scholarly journals Triterpenoids manipulate a broad range of virus-host fusion via wrapping the HR2 domain prevalent in viral envelopes

2018 ◽  
Vol 4 (11) ◽  
pp. eaau8408 ◽  
Author(s):  
Longlong Si ◽  
Kun Meng ◽  
Zhenyu Tian ◽  
Jiaqi Sun ◽  
Huiqiang Li ◽  
...  
Keyword(s):  
Influenza A ◽  
Viral Envelope ◽  
Heptad Repeat ◽  
Viral Fusion ◽  
Influenza A Viruses ◽  
Fusion Inhibitors ◽  
Distinct Structure ◽  
Viral Envelopes ◽  
Hydrophobic Helix ◽  
Insight Into

A trimer-of-hairpins motif has been identified in triggering virus-cell fusion within a variety of viral envelopes. Chemically manipulating such a motif represents current repertoire of viral fusion inhibitors. Here, we report that triterpenoids, a class of natural products, antagonize this trimer-of-hairpins via its constitutive heptad repeat-2 (HR2), a prevalent α-helical coil in class I viral fusion proteins. Triterpenoids inhibit the entry of Ebola, Marburg, HIV, and influenza A viruses with distinct structure-activity relationships. Specifically, triterpenoid probes capture the viral envelope via photocrosslinking HR2. Profiling the Ebola HR2-triterpenoid interactions using amino acid substitution, surface plasmon resonance, and nuclear magnetic resonance revealed six residues accessible to triterpenoids, leading to wrapping of the hydrophobic helix and blocking of the HR1-HR2 interaction critical in the trimer-of-hairpins formation. This finding was also observed in the envelopes of HIV and influenza A viruses and might potentially extend to a broader variety of viruses, providing a mechanistic insight into triterpenoid-mediated modulation of viral fusion.

scholarly journals Triterpenoids Manipulate a Broad Range of Virus-Host Fusion via Wrapping the HR2 Domain Prevalent in Viral Envelopes

2018 ◽  
Author(s):  
Longlong Si ◽  
Kun Meng ◽  
Zhenyu Tian ◽  
Ziwei Zhang ◽  
Veronica Soloveva ◽  
...  
Keyword(s):  
Influenza A ◽  
Antiviral Agents ◽  
Repeat Sequence ◽  
Viral Envelope ◽  
Heptad Repeat ◽  
Influenza A Viruses ◽  
Distinct Structure ◽  
Viral Envelopes ◽  
Multiple Conformations ◽  
Hydrophobic Helix

Recent years have witnessed a breakthrough in identification of a trimer-of-hairpins motif within viral envelopes that triggers a broad range of virus-host fusion. Identifying a domain capable of controlling virus-host fusion remains a challenge due to sequence diversity, heavy glycan shielding and multiple conformations. Here, we report that HR2, a prevalent heptad repeat sequence comprising an alpha-helical coil anchored in viral membranes, is an accessible site to triterpenes, a class of widely distributed natural products. Triterpenes and their derivatives inhibit the entry of Ebola, HIV, and influenza A viruses with distinct structure-activity relationships. Specifically, triterpenoid probes, upon activation by ultraviolet light, capture the viral envelope via crosslinking the HR2 coil. Profiling the Ebola HR2 sequence using amino acid substitution, surface plasmon resonance (SPR) and nuclear magnetic resonance (NMR) spectroscopy disclosed six constitutive residues that are accessible to triterpenoids, leading to wrapping of the hydrophobic helix by triterpenoids and blocking of the HR1-HR2 interaction, which is critical in the trimer-of-hairpins formation. This finding was also observed in the envelopes of HIV and influenza A viruses and might potentially extend to a broader variety of viruses. Our findings might translate into a shared mechanism that host utilize natural product triterpenoids to antagonize membrane fusion of respective viruses, complementing the current repertoire of antiviral agents.

scholarly journals Structural and Functional Characterization of the Secondary Mutation N126K Selected by Various HIV-1 Fusion Inhibitors

Viruses ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 326
Author(s):  
Danwei Yu ◽  
Yang Su ◽  
Xiaohui Ding ◽  
Yuanmei Zhu ◽  
Bo Qin ◽  
...  
Keyword(s):  
Cell Fusion ◽  
Functional Characterization ◽  
Glycosylation Site ◽  
Heptad Repeat ◽  
Viral Fusion ◽  
Secondary Mutation ◽  
Fusion Inhibitors ◽  
Hiv 1

Peptides derived from the C-terminal heptad repeat (CHR) region of HIV-1 gp41 is potent viral membrane fusion inhibitors, such as the first clinically approved peptide drug T20 and a group of newly-designed peptides. The resistance profiles of various HIV-1 fusion inhibitors were previously characterized, and the secondary mutation N126K in the gp41 CHR was routinely identified during the in vitro and in vivo selections. In this study, the functional and structural relevance of the N126K mutation has been characterized from multiple angles. First, we show that a single N126K mutation across several HIV-1 isolates conferred mild to moderate cross-resistances. Second, the N126K mutation exerted different effects on Env-mediated HIV-1 entry and cell-cell fusion. Third, the N126K mutation did not interfere with the expression and processing of viral Env glycoproteins, but it disrupted the Asn126-based glycosylation site in gp41. Fourth, the N126K mutation was verified to enhance the thermal stability of 6-HB conformation. Fifth, we determined the crystal structure of a 6-HB bearing the N126K mutation, which revealed the interhelical and intrahelical interactions underlying the increased thermostability. Therefore, our data provide new information to understand the mechanism of HIV-1 gp41-mediated cell fusion and its resistance mode to viral fusion inhibitors.

scholarly journals pH Optimum of Hemagglutinin-Mediated Membrane Fusion Determines Sensitivity of Influenza A Viruses to the Interferon-Induced Antiviral State and IFITMs

2017 ◽  
Vol 91 (11) ◽  
Author(s):  
Thomas Gerlach ◽  
Luca Hensen ◽  
Tatyana Matrosovich ◽  
Janina Bergmann ◽  
Michael Winkler ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Membrane Fusion ◽  
Influenza A ◽  
Target Cells ◽  
Viral Fusion ◽  
Influenza A Viruses ◽  
Ph Optimum ◽  
High Ph ◽  
Innate Defense ◽  
Antiviral State

ABSTRACT The replication and pathogenicity of influenza A viruses (IAVs) critically depend on their ability to tolerate the antiviral interferon (IFN) response. To determine a potential role for the IAV hemagglutinin (HA) in viral sensitivity to IFN, we studied the restriction of IAV infection in IFN-β-treated human epithelial cells by using 2:6 recombinant IAVs that shared six gene segments of A/Puerto Rico/8/1934 virus (PR8) and contained HAs and neuraminidases of representative avian, human, and zoonotic H5N1 and H7N9 viruses. In A549 and Calu-3 cells, viruses displaying a higher pH optimum of HA-mediated membrane fusion, H5N1-PR8 and H7N9-PR8, were less sensitive to the IFN-induced antiviral state than their counterparts with HAs from duck and human viruses, which fused at a lower pH. The association between a high pH optimum of fusion and reduced IFN sensitivity was confirmed by using HA point mutants of A/Hong Kong/1/1968-PR8 that differed solely by their fusion properties. Furthermore, similar effects of the viral fusion pH on IFN sensitivity were observed in experiments with (i) primary human type II alveolar epithelial cells and differentiated cultures of human airway epithelial cells, (ii) nonrecombinant zoonotic and pandemic IAVs, and (iii) preparations of IFN-α and IFN-λ1. A higher pH of membrane fusion and reduced sensitivity to IFN correlated with lower restriction of the viruses in MDCK cells stably expressing the IFN-inducible transmembrane proteins IFITM2 and IFITM3, which are known to inhibit viral fusion. Our results reveal that the pH optimum of HA-driven membrane fusion of IAVs is a determinant of their sensitivity to IFN and IFITM proteins. IMPORTANCE The IFN system constitutes an important innate defense against viral infection. Substantial information is available on how IAVs avoid detection by sensors of the IFN system and disable IFN signaling pathways. Much less is known about the ability of IAVs to tolerate the antiviral activity of IFN-induced cellular proteins. The IFN-induced proteins of the IFITM family block IAV entry into target cells and can restrict viral spread and pathogenicity. Here we show for the first time that the sensitivity of IAVs to the IFN-induced antiviral state and IFITM2 and IFITM3 proteins depends on the pH value at which the viral HA undergoes a conformational transition and mediates membrane fusion. Our data imply that the high pH optimum of membrane fusion typical of zoonotic IAVs of gallinaceous poultry, such as H5N1 and H7N9, may contribute to their enhanced virulence in humans.

scholarly journals Mutations flanking the carbohydrate binding site of surfactant protein D confer antiviral activity for pandemic influenza A viruses

2014 ◽  
Vol 306 (11) ◽  
pp. L1036-L1044 ◽  
Author(s):  
Nikolaos M. Nikolaidis ◽  
Mitchell R. White ◽  
Kimberly Allen ◽  
Shweta Tripathi ◽  
Li Qi ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Host Defense ◽  
Influenza A ◽  
Surfactant Protein ◽  
Viral Envelope ◽  
Surfactant Protein D ◽  
Carbohydrate Binding ◽  
Influenza A Viruses ◽  
Head Region ◽  
Pandemic Strains

We recently reported that a trimeric neck and carbohydrate recognition domain (NCRD) fragment of human surfactant protein D (SP-D), a host defense lectin, with combinatorial substitutions at the 325 and 343 positions (D325A+R343V) exhibits markedly increased antiviral activity for seasonal strains of influenza A virus (IAV). The NCRD binds to glycan-rich viral envelope proteins including hemagglutinin (HA). We now show that replacement of D325 with serine to create D325S+R343V provided equal or increased neutralizing activity compared with D325A+R343V. The activity of the double mutants was significantly greater than that of either single mutant (D325A/S or R343V). D325A+R343V and D325S+R343V also strongly inhibited HA activity, and markedly aggregated, the 1968 pandemic H3N2 strain, Aichi68. D325S+R343V significantly reduced viral loads and mortality of mice infected with Aichi68, whereas wild-type SP-D NCRD did not. The pandemic H1N1 strains of 1918 and 2009 have only one N-linked glycan side on the head region of the HA and are fully resistant to inhibition by native SP-D. Importantly, we now show that D325A+R343V and D325S+R343V inhibited Cal09 H1N1 and related strains, and reduced uptake of Cal09 by epithelial cells. Inhibition of Cal09 was mediated by the lectin activity of the NCRDs. All known human pandemic strains have at least one glycan attachment on the top or side of the HA head, and our results indicate that they may be susceptible to inhibition by modified host defense lectins.

scholarly journals Electron tomography visualization of HIV-1 fusion with target cells using fusion inhibitors to trap the pre-hairpin intermediate

2020 ◽  
Author(s):  
Mark S. Ladinsky ◽  
Priyanthi N.P. Gnanapragasam ◽  
Zhi Yang ◽  
Anthony P. West ◽  
Michael S Kay ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Intermediate Stage ◽  
Viral Envelope ◽  
Host Cells ◽  
Target Cells ◽  
Virus Infectivity ◽  
Viral Fusion ◽  
Fusion Inhibitors ◽  
Viral Egress ◽  
Hiv 1

AbstractFusion of HIV-1 with the membrane of its target cell, an obligate first step in virus infectivity, is mediated by binding of the viral envelope (Env) spike protein to its receptors, CD4 and CCR5/CXCR4, on the cell surface. The process of viral fusion appears to be fast compared with viral egress and has not been visualized by electron microscopy (EM). To capture fusion events for EM, the process must be slowed or stopped by trapping Env-receptor binding at an intermediate stage. Here we describe using fusion inhibitors to trap HIV-1 virions attached to target cells by Envs in an extended pre-hairpin intermediate state. Electron tomography revealed HIV-1 virions bound to TZM-bl cells by 2-4 narrow spokes, with slightly more spokes present when evaluated with mutant virions that lacked the Env cytoplasmic tail. These results represent the first direct visualization of the hypothesized pre-hairpin intermediate and improve our understanding of Env-mediated HIV-1 fusion and infection of host cells.

scholarly journals Discovery of New Potent anti-MERS CoV Fusion Inhibitors

2021 ◽  
Vol 12 ◽  
Author(s):  
Mahmoud Kandeel ◽  
Mizuki Yamamoto ◽  
Byoung Kwon Park ◽  
Abdulla Al-Taher ◽  
Aya Watanabe ◽  
...  
Keyword(s):  
Antiviral Agents ◽  
Heptad Repeat ◽  
Viral Fusion ◽  
Binding Affinities ◽  
Strong Inhibition ◽  
Wild Type ◽  
Fusion Inhibitors ◽  
Repeat Domain ◽  
Ic50 Values ◽  
Nanomolar Range

Middle East respiratory syndrome coronavirus (MERS-CoV), capable of zoonotic transmission, has been associated with emerging viral pneumonia in humans. In this study, a set of highly potent peptides were designed to prevent MERS-CoV fusion through competition with heptad repeat domain 2 (HR2) at its HR1 binding site. We designed eleven peptides with stronger estimated HR1 binding affinities than the wild-type peptide to prevent viral fusion with the cell membrane. Eight peptides showed strong inhibition of spike-mediated MERS-CoV cell-cell fusion with IC50 values in the nanomolar range (0.25–2.3 µM). Peptides #4–6 inhibited 95–98.3% of MERS-CoV plaque formation. Notably, peptide four showed strong inhibition of MERS-CoV plaques formation with EC50 = 0.302 µM. All peptides demonstrated safe profiles without cytotoxicity up to a concentration of 10 μM, and this cellular safety, combined with their anti-MERS-CoV antiviral activity, indicate all peptides can be regarded as potential promising antiviral agents.

scholarly journals Kinetic Dependence of Paramyxovirus Entry Inhibition

2009 ◽  
Vol 83 (13) ◽  
pp. 6947-6951 ◽  
Author(s):  
Matteo Porotto ◽  
Christine C. Yokoyama ◽  
Gianmarco Orefice ◽  
Han-Sung Kim ◽  
Mohamed Aljofan ◽  
...  
Keyword(s):  
Heptad Repeat ◽  
Viral Fusion ◽  
Attachment Protein ◽  
Kinetic Dependence ◽  
Inhibitory Peptides ◽  
Fusion Inhibitors ◽  
Intermediate States ◽  
Kinetics Of ◽  
Entry Inhibition ◽  
Bundle Structure

ABSTRACT Peptides derived from conserved heptad repeat (HR) regions of paramyxovirus fusion (F) proteins inhibit viral fusion by interfering with the formation of the fusogenic six-helix bundle structure. Peptide efficacy is affected by the strength of the peptide association with the target virus's complementary HR region. Here, we show that a second basis for peptide efficacy lies in the kinetics of F activation by the homotypic attachment protein: efficient F activation by the attachment protein shortens the period during which antiviral molecules targeting intermediate states of F may act, thereby modulating the effectiveness of inhibitory peptides. These results highlight new issues to be considered in developing strategies for fusion inhibitors.

scholarly journals Identification of Novel Fusion Inhibitors of Influenza A Virus by Chemical Genetics

2015 ◽  
Vol 90 (5) ◽  
pp. 2690-2701 ◽  
Author(s):  
Kin Kui Lai ◽  
Nam Nam Cheung ◽  
Fang Yang ◽  
Jun Dai ◽  
Li Liu ◽  
...  
Keyword(s):  
Molecular Docking ◽  
Influenza A Virus ◽  
Influenza A ◽  
Chemical Genetics ◽  
Docking Studies ◽  
Influenza A Viruses ◽  
Fusion Inhibitors ◽  
Close Proximity ◽  
Binding Pockets ◽  
Group 1

ABSTRACTA previous screening of more than 50,000 compounds led to the identification of a pool of bioactive small molecules with inhibitory effect on the influenza A virus. One of these compounds, now widely known as nucleozin, is a small molecule that targets the influenza A virus nucleoprotein. Here we identify and characterize two structurally different novel fusion inhibitors of the influenza A virus group 1 hemagglutinin (HA), FA-583 and FA-617, with low nanomolar activities. Escape mutants that are highly resistant to each of these compounds were generated, and both were found to carry mutations localized in close proximity to the B-loop of the hemagglutinin 2 protein, which plays a crucial role in the virion-host cell fusion process. Recombinant virus, generated through reverse genetics, confirmed the resistance phenotype. In addition, the proposed binding pockets predicted by molecular docking studies are in accordance with the resistance-bearing mutation sites. We show through mechanistic studies that FA-583 and FA-617 act as fusion inhibitors by prohibiting the low-pH-induced conformational change of hemagglutinin. Our study has offered concrete biological and mechanistic explorations for the strategic development of novel fusion inhibitors of influenza A viruses.IMPORTANCEHere we report two structurally distinctive novel fusion inhibitors of influenza A virus that act by interfering with the structural change of HA at acidic pH, a process necessary for successful entry of the virus. Mutational and molecular docking studies have identified their binding pockets situated in close proximity to the B-loop region of hemagglutinin 2. The reduced sensitivity of FA-583- or FA-617-associated mutants to another compound suggests a close proximity and even partial overlap of their binding sites on hemagglutinin. Amino acid sequence alignments and crystal structure analyses of group 1 and group 2 hemagglutinins have shed light on the possible binding mode of these two compounds. This report offers new lead compounds for the design of fusion inhibitors for influenza A viruses and further shows that analysis by forward chemical genetics is a highly effective approach for the identification of novel compounds that can perturb the infectivity of viruses and to probe new druggable targets or druggable domains in various viruses.

scholarly journals Examination of a Fusogenic Hexameric Core from Human Metapneumovirus and Identification of a Potent Synthetic Peptide Inhibitor from the Heptad Repeat 1 Region

2006 ◽  
Vol 81 (1) ◽  
pp. 141-149 ◽  
Author(s):  
Scott A. Miller ◽  
Sharon Tollefson ◽  
James E. Crowe ◽  
John V. Williams ◽  
David W. Wright
Keyword(s):  
Membrane Proteins ◽  
Coiled Coil ◽  
Helical Structure ◽  
Human Metapneumovirus ◽  
Integral Membrane Proteins ◽  
Heptad Repeat ◽  
Type I ◽  
Viral Fusion ◽  
Fusion Inhibitors ◽  
Major Player

ABSTRACT Paramyxoviruses are a leading cause of childhood illness worldwide. A recently discovered paramyxovirus, human metapneumovirus (hMPV), has been studied by our group in order to determine the structural relevance of its fusion (F) protein to other well-characterized viruses utilizing type I integral membrane proteins as fusion aids. Sequence analysis and homology models suggested the presence of requisite heptad repeat (HR) regions. Synthetic peptides from HR regions 1 and 2 (HR-1 and -2, respectively) were induced to form a thermostable (melting temperature, ∼90°C) helical structure consistent in mass with a hexameric coiled coil. Inhibitory studies of hMPV HR-1 and -2 indicated that the synthetic HR-1 peptide was a significant fusion inhibitor with a 50% inhibitory concentration and a 50% effective concentration of ∼50 nM. Many viral fusion proteins are type I integral membrane proteins utilizing the formation of a hexameric coiled coil of HR peptides as a major driving force for fusion. Our studies provide evidence that hMPV also uses a coiled-coil structure as a major player in the fusion process. Additionally, viral HR-1 peptide sequences may need further investigation as potent fusion inhibitors.

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