scholarly journals Hemagglutinin stability determines influenza A virus susceptibility to a broad-spectrum fusion inhibitor Arbidol

2022 ◽  
Author(s):  
Zhenyu Li ◽  
Tian Li ◽  
Meisui Liu ◽  
Tijana Ivanovic

Understanding mechanisms of resistance to antiviral inhibitors can reveal nuanced features of targeted viral mechanisms and, in turn, lead to improved strategies for inhibitor design. Arbidol is a broad-spectrum antiviral which binds to and prevents the fusion-associated conformational changes in the trimeric influenza hemagglutinin (HA). The rate-limiting step during HA-mediated membrane fusion is the release of the hydrophobic fusion peptides from a conserved pocket on HA. Here, we investigated how destabilizing or stabilizing mutations in or near the fusion peptide affect viral sensitivity to Arbidol. The degree of sensitivity was proportional to the extent of fusion peptide stability on the pre-fusion HA: stabilized mutants were more sensitive, and destabilized ones resistant to Arbidol. Single-virion membrane fusion experiments for representative Wild Type and mutant viruses demonstrated that resistance is a direct consequence of fusion-peptide destabilization not dependent on reduced Arbidol binding to HA at neutral pH. Our results support the model whereby the probability of individual HAs extending to engage the target membrane is determined by the composite of two critical forces: a "tug" on the fusion peptide by the extension of the central coiled-coil on HA, and the key interactions stabilizing fusion peptide in the pre-fusion pocket. Arbidol increases the free-energy penalty for coiled-coil extension, but destabilizing mutations decrease the free-energy cost for fusion peptide release, accounting for the observed resistance. Our findings have broad implications for fusion-inhibitor design, viral mechanisms of resistance, and our basic understanding of HA-mediated membrane fusion.

2002 ◽  
Vol 76 (9) ◽  
pp. 4456-4466 ◽  
Author(s):  
Jennifer A. Gruenke ◽  
R. Todd Armstrong ◽  
William W. Newcomb ◽  
Jay C. Brown ◽  
Judith M. White

ABSTRACT Influenza virus hemagglutinin undergoes a conformational change in which a loop-to-helix “spring-loaded” conformational change forms a coiled coil that positions the fusion peptide for interaction with the target bilayer. Previous work has shown that two proline mutations designed to disrupt this change disrupt fusion but did not determine the basis for the fusion defect. In this work, we made six additional mutants with single proline substitutions in the region that undergoes the spring-loaded conformational change and two additional mutants with double proline substitutions in this region. All double mutants were fusion inactive. We analyzed one double mutant, F63P/F70P, as an example. We observed that F63P/F70P undergoes key low-pH-induced conformational changes and binds tightly to target membranes. However, limited proteolysis and electron microscopy observations showed that the mutant forms a coiled coil that is only ∼50% the length of the wild type, suggesting that it is splayed in its N-terminal half. This work further supports the hypothesis that the spring-loaded conformational change is necessary for fusion. Our data also indicate that the spring-loaded conformational change has another role beyond presenting the fusion peptide to the target membrane.


2015 ◽  
Vol 89 (8) ◽  
pp. 4504-4516 ◽  
Author(s):  
Lauren Byrd-Leotis ◽  
Summer E. Galloway ◽  
Evangeline Agbogu ◽  
David A. Steinhauer

ABSTRACTInfluenza A viruses enter host cells through endosomes, where acidification induces irreversible conformational changes of the viral hemagglutinin (HA) that drive the membrane fusion process. The prefusion conformation of the HA is metastable, and the pH of fusion can vary significantly among HA strains and subtypes. Furthermore, an accumulating body of evidence implicates HA stability properties as partial determinants of influenza host range, transmission phenotype, and pathogenic potential. Although previous studies have identified HA mutations that can affect HA stability, these have been limited to a small selection of HA strains and subtypes. Here we report a mutational analysis of HA stability utilizing a panel of expressed HAs representing a broad range of HA subtypes and strains, including avian representatives across the phylogenetic spectrum and several human strains. We focused on two highly conserved residues in the HA stem region: HA2 position 58, located at the membrane distal tip of the short helix of the hairpin loop structure, and HA2 position 112, located in the long helix in proximity to the fusion peptide. We demonstrate that a K58I mutation confers an acid-stable phenotype for nearly all HAs examined, whereas a D112G mutation consistently leads to elevated fusion pH. The results enhance our understanding of HA stability across multiple subtypes and provide an additional tool for risk assessment for circulating strains that may have other hallmarks of human adaptation. Furthermore, the K58I mutants, in particular, may be of interest for potential use in the development of vaccines with improved stability profiles.IMPORTANCEThe influenza A hemagglutinin glycoprotein (HA) mediates the receptor binding and membrane fusion functions that are essential for virus entry into host cells. While receptor binding has long been recognized for its role in host species specificity and transmission, membrane fusion and associated properties of HA stability have only recently been appreciated as potential determinants. We show here that mutations can be introduced at highly conserved positions to stabilize or destabilize the HA structure of multiple HA subtypes, expanding our knowledge base for this important phenotype. The practical implications of these findings extend to the field of vaccine design, since the HA mutations characterized here could potentially be utilized across a broad spectrum of influenza virus subtypes to improve the stability of vaccine strains or components.


2006 ◽  
Vol 87 (6) ◽  
pp. 1649-1658 ◽  
Author(s):  
Diana Martín ◽  
Lesley J. Calder ◽  
Blanca García-Barreno ◽  
John J. Skehel ◽  
José A. Melero

We have reported previously the expression and purification of an anchorless form of the human respiratory syncytial virus (HRSV) F protein () representing the ectodomain of the full-length F. molecules are seen as unaggregated cones by electron microscopy but completion of proteolytic cleavage of the F0 monomers in the trimer leads to a change in shape from cones to lollipops that aggregate into rosettes. This aggregation apparently occurs by interaction of the fusion peptides of molecules that are exposed after cleavage. Since exposure of the fusion peptide is a key event in the process of membrane fusion, changes associated with cleavage may reflect those occurring in full-length F during membrane fusion. Deletions or substitutions that changed either the length, charge or hydrophobicity of the fusion peptide inhibited aggregation of , and these mutants remained as unaggregated cones after cleavage. In contrast, more conservative changes did not inhibit the change of shape and aggregation of . When the same changes were introduced in the fusion peptide of full-length F, only the mutations that inhibited aggregation of prevented membrane fusion. Thus, the conformational changes that follow completion of cleavage of the protein require a functional fusion peptide. These sequence constraints may restrict accumulation of sequence changes in the fusion peptide of HRSV F when compared with other hydrophobic regions of the molecule.


2009 ◽  
Vol 83 (8) ◽  
pp. 3568-3580 ◽  
Author(s):  
Mark L. Reed ◽  
Hui-Ling Yen ◽  
Rebecca M. DuBois ◽  
Olga A. Bridges ◽  
Rachelle Salomon ◽  
...  

ABSTRACT The receptor specificity and cleavability of the hemagglutinin (HA) protein have been shown to regulate influenza A virus transmissibility and pathogenicity, but little is known about how its pH of activation contributes to these important biological properties. To identify amino acid residues that regulate the acid stability of the HA protein of H5N1 influenza viruses, we performed a mutational analysis of the HA protein of the moderately pathogenic A/chicken/Vietnam/C58/04 (H5N1) virus. Nineteen HA proteins containing point mutations in the HA2 coiled-coil domain or in an HA1 histidine or basic patch were generated. Wild-type and mutant HA plasmids were transiently transfected in cell culture and analyzed for total protein expression, surface expression, cleavage efficiency, pH of fusion, and pH of conformational change. Four mutations to residues in the fusion peptide pocket, Y23H and H24Q in the HA1 subunit and E105K and N114K in the HA2 subunit, and a K58I mutation in the HA2 coiled-coil domain significantly altered the pH of activation of the H5 HA protein. In some cases, the magnitude and direction of changes of individual mutations in the H5 HA protein differed considerably from similar mutations in other influenza A virus HA subtypes. Introduction of Y23H, H24Q, K58I, and N114K mutations into recombinant viruses resulted in virus-expressed HA proteins with similar shifts in the pH of fusion. Overall, the data show that residues comprising the fusion peptide pocket are important in triggering pH-dependent activation of the H5 HA protein.


2007 ◽  
Vol 81 (7) ◽  
pp. 3130-3141 ◽  
Author(s):  
Laura E. Luque ◽  
Charles J. Russell

ABSTRACT During viral entry, the paramyxovirus fusion (F) protein fuses the viral envelope to a cellular membrane. Similar to other class I viral fusion glycoproteins, the F protein has two heptad repeat regions (HRA and HRB) that are important in membrane fusion and can be targeted by antiviral inhibitors. Upon activation of the F protein, HRA refolds from a spring-loaded, crumpled structure into a coiled coil that inserts a hydrophobic fusion peptide into the target membrane and binds to the HRB helices to form a fusogenic hairpin. To investigate how F protein conformational changes are regulated, we mutated in the Sendai virus F protein a highly conserved 10-residue sequence in HRA that undergoes major structural changes during protein refolding. Nine of the 15 mutations studied caused significant defects in F protein expression, processing, and fusogenicity. Conversely, the remaining six mutations enhanced the fusogenicity of the F protein, most likely by helping spring the HRA coil. Two of the residues that were neither located at “a” or “d” positions in the heptad repeat nor conserved among the paramyxoviruses were key regulators of the folding and fusion activity of the F protein, showing that residues not expected to be important in coiled-coil formation may play important roles in regulating membrane fusion. Overall, the data support the hypothesis that regions in the F protein that undergo dramatic changes in secondary and tertiary structure between the prefusion and hairpin conformations regulate F protein expression and activation.


Viruses ◽  
2020 ◽  
Vol 12 (2) ◽  
pp. 225 ◽  
Author(s):  
Mengdie Ye ◽  
Yixian Liao ◽  
Li Wu ◽  
Wenbao Qi ◽  
Namrta Choudhry ◽  
...  

Influenza A viruses (IAV) have been a major public health threat worldwide, and options for antiviral therapy become increasingly limited with the emergence of drug-resisting virus strains. New and effective anti-IAV drugs, especially for highly pathogenic influenza, with different modes of action, are urgently needed. The influenza virus glycoprotein hemagglutinin (HA) plays critical roles in the early stage of virus infection, including receptor binding and membrane fusion, making it a potential target for the development of anti-influenza drugs. In this study, we show that OA-10, a newly synthesized triterpene out of 11 oleanane-type derivatives, exhibited significant antiviral activity against four different subtypes of IAV (H1N1, H5N1, H9N2 and H3N2) replications in A549 cell cultures with EC50 ranging from 6.7 to 19.6 μM and a negligible cytotoxicity (CC50 > 640 μM). It inhibited acid-induced hemolysis in a dose-dependent manner, with an IC50 of 26 µM, and had a weak inhibition on the adsorption of H5 HA to chicken erythrocytes at higher concentrations (≥40 µM). Surface plasmon resonance (SPR) analysis showed that OA-10 interacted with HA in a dose-dependent manner with the equilibrium dissociation constants (KD) of the interaction of 2.98 × 10−12 M. Computer-aided molecular docking analysis suggested that OA-10 might bind to the cavity in HA stem region which is known to undergo significant rearrangement during membrane fusion. Our results demonstrate that OA-10 inhibits H5N1 IAV replication mainly by blocking the conformational changes of HA2 subunit required for virus fusion with endosomal membrane. These findings suggest that OA-10 could serve as a lead for further development of novel virus entry inhibitors to prevent and treat IAV infections.


2021 ◽  
Author(s):  
Xiaoyu Sun ◽  
Chunyan Yi ◽  
Yuanfei Zhu ◽  
Longfei Ding ◽  
Shuai Xia ◽  
...  

Abstract The recurrent outbreak of coronaviruses and variants underscores the need for broadly reactive antivirals and vaccines. Here, a novel broad-spectrum human antibody named 76E1 was isolated from a COVID-19 convalescent patient and showed broad neutralization activity against multiple α- and β-coronaviruses, including the SARS-CoV-2 variants and also exhibited the binding breath to peptides containing the epitope from γ- and δ- coronaviruses. 76E1 cross-protects mice from SARS-CoV-2 and HCoV-OC43 infection in both prophylactic and treatment models. The epitope including the fusion peptide and S2’ cleavage site recognized by 76E1 was significantly conserved among α-, β-, γ- and δ- coronaviruses. We uncovered a novel mechanism of antibody neutralization that the epitope of 76E1 was proportionally less exposed in the prefusion trimeric structure of spike protein but could be unmasked by binding to the receptor ACE2. Once the epitope exposed, 76E1 inhibited S2’ cleavage, thus blocked the membrane fusion process. Our data demonstrate a key epitope targeted by broadly-neutralizing antibodies and will guide next-generation epitope-based pan-coronavirus vaccine design.


2008 ◽  
Vol 82 (13) ◽  
pp. 6337-6348 ◽  
Author(s):  
Zhu-Nan Li ◽  
Byeong-Jae Lee ◽  
William A. Langley ◽  
Konrad C. Bradley ◽  
Rupert J. Russell ◽  
...  

ABSTRACT During membrane fusion, the influenza A virus hemagglutinin (HA) adopts an extended helical structure that contains the viral transmembrane and fusion peptide domains at the same end of the molecule. The peptide segments that link the end of this rod-like structure to the membrane-associating domains are approximately 10 amino acids in each case, and their structure at the pH of fusion is currently unknown. Here, we examine mutant HAs and influenza viruses containing such HAs to determine whether these peptide linkers are subject to specific length requirements for the proper folding of native HA and for membrane fusion function. Using pairwise deletions and insertions, we show that the region flanking the fusion peptide appears to be important for the folding of the native HA structure but that mutant proteins with small insertions can be expressed on the cell surface and are functional for membrane fusion. HA mutants with deletions of up to 10 residues and insertions of as many as 12 amino acids were generated for the peptide linker to the viral transmembrane domain, and all folded properly and were expressed on the cell surface. For these mutants, it was possible to designate length restrictions for efficient membrane fusion, as functional activity was observed only for mutants containing linkers with insertions or deletions of eight residues or less. The linker peptide mutants are discussed with respect to requirements for the folding of native HAs and length restrictions for membrane fusion activity.


2005 ◽  
Vol 79 (7) ◽  
pp. 4533-4539 ◽  
Author(s):  
Kirilee A. Wilson ◽  
Séverine Bär ◽  
Anne L. Maerz ◽  
Marc Alizon ◽  
Pantelis Poumbourios

ABSTRACT Retroviral transmembrane proteins (TMs) contain an N-terminal fusion peptide that initiates virus-cell membrane fusion. The fusion peptide is linked to the coiled-coil core through a conserved sequence that is often rich in glycines. We investigated the functional role of the glycine-rich segment, Met-326 to Ser-337, of the human T-cell leukemia virus type 1 (HTLV-1) TM, gp21, by alanine and proline scanning mutagenesis. Alanine substitution for the hydrophobic residue Ile-334 caused an ∼90% reduction in cell-cell fusion activity without detectable effects on the lipid-mixing and pore formation phases of fusion. Alanine substitutions at other positions had smaller effects (Gly-329, Val-330, and Gly-332) or no effect on fusion function. Proline substitution for glycine residues inhibited cell-cell fusion function with position-dependent effects on the three phases of fusion. Retroviral glycoprotein fusion function thus appears to require flexibility within the glycine-rich segment and hydrophobic contacts mediated by this segment.


2020 ◽  
Author(s):  
Avijit Sardar ◽  
Aritraa Lahiri ◽  
Amirul Islam Mallick ◽  
Pradip Kumar Tarafdar

The entry of enveloped viruses requires fusion of viral and host cell membranes. An effective fusion inhibitor aiming at impeding such virus-host cell membrane fusion may emerge as a broad-spectrum antiviral agent to neutralize the infection from an increasing diversity of harmful new viruses. Mycobacterium survives inside the phagosome of the host cells by inhibiting phagosome-lysosome fusion with the help of a coat protein coronin 1. Structural analysis of coronin 1 and other WD40-repeat containing protein suggest that the tryptophan-aspartic acid (WD) sequence is placed at distorted β-meander motif (more exposed) whereas the WD resides in regular β-meander motif in other WD40 proteins. The unique structural feature of coronin 1 was explored to identify a simple lipo-peptide sequence (lipid-WD), which effectively inhibit the membrane fusion by increasing interfacial order and decreasing water penetration, surface potential. The effective fusion inhibitory role of mycobacterium inspired lipo-dipeptide was applied to combat type 1 influenza virus (H1N1) infection as a ‘broad spectrum’ antiviral agent.<br>


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