scholarly journals The Core of the Respiratory Syncytial Virus Fusion Protein Is a Trimeric Coiled Coil

2000 ◽  
Vol 74 (13) ◽  
pp. 5911-5920 ◽  
Author(s):  
Jacqueline M. Matthews ◽  
Thomas F. Young ◽  
Simon P. Tucker ◽  
Joel P. Mackay
Keyword(s):  
Respiratory Syncytial Virus ◽  
Fusion Protein ◽  
Limited Proteolysis ◽  
Coiled Coil ◽  
Heptad Repeat ◽  
Sedimentation Equilibrium ◽  
Resonance Spectroscopy ◽  
The Core ◽  
Syncytial Virus ◽  
Tract Infections

ABSTRACT Entry into the host cell by enveloped viruses is mediated by fusion (F) or transmembrane glycoproteins. Many of these proteins share a fold comprising a trimer of antiparallel coiled-coil heterodimers, where the heterodimers are formed by two discontinuous heptad repeat motifs within the proteolytically processed chain. The F protein of human respiratory syncytial virus (RSV; the major cause of lower respiratory tract infections in infants) contains two corresponding regions that are predicted to form coiled coils (HR1 and HR2), together with a third predicted heptad repeat (HR3) located in a nonhomologous position. In order to probe the structures of these three domains and ascertain the nature of the interactions between them, we have studied the isolated HR1, HR2, and HR3 domains of RSV F by using a range of biophysical techniques, including circular dichroism, nuclear magnetic resonance spectroscopy, and sedimentation equilibrium. HR1 forms a symmetrical, trimeric coiled coil in solution (K 3 ≈ 2.2 × 1011 M−2) which interacts with HR2 to form a 3:3 hexamer. The HR1-HR2 interaction domains have been mapped using limited proteolysis, reversed-phase high-performance liquid chromatography, and electrospray-mass spectrometry. HR2 in isolation exists as a largely unstructured monomer, although it exhibits a tendency to form aggregates with β-sheet-like characteristics. Only a small increase in α-helical content was observed upon the formation of the hexamer. This suggests that the RSV F glycoprotein contains a domain that closely resembles the core structure of the simian parainfluenza virus 5 fusion protein (K. A. Baker, R. E. Dutch, R. A. Lamb, and T. S. Jardetzky, Mol. Cell 3:309–319, 1999). Finally, HR3 forms weak α-helical homodimers that do not appear to interact with HR1, HR2, or the HR1-HR2 complex. The results of these studies support the idea that viral fusion proteins have a common core architecture.

scholarly journals Identification and Evaluation of a Highly Effective Fusion Inhibitor for Human Metapneumovirus

2007 ◽  
Vol 52 (1) ◽  
pp. 279-287 ◽  
Author(s):  
Céline Deffrasnes ◽  
Marie-Ève Hamelin ◽  
Gregory A. Prince ◽  
Guy Boivin
Keyword(s):  
Fusion Protein ◽  
Clinical Symptoms ◽  
Pulmonary Inflammation ◽  
Human Metapneumovirus ◽  
Heptad Repeat ◽  
Elderly Subjects ◽  
Monocyte Chemoattractant Protein 1 ◽  
Syncytial Virus ◽  
Tract Infections

ABSTRACT Human metapneumovirus (hMPV) can cause acute upper and lower respiratory tract infections that are particularly severe in young children, elderly subjects, and immunocompromised patients. To date, no treatments or vaccines are available for hMPV infections. Our objective was to assess the inhibitory potential of several peptides derived from the heptad repeat A and B (HRA and HRB) domains of the hMPV fusion protein. Nine candidate peptides were expressed in Escherichia coli or obtained synthetically and tested in vitro and in an animal model. Excellent in vitro inhibition of an hMPV strain of the A1 subgroup was obtained with five peptides, with 50% inhibitory concentrations ranging from 1.4 nM to 3.3 μM. One peptide, HRA2, displayed very potent activity against all four hMPV subgroups. It was also moderately active against human respiratory syncytial virus (strain A2) but displayed no activity against human parainfluenza virus type 3. BALB/c mice that received the HRA2 peptide and a lethal hMPV intranasal challenge simultaneously were completely protected from clinical symptoms and mortality. On day 5 postinfection, HRA2-treated mice had undetectable lung viral loads which were significantly less than those of untreated mice (3 × 104 50% tissue culture infective doses/lung). Pulmonary inflammation, levels of proinflammatory cytokines/chemokines (RANTES, gamma interferon, and monocyte chemoattractant protein 1) and airway obstruction were also significantly decreased in HRA2-treated mice. The results of this study demonstrate that potent antivirals can be derived from the hMPV fusion protein HR domains. Moreover, hMPV, compared to other paramyxoviruses and to the human immunodeficiency virus, seems to be more susceptible to HRA- than HRB-derived peptides.

scholarly journals Potent single-domain antibodies that arrest respiratory syncytial virus fusion protein in its prefusion state

2017 ◽  
Vol 8 (1) ◽  
Author(s):  
Iebe Rossey ◽  
Morgan S. A. Gilman ◽  
Stephanie C. Kabeche ◽  
Koen Sedeyn ◽  
Daniel Wrapp ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Fusion Protein ◽  
Respiratory Tract Infections ◽  
Antiviral Agents ◽  
Single Domain ◽  
Neutralizing Activity ◽  
Inflammatory Monocytes ◽  
Single Domain Antibodies ◽  
Syncytial Virus ◽  
Tract Infections

Abstract Human respiratory syncytial virus (RSV) is the main cause of lower respiratory tract infections in young children. The RSV fusion protein (F) is highly conserved and is the only viral membrane protein that is essential for infection. The prefusion conformation of RSV F is considered the most relevant target for antiviral strategies because it is the fusion-competent form of the protein and the primary target of neutralizing activity present in human serum. Here, we describe two llama-derived single-domain antibodies (VHHs) that have potent RSV-neutralizing activity and bind selectively to prefusion RSV F with picomolar affinity. Crystal structures of these VHHs in complex with prefusion F show that they recognize a conserved cavity formed by two F protomers. In addition, the VHHs prevent RSV replication and lung infiltration of inflammatory monocytes and T cells in RSV-challenged mice. These prefusion F-specific VHHs represent promising antiviral agents against RSV.

scholarly journals Five Residues in the Apical Loop of the Respiratory Syncytial Virus Fusion Protein F2 Subunit Are Critical for Its Fusion Activity

2018 ◽  
Vol 92 (15) ◽  
Author(s):  
Stephanie N. Hicks ◽  
Supranee Chaiwatpongsakorn ◽  
Heather M. Costello ◽  
Jason S. McLellan ◽  
William Ray ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Target Cell ◽  
Antiviral Drug ◽  
High Energy ◽  
Heptad Repeat ◽  
Alanine Scanning Mutagenesis ◽  
F Protein ◽  
Syncytial Virus ◽  
Apical Loop ◽  
Tract Infections

ABSTRACT The respiratory syncytial virus (RSV) fusion (F) protein is a trimeric, membrane-anchored glycoprotein capable of mediating both virus-target cell membrane fusion to initiate infection and cell-cell fusion, even in the absence of the attachment glycoprotein. The F protein is initially expressed in a precursor form, whose functional capabilities are activated by proteolysis at two sites between the F1 and F2 subunits. This cleavage results in expression of the metastable and high-energy prefusion conformation. To mediate fusion, the F protein is triggered by an unknown stimulus, causing the F1 subunit to refold dramatically while F2 changes minimally. Hypothesizing that the most likely site for interaction with a target cell component would be the top, or apex, of the protein, we determined the importance of the residues in the apical loop of F2 by alanine scanning mutagenesis analysis. Five residues were not important, two were of intermediate importance, and all four lysines and one isoleucine were essential. Alanine replacement did not result in the loss of the pre-F conformation for any of these mutants. Each of the four lysines required its specific charge for fusion function. Alanine replacement of the three essential lysines on the ascent to the apex hindered fusion following a forced fusion event, suggesting that these residues are involved in refolding. Alanine mutations at Ile64, also on the ascent to the apex, and Lys75 did not prevent fusion following forced triggering, suggesting that these residues are not involved in refolding and may instead be involved in the natural triggering of the F protein. IMPORTANCE RSV infects virtually every child by the age of 3 years, causing nearly 33 million acute lower respiratory tract infections (ALRI) worldwide each year in children younger than 5 years of age (H. Nair et al., Lancet 375:1545–1555, 2010). RSV is also the second leading cause of respiratory system-related death in the elderly (A. R. Falsey and E. E. Walsh, Drugs Aging 22:577–587, 2005; A. R. Falsey, P. A. Hennessey, M. A. Formica, C. Cox, and E. E. Walsh, N Engl J Med 352:1749–1759, 2005). The monoclonal antibody palivizumab is approved for prophylactic use in some at-risk infants, but healthy infants remain unprotected. Furthermore, its expense limits its use primarily to developed countries. No vaccine or effective small-molecule drug is approved for preventing disease or treating infection (H. M. Costello, W. Ray, S. Chaiwatpongsakorn, and M. E. Peeples, Infect Disord Drug Targets, 12:110–128, 2012). The essential residues identified in the apical domain of F2 are adjacent to the apical portion of F1, which, upon triggering, refolds into a long heptad repeat A (HRA) structure with the fusion peptide at its N terminus. These essential residues in F2 are likely involved in triggering and/or refolding of the F protein and, as such, may be ideal targets for antiviral drug development.

scholarly journals Pharmacological Characterization of TP0591816, a Novel Macrocyclic Respiratory Syncytial Virus Fusion Inhibitor with Antiviral Activity against F Protein Mutants

2020 ◽  
Vol 65 (1) ◽  
pp. e01407-20
Author(s):  
Ippei Yoshida ◽  
Kaho Arikawa ◽  
Yusuke Honma ◽  
Shoko Inatani ◽  
Mitsukane Yoshinaga ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Antiviral Activity ◽  
Heptad Repeat ◽  
Fusion Inhibitor ◽  
Rich Region ◽  
F Protein ◽  
Protein Mutants ◽  
Rsv Infection ◽  
Syncytial Virus ◽  
Tract Infections

ABSTRACTHuman respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infections in early childhood. However, no vaccines have yet been approved for prevention of RSV infection, and the treatment options are limited. Therefore, development of effective and safe anti-RSV drugs is needed. In this study, we evaluated the antiviral activity and mechanism of action of a novel macrocyclic anti-RSV compound, TP0591816. TP0591816 showed significant antiviral activities against both subgroup A and subgroup B RSV, while exerting no cytotoxicity. Notably, the antiviral activity of TP0591816 was maintained against a known fusion inhibitor-resistant RSV strain with a mutation in the cysteine-rich region or in heptad repeat B. Results of a time-of-addition assay and a temperature shift assay indicated that TP0591816 inhibited fusion of RSV with the cell membrane during viral entry. In addition, TP0591816 added after cell infection also inhibited cell-cell fusion. A TP0591816-resistant virus strain selected by serial passage had an L141F mutation, but no mutation in the cysteine-rich region or in heptad repeat B in the fusion (F) protein. Treatment with TP0591816 reduced lung virus titers in a dose-dependent manner in a mouse model of RSV infection. Furthermore, the estimated effective dose of TP0591816 for use against F protein mutants was thought to be clinically realistic and potentially tolerable. Taken together, these findings suggest that TP0591816 is a promising novel candidate for the treatment of resistant RSV infection.

Heptad-Repeat Regions of Respiratory Syncytial Virus F1Protein Form a Six-Membered Coiled-Coil Complex

Biochemistry ◽  
2000 ◽  
Vol 39 (38) ◽  
pp. 11684-11695 ◽  
Author(s):  
Mary K. Lawless-Delmedico ◽  
Prakash Sista ◽  
Ratna Sen ◽  
Nicole C. Moore ◽  
James B. Antczak ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Coiled Coil ◽  
Heptad Repeat ◽  
Syncytial Virus

scholarly journals Genetic Characterization of Fusion Protein of Human Respiratory Syncytial Virus: Beijing

2012 ◽  
Vol 1 (2) ◽  
pp. 74-79
Author(s):  
Qi Lu ◽  
Chun-xia Zhao ◽  
Kun-ling Shen ◽  
Wen-bo Xu ◽  
Yan Zhang ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Amino Acid ◽  
Fusion Protein ◽  
Human Respiratory Syncytial Virus ◽  
Genetic Characteristics ◽  
Sequence Identity ◽  
Pcr Products ◽  
A Subunit ◽  
Syncytial Virus ◽  
Tract Infections

Abstract Objective Fusion protein is a subunit of the human respiratory syncytial virus (HRSV) and a potential vaccine candidate. Thus, a study on the genetic characteristics of F protein was considered important for further investigations in this field. The aim of this study was to determine the prevalence and genetic diversity of the F gene of HRSV infections in hospitalized pediatric patients in Beijing with acute lower respiratory tract infections and to compare the circulating genotypes that are currently found worldwide. Methods HRSV particles were amplified by RT-PCR and the PCR products were purified for sequencing. Further analysis was carried out by Bioedit and MEGA 3.0 biological software programs. Results Seventy-six samples (23.1%) were positive for HRSV. The percentage of cases in patients younger than 1 year was 84.21%. Among the six Beijing isolates, four belonged to subgroup A, whose respective F genes shared 97.0%-97.4% nucleotide sequence identity and 92.1%-93.0% amino acid sequence identity. The other two isolates belonged to subgroup B. Here, 97.3% and 98.2% sequence identity were found at nucleotide and amino acid levels, respectively. Conclusions Phylogenetic analysis of nucleotide sequences revealed that those four isolates within subgroup A were monophyletic and closely related to each other, but those two within subgroup B distributed in two distinct clusters. Subgroup A and B strains co-circulated, indicating that two different transmission chains occurred in Beijing from 2003-2004.

scholarly journals Inhibition of Respiratory Syncytial Virus Fusion by the Small Molecule VP-14637 via Specific Interactions with F Protein

2003 ◽  
Vol 77 (9) ◽  
pp. 5054-5064 ◽  
Author(s):  
Janet L. Douglas ◽  
Marites L. Panis ◽  
Edmund Ho ◽  
Kuei-Ying Lin ◽  
Steve H. Krawczyk ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Small Molecule ◽  
Respiratory Tract Infections ◽  
Heptad Repeat ◽  
Resistance Mutations ◽  
F Protein ◽  
Infected Cells ◽  
Syncytial Virus ◽  
Tract Infections ◽  
Virus System

ABSTRACT Human respiratory syncytial virus (RSV) is a major cause of respiratory tract infections worldwide. Several novel small-molecule inhibitors of RSV have been identified, but they are still in preclinical or early clinical evaluation. One such inhibitor is a recently discovered triphenol-based molecule, VP-14637 (ViroPharma). Initial experiments suggested that VP-14637 acted early and might be an RSV fusion inhibitor. Here we present studies demonstrating that VP-14637 does not block RSV adsorption but inhibits RSV-induced cell-cell fusion and binds specifically to RSV-infected cells with an affinity corresponding to its inhibitory potency. VP-14637 is capable of specifically interacting with the RSV fusion protein expressed by a T7 vaccinia virus system. RSV variants resistant to VP-14637 were selected; they had mutations localized to two distinct regions of the RSV F protein, heptad repeat 2 (HR2) and the intervening domain between heptad repeat 1 (HR1) and HR2. No mutations arose in HR1, suggesting a mechanism other than direct disruption of the heptad repeat interaction. The F proteins containing the resistance mutations exhibited greatly reduced binding of VP-14637. Despite segregating with the membrane fraction following incubation with intact RSV-infected cells, the compound did not bind to membranes isolated from RSV-infected cells. In addition, binding of VP-14637 was substantially compromised at temperatures of ≤22°C. Therefore, we propose that VP-14637 inhibits RSV through a novel mechanism involving an interaction between the compound and a transient conformation of the RSV F protein.

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