sequence RAR/KR109 and Presence of the Intervening Peptide of the Respiratory Syncytial Virus Fusion Protein Are Dispensable for Virus Replication in Cell Culture

Journal of Virology ◽

10.1128/jvi.76.18.9218-9224.2002 ◽

2002 ◽

Vol 76 (18) ◽

pp. 9218-9224 ◽

Cited By ~ 25

Author(s):

Gert Zimmer ◽

Karl-Klaus Conzelmann ◽

Georg Herrler

Keyword(s):

Cell Culture ◽

Fusion Protein ◽

Reverse Genetics ◽

Consensus Sequence ◽

Vero Cells ◽

Proteolytic Processing ◽

Furin Cleavage ◽

F Protein ◽

ABSTRACT Proteolytic processing of the respiratory syncytial virus F (fusion) protein results in the generation of the disulfide-linked subunits F1 and F2 and in the release of pep27, a glycopeptide originally located between the two furin cleavage sites FCS-1 (RKRR136) and FCS-2 (RAR/KR109). We made use of reverse genetics to study the importance of FCS-2 and of pep27 for BRSV replication in cell culture. Replacement of FCS-2 in the F protein of recombinant viruses by either of the sequences NANR109, RANN109 or SANN109, respectively, abolished proteolytic processing at this position, whereas the cleavage of FCS-1 was not affected. All mutants replicated in calf kidney and Vero cells in the absence of exogenous trypsin, although somewhat higher titers of BRSV containing the NANR109 or the RANN109 motif were achieved in the presence of trypsin. The virus mutants showed a reduced cytopathic effect which was lowest in the case of the SANN109 mutant. These findings demonstrate that cleavage at FCS-2 is dispensable for replication of respiratory syncytial virus in cell culture. A deletion mutant containing FCS-1 but lacking FCS-2 and most of pep27 replicated in cell culture as efficiently as the parental virus, indicating that this domain of the F protein is not essential for virus maturation and infectivity.

The Fusion Protein of Respiratory Syncytial Virus Triggers p53-Dependent Apoptosis

Journal of Virology ◽

10.1128/jvi.01887-07 ◽

2008 ◽

Vol 82 (7) ◽

pp. 3236-3249 ◽

Cited By ~ 41

Author(s):

Julia Eckardt-Michel ◽

Markus Lorek ◽

Diane Baxmann ◽

Thomas Grunwald ◽

Günther M. Keil ◽

...

Keyword(s):

Epithelial Cells ◽

Cell Monolayer ◽

A549 Cells ◽

Viral Envelope ◽

Dominant Negative ◽

Lung Epithelial Cells ◽

Small Airways ◽

F Protein ◽

ABSTRACT Infection with respiratory syncytial virus (RSV) frequently causes inflammation and obstruction of the small airways, leading to severe pulmonary disease in infants. We show here that the RSV fusion (F) protein, an integral membrane protein of the viral envelope, is a strong elicitor of apoptosis. Inducible expression of F protein in polarized epithelial cells triggered caspase-dependent cell death, resulting in rigorous extrusion of apoptotic cells from the cell monolayer and transient loss of epithelial integrity. A monoclonal antibody directed against F protein inhibited apoptosis and was also effective if administered to A549 lung epithelial cells postinfection. F protein expression in epithelial cells caused phosphorylation of tumor suppressor p53 at serine 15, activation of p53 transcriptional activity, and conformational activation of proapoptotic Bax. Stable expression of dominant-negative p53 or p53 knockdown by RNA interference inhibited the apoptosis of RSV-infected A549 cells. HEp-2 tumor cells with low levels of p53 were not sensitive to RSV-triggered apoptosis. We propose a new model of RSV disease with the F protein as an initiator of epithelial cell shedding, airway obstruction, secondary necrosis, and consequent inflammation. This makes the RSV F protein a key target for the development of effective postinfection therapies.

Comparison of Immune Responses to Different Versions of VLP Associated Stabilized RSV Pre-Fusion F Protein

Vaccines ◽

10.3390/vaccines7010021 ◽

2019 ◽

Vol 7 (1) ◽

pp. 21 ◽

Cited By ~ 4

Author(s):

Lori Cullen ◽

Madelyn Schmidt ◽

Gretel Torres ◽

Adam Capoferri ◽

Trudy Morrison

Keyword(s):

Monoclonal Antibodies ◽

Immune Responses ◽

Fusion Protein ◽

Neutralizing Antibodies ◽

Vaccine Development ◽

Vaccine Candidates ◽

F Protein ◽

Syncytial Virus ◽

Conformation Stability

Efforts to develop a vaccine for respiratory syncytial virus (RSV) have primarily focused on the RSV fusion protein. The pre-fusion conformation of this protein induces the most potent neutralizing antibodies and is the focus of recent efforts in vaccine development. Following the first identification of mutations in the RSV F protein (DS-Cav1 mutant protein) that stabilized the pre-fusion conformation, other mutant stabilized pre-fusion F proteins have been described. To determine if there are differences in alternate versions of stabilized pre-fusion F proteins, we explored the use, as vaccine candidates, of virus-like particles (VLPs) containing five different pre-fusion F proteins, including the DS-Cav1 protein. The expression of these five pre-F proteins, their assembly into VLPs, their pre-fusion conformation stability in VLPs, their reactivity with anti-F monoclonal antibodies, and their induction of immune responses after the immunization of mice, were characterized, comparing VLPs containing the DS-Cav1 pre-F protein with VLPs containing four alternative pre-fusion F proteins. The concentrations of anti-F IgG induced by each VLP that blocked the binding of prototype monoclonal antibodies using two different soluble pre-fusion F proteins as targets were measured. Our results indicate that both the conformation and immunogenicity of alternative VLP associated stabilized pre-fusion RSV F proteins are different from those of DS-Cav1 VLPs.

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

Journal of Virology ◽

10.1128/jvi.00078-08 ◽

2008 ◽

Vol 82 (12) ◽

pp. 5986-5998 ◽

Cited By ~ 22

Author(s):

Joanna Rawling ◽

Blanca García-Barreno ◽

José A. Melero

Keyword(s):

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.