scholarly journals Mutations in the Fusion Protein of Measles Virus That Confer Resistance to the Membrane Fusion Inhibitors Carbobenzoxy-d-Phe-l-Phe-Gly and 4-Nitro-2-Phenylacetyl Amino-Benzamide

2017 ◽  
Vol 91 (23) ◽  
Author(s):  
Michael N. Ha ◽  
Sébastien Delpeut ◽  
Ryan S. Noyce ◽  
Gary Sisson ◽  
Karen M. Black ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Cell Lines ◽  
Measles Virus ◽  
Structural Changes ◽  
3D Structure ◽  
Syncytium Formation ◽  
Heptad Repeat ◽  
Fusion Inhibitors ◽  
Site Of Action

ABSTRACT The inhibitors carbobenzoxy (Z)-d-Phe-l-Phe-Gly (fusion inhibitor peptide [FIP]) and 4-nitro-2-phenylacetyl amino-benzamide (AS-48) have similar efficacies in blocking membrane fusion and syncytium formation mediated by measles virus (MeV). Other homologues, such as Z-d-Phe, are less effective but may act through the same mechanism. In an attempt to map the site of action of these inhibitors, we generated mutant viruses that were resistant to the inhibitory effects of Z-d-Phe-l-Phe-Gly. These 10 mutations were localized to the heptad repeat B (HRB) region of the fusion protein, and no changes were observed in the viral hemagglutinin, which is the receptor attachment protein. Mutations were validated in a luciferase-based membrane fusion assay, using transfected fusion and hemagglutinin expression plasmids or with syncytium-based assays in Vero, Vero-SLAM, and Vero-Nectin 4 cell lines. The changes I452T, D458N, D458G/V459A, N462K, N462H, G464E, and I483R conferred resistance to both FIP and AS-48 without compromising membrane fusion. The inhibitors did not block hemagglutinin protein-mediated binding to the target cell. Edmonston vaccine/laboratory and IC323 wild-type strains were equally affected by the inhibitors. Escape mutations were mapped upon a three-dimensional (3D) structure modeled from the published crystal structure of parainfluenzavirus 5 fusion protein. The most effective mutations were situated in a region located near the base of the globular head and its junction with the alpha-helical stalk of the prefusion protein. We hypothesize that the fusion inhibitors could interfere with the structural changes that occur between the prefusion and postfusion conformations of the fusion protein. IMPORTANCE Due to lapses in vaccination worldwide that have caused localized outbreaks, measles virus (MeV) has regained importance as a pathogen. Antiviral agents against measles virus are not commercially available but could be useful in conjunction with MeV eradication vaccine programs and as a safeguard in oncolytic viral therapy. Three decades ago, the small hydrophobic peptide Z-d-Phe-l-Phe-Gly (FIP) was shown to block MeV infections and syncytium formation in monkey kidney cell lines. The exact mechanism of its action has yet to be determined, but it does appear to have properties similar to those of another chemical inhibitor, AS-48, which appears to interfere with the conformational change in the viral F protein that is required to elicit membrane fusion. Escape mutations were used to map the site of action for FIP. Knowledge gained from these studies could help in the design of new inhibitors against morbilliviruses and provide additional knowledge concerning the mechanism of virus-mediated membrane fusion.

scholarly journals Design of Potent Membrane Fusion Inhibitors against SARS-CoV-2, an Emerging Coronavirus with High Fusogenic Activity

2020 ◽  
Vol 94 (14) ◽  
Author(s):  
Yuanmei Zhu ◽  
Danwei Yu ◽  
Hongxia Yan ◽  
Huihui Chong ◽  
Yuxian He
Keyword(s):  
Public Health ◽  
Severe Acute Respiratory Syndrome ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Heptad Repeat ◽  
Global Public Health ◽  
S Protein ◽  
Fusion Inhibitors ◽  
Cell Cell

ABSTRACT The 2019 coronavirus disease (COVID-19), caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has posed serious threats to global public health and economic and social stabilities, calling for the prompt development of therapeutics and prophylactics. In this study, we first verified that SARS-CoV-2 uses human angiotensin-converting enzyme 2 (ACE2) as a cell receptor and that its spike (S) protein mediates high membrane fusion activity. The heptad repeat 1 (HR1) sequence in the S2 fusion protein of SARS-CoV-2 possesses markedly increased α-helicity and thermostability, as well as a higher binding affinity with its corresponding heptad repeat 2 (HR2) site, than the HR1 sequence in S2 of severe acute respiratory syndrome coronavirus (SARS-CoV). Then, we designed an HR2 sequence-based lipopeptide fusion inhibitor, termed IPB02, which showed highly potent activities in inhibiting SARS-CoV-2 S protein-mediated cell-cell fusion and pseudovirus transduction. IPB02 also inhibited the SARS-CoV pseudovirus efficiently. Moreover, the structure-activity relationship (SAR) of IPB02 was characterized with a panel of truncated lipopeptides, revealing the amino acid motifs critical for its binding and antiviral capacities. Therefore, the results presented here provide important information for understanding the entry pathway of SARS-CoV-2 and the design of antivirals that target the membrane fusion step. IMPORTANCE The COVID-19 pandemic, caused by SARS-CoV-2, presents a serious global public health emergency in urgent need of prophylactic and therapeutic interventions. The S protein of coronaviruses mediates viral receptor binding and membrane fusion, thus being considered a critical target for antivirals. Herein, we report that the SARS-CoV-2 S protein has evolved a high level of activity to mediate cell-cell fusion, significantly differing from the S protein of SARS-CoV that emerged previously. The HR1 sequence in the fusion protein of SARS-CoV-2 adopts a much higher helical stability than the HR1 sequence in the fusion protein of SARS-CoV and can interact with the HR2 site to form a six-helical bundle structure more efficiently, underlying the mechanism of the enhanced fusion capacity. Also, importantly, the design of membrane fusion inhibitors with high potencies against both SARS-CoV-2 and SARS-CoV has provided potential arsenals to combat the pandemic and tools to exploit the fusion mechanism.

scholarly journals A Functional F Analogue of Autographa californica Nucleopolyhedrovirus GP64 from the Agrotis segetum Granulovirus

2008 ◽  
Vol 82 (17) ◽  
pp. 8922-8926 ◽  
Author(s):  
Feifei Yin ◽  
Manli Wang ◽  
Ying Tan ◽  
Fei Deng ◽  
Just M. Vlak ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Plutella Xylostella ◽  
Syncytium Formation ◽  
Low Ph ◽  
Autographa Californica ◽  
Agrotis Segetum ◽  
Induced Membrane ◽  
F Protein ◽  
Autographa Californica Nucleopolyhedrovirus

ABSTRACT The envelope fusion protein F of Plutella xylostella granulovirus is a computational analogue of the GP64 envelope fusion protein of Autographa californica nucleopolyhedrovirus (AcMNPV). Granulovirus (GV) F proteins were thought to be unable to functionally replace GP64 in the AcMNPV pseudotyping system. In the present study the F protein of Agrotis segetum GV (AgseGV) was identified experimentally as the first functional GP64 analogue from GVs. AgseF can rescue virion propagation and infectivity of gp64-null AcMNPV. The AgseF-pseudotyped AcMNPV also induced syncytium formation as a consequence of low-pH-induced membrane fusion.

scholarly journals Prevention of Measles Virus Infection by Intranasal Delivery of Fusion Inhibitor Peptides

2014 ◽  
Vol 89 (2) ◽  
pp. 1143-1155 ◽  
Author(s):  
C. Mathieu ◽  
D. Huey ◽  
E. Jurgens ◽  
J. C. Welsch ◽  
I. DeVito ◽  
...  
Keyword(s):  
Animal Models ◽  
Measles Virus ◽  
Structural Transition ◽  
Specific Treatment ◽  
Heptad Repeat ◽  
Receptor Interaction ◽  
Target Cells ◽  
Inhibitory Peptides ◽  
Fusion Inhibitors

ABSTRACTMeasles virus (MV) infection is undergoing resurgence and remains one of the leading causes of death among young children worldwide despite the availability of an effective measles vaccine. MV infects its target cells by coordinated action of the MV H and the fusion (F) envelope glycoprotein; upon receptor engagement by H, the prefusion F undergoes a structural transition, extending and inserting into the target cell membrane and then refolding into a postfusion structure that fuses the viral and cell membranes. By interfering with this structural transition of F, peptides derived from the heptad-repeat (HR) regions of F can potently inhibit MV infection at the entry stage. We show here that specific features of H's interaction with its receptors modulate the susceptibility of MV F to peptide fusion inhibitors. A higher concentration of inhibitory peptides is required to inhibit F-mediated fusion when H is engaged to its nectin-4 receptor than when H is engaged to its CD150 receptor. Peptide inhibition of F may be subverted by continued engagement of receptor by H, a finding that highlights the ongoing role of H-receptor interaction after F has been activated and that helps guide the design of more potent inhibitory peptides. Intranasal administration of these peptides results in peptide accumulation in the airway epithelium with minimal systemic levels of peptide and efficiently prevents MV infectionin vivoin animal models. The results suggest an antiviral strategy for prophylaxis in vulnerable and/or immunocompromised hosts.IMPORTANCEMeasles virus (MV) infection causes an acute illness that may be associated with infection of the central nervous system (CNS) and severe neurological disease. No specific treatment is available. We have shown that parenterally delivered fusion-inhibitory peptides protect mice from lethal CNS MV disease. Here we show, using established small-animal models of MV infection, that fusion-inhibitory peptides delivered intranasally provide effective prophylaxis against MV infection. Since the fusion inhibitors are stable at room temperature, this intranasal strategy is feasible even outside health care settings, could be used to protect individuals and communities in case of MV outbreaks, and could complement global efforts to control measles.

scholarly journals Cell-to-Cell Measles Virus Spread between Human Neurons Is Dependent on Hemagglutinin and Hyperfusogenic Fusion Protein

2018 ◽  
Vol 92 (6) ◽  
Author(s):  
Yuma Sato ◽  
Shumpei Watanabe ◽  
Yoshinari Fukuda ◽  
Takao Hashiguchi ◽  
Yusuke Yanagi ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Measles Virus ◽  
Subacute Sclerosing Panencephalitis ◽  
Acute Infection ◽  
Syncytium Formation ◽  
Wild Type ◽  
F Protein ◽  
Human Neurons ◽  
A Cell ◽  
Nt2 Neurons

ABSTRACTMeasles virus (MV) usually causes acute infection but in rare cases persists in the brain, resulting in subacute sclerosing panencephalitis (SSPE). Since human neurons, an important target affected in the disease, do not express the known MV receptors (signaling lymphocyte activation molecule [SLAM] and nectin 4), how MV infects neurons and spreads between them is unknown. Recent studies have shown that many virus strains isolated from SSPE patients possess substitutions in the extracellular domain of the fusion (F) protein which confer enhanced fusion activity. Hyperfusogenic viruses with such mutations, unlike the wild-type MV, can induce cell-cell fusion even in SLAM- and nectin 4-negative cells and spread efficiently in human primary neurons and the brains of animal models. We show here that a hyperfusogenic mutant MV, IC323-F(T461I)-EGFP (IC323 with a fusion-enhancing T461I substitution in the F protein and expressing enhanced green fluorescent protein), but not the wild-type MV, spreads in differentiated NT2 cells, a widely used human neuron model. Confocal time-lapse imaging revealed the cell-to-cell spread of IC323-F(T461I)-EGFP between NT2 neurons without syncytium formation. The production of virus particles was strongly suppressed in NT2 neurons, also supporting cell-to-cell viral transmission. The spread of IC323-F(T461I)-EGFP was inhibited by a fusion inhibitor peptide as well as by some but not all of the anti-hemagglutinin antibodies which neutralize SLAM- or nectin-4-dependent MV infection, suggesting the presence of a distinct neuronal receptor. Our results indicate that MV spreads in a cell-to-cell manner between human neurons without causing syncytium formation and that the spread is dependent on the hyperfusogenic F protein, the hemagglutinin, and the putative neuronal receptor for MV.IMPORTANCEMeasles virus (MV), in rare cases, persists in the human central nervous system (CNS) and causes subacute sclerosing panencephalitis (SSPE) several years after acute infection. This neurological complication is almost always fatal, and there is currently no effective treatment for it. Mechanisms by which MV invades the CNS and causes the disease remain to be elucidated. We have previously shown that fusion-enhancing substitutions in the fusion protein of MVs isolated from SSPE patients contribute to MV spread in neurons. In this study, we demonstrate that MV bearing the hyperfusogenic mutant fusion protein spreads between human neurons in a cell-to-cell manner. Spread of the virus was inhibited by a fusion inhibitor peptide and antibodies against the MV hemagglutinin, indicating that both the hemagglutinin and hyperfusogenic fusion protein play important roles in MV spread between human neurons. The findings help us better understand the disease process of SSPE.

scholarly journals Therapeutic Efficacy and Resistance Selection of a Lipopeptide Fusion Inhibitor in Simian Immunodeficiency Virus-Infected Rhesus Macaques

2020 ◽  
Vol 94 (15) ◽  
Author(s):  
Danwei Yu ◽  
Jing Xue ◽  
Huamian Wei ◽  
Zhe Cong ◽  
Ting Chen ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Therapeutic Efficacy ◽  
Rhesus Macaques ◽  
Heptad Repeat ◽  
Fusion Inhibitor ◽  
Resistance Mutations ◽  
Fusion Inhibitors ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT We recently reported a group of lipopeptide-based membrane fusion inhibitors with potent antiviral activities against human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). In this study, the in vivo therapeutic efficacy of such a lipopeptide, LP-52, was evaluated in rhesus macaques chronically infected with pathogenic SIVmac239. In a pilot study with one monkey, monotherapy with low-dose LP-52 rapidly reduced the plasma viral loads to below the limit of detection and maintained viral suppression during three rounds of structurally interrupted treatment. The therapeutic efficacy of LP-52 was further verified in four infected monkeys; however, three out of the monkeys had viral rebounds under the LP-52 therapy. We next focused on characterizing SIV mutants responsible for the in vivo resistance. Sequence analyses revealed that a V562A or V562M mutation in the N-terminal heptad repeat (NHR) and a E657G mutation in the C-terminal heptad repeat (CHR) of SIV gp41 conferred high resistance to LP-52 and cross-resistance to the peptide drug T20 and two newly designed lipopeptides (LP-80 and LP-83). Moreover, we showed that the resistance mutations greatly reduced the stability of diverse fusion inhibitors with the NHR site, and V562A or V562M in combination with E657G could significantly impair the functionality of viral envelopes (Envs) to mediate SIVmac239 infection and decrease the thermostability of viral six-helical bundle (6-HB) core structure. In conclusion, the present data have not only facilitated the development of novel anti-HIV drugs that target the membrane fusion step, but also help our understanding of the mechanism of viral evolution to develop drug resistance. IMPORTANCE The anti-HIV peptide drug T20 (enfuvirtide) is the only membrane fusion inhibitor available for treatment of viral infection; however, it exhibits relatively weak antiviral activity, short half-life, and a low genetic barrier to inducing drug resistance. Design of lipopeptide-based fusion inhibitors with extremely potent and broad antiviral activities against divergent HIV-1, HIV-2, and SIV isolates have provided drug candidates for clinical development. Here, we have verified a high therapeutic efficacy for the lipopeptide LP-52 in SIVmac239-infected rhesus monkeys. The resistance mutations selected in vivo have also been characterized, providing insights into the mechanism of action of newly designed fusion inhibitors with a membrane-anchoring property. For the first time, the data show that HIV-1 and SIV can share a similar genetic pathway to develop resistance, and that a lipopeptide fusion inhibitor could have a same resistance profile as its template peptide.

scholarly journals An effective strategy for recapitulating N-terminal heptad repeat trimers in enveloped virus surface glycoproteins for therapeutic applications

2016 ◽  
Vol 7 (3) ◽  
pp. 2145-2150 ◽  
Author(s):  
Wenqing Lai ◽  
Chao Wang ◽  
Fei Yu ◽  
Lu Lu ◽  
Qian Wang ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Membrane Fusion ◽  
Heptad Repeat ◽  
Effective Strategy ◽  
Virus Surface ◽  
Efficient Strategy ◽  
Therapeutic Applications ◽  
Enveloped Virus ◽  
Membrane Fusion Protein ◽  
Hiv 1

We report an efficient strategy to recapitulate NHR α-helical trimers in the HIV-1 membrane fusion protein as promising antiviral therapeutics.

scholarly journals Mutations in Pseudorabies Virus Glycoproteins gB, gD, and gH Functionally Compensate for the Absence of gL

2015 ◽  
Vol 90 (5) ◽  
pp. 2264-2272 ◽  
Author(s):  
Christina Schröter ◽  
Melina Vallbracht ◽  
Jan Altenschmidt ◽  
Sabrina Kargoll ◽  
Walter Fuchs ◽  
...  
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Pseudorabies Virus ◽  
Syncytium Formation ◽  
Single Amino Acid ◽  
Independent Experiment ◽  
Viral Glycoprotein ◽  
Bona Fide ◽  
Infectious Entry

ABSTRACTEntry of herpesviruses depends on the combined action of viral glycoprotein B (gB) and the heterodimeric gH/gL complex, which are activated by binding of the virion to specific cellular receptors. While gB carries signatures of a bona fide fusion protein, efficient membrane fusion requires gH/gL. However, although gB and gH/gL are essential for entry, the alphaherpesvirus pseudorabies virus (PrV) is capable of limited cell-to-cell spread in the absence of gL. To understand gH/gL function in more detail, the limited spread of PrV-ΔgL was used for reversion analyses by serial cell culture passages. In a first experiment, an infectious gL-negative mutant in which gL function was replaced by generation of a gD-gH hybrid protein was isolated (B. G. Klupp and T. C. Mettenleiter, J Virol 73:3014–3022, 1999). In a second, independent experiment PrV-ΔgLPassB4.1, which also replicated productively without gL, was isolated. Sequence analysis revealed mutations in gH but also in gB and gD. In a transfection-based fusion assay, two amino acid substitutions in the N-terminal part of gHB4.1(L70P and W103R) were found to be sufficient to compensate for lack of gL, while mutations present in gBB4.1enhanced fusogenicity. Coexpression of gBB4.1with the homologous gHB4.1resulted in strongly increased syncytium formation, which was further augmented by truncation of the gBB4.1C-terminal 29 amino acids. Nevertheless, gH was still required for membrane fusion. Surprisingly, coexpression of gDB4.1blocked syncytium formation in the fusion assays, which could be attributed to a V106A substitution within the ectodomain of gDB4.1.IMPORTANCEIn contrast to many other enveloped viruses, herpesviruses rely on the concerted action of four viral glycoproteins for membrane fusion during infectious entry. Although the highly conserved gB shows signatures of a fusion protein, for fusion induction it requires the gH/gL complex, whose role is still elusive. Here we demonstrated fusion activation by gH in the absence of gL after reversion analysis of gL-deleted pseudorabies virus. This gL-independent fusion activity depended on single amino acid exchanges affecting the gL-binding domain in gH, increasing fusogenicity in gB and allowing negative fusion regulation by gD. Thus, our results provide novel information on the interplay in the fusion machinery of herpesviruses.

scholarly journals Design of potent membrane fusion inhibitors against SARS-CoV-2, an emerging coronavirus with high fusogenic activity

2020 ◽  
Author(s):  
Yuanmei Zhu ◽  
Danwei Yu ◽  
Hongxia Yan ◽  
Huihui Chong ◽  
Yuxian He
Keyword(s):  
Membrane Fusion ◽  
Cell Receptor ◽  
Heptad Repeat ◽  
Fusion Inhibitor ◽  
Fusion Activity ◽  
Global Public Health ◽  
S Protein ◽  
Entry Pathway ◽  
Fusion Inhibitors ◽  
A Cell

AbstractThe coronavirus disease COVID-19, caused by emerging SARS-CoV-2, has posed serious threats to global public health, economic and social stabilities, calling for the prompt development of therapeutics and prophylactics. In this study, we firstly verified that SARS-CoV-2 uses human ACE2 as a cell receptor and its spike (S) protein mediates high membrane fusion activity. Comparing to that of SARS-CoV, the heptad repeat 1 (HR1) sequence in the S2 fusion protein of SARS-CoV-2 possesses markedly increased α-helicity and thermostability, as well as a higher binding affinity with its corresponding heptad repeat 2 (HR1) site. Then, we designed a HR2 sequence-based lipopeptide fusion inhibitor, termed IPB02, which showed highly poent activities in inibibiting the SARS-CoV-2 S protein-mediated cell-cell fusion and pseudovirus infection. IPB02 also inhibited the SARS-CoV pseudovirus efficiently. Moreover, the strcuture and activity relationship (SAR) of IPB02 were characterzized with a panel of truncated lipopeptides, revealing the amino acid motifs critical for its binding and antiviral capacities. Therefore, the presented results have provided important information for understanding the entry pathway of SARS-CoV-2 and the design of antivirals that target the membrane fusion step.

scholarly journals Residues in the Heptad Repeat A Region of the Fusion Protein Modulate the Virulence of Sendai Virus in Mice

2009 ◽  
Vol 84 (2) ◽  
pp. 810-821 ◽  
Author(s):  
Laura E. Luque ◽  
Olga A. Bridges ◽  
John N. Mason ◽  
Kelli L. Boyd ◽  
Allen Portner ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Sendai Virus ◽  
Syncytium Formation ◽  
Heptad Repeat ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
F Protein

ABSTRACT While the molecular basis of fusion (F) protein refolding during membrane fusion has been studied extensively in vitro, little is known about the biological significance of membrane fusion activity in parainfluenza virus replication and pathogenesis in vivo. Two recombinant Sendai viruses, F-L179V and F-K180Q, were generated that contain F protein mutations in the heptad repeat A region of the ectodomain, a region of the protein known to regulate F protein activation. In vitro, the F-L179V virus caused increased syncytium formation (cell-cell membrane fusion) yet had a rate of replication and levels of F protein expression and cleavage similar to wild-type virus. The F-K180Q virus had a reduced replication rate along with reduced levels of F protein expression, cleavage, and fusogenicity. In DBA/2 mice, the hyperfusogenic F-L179V virus induced greater morbidity and mortality than wild-type virus, while the attenuated F-K180Q virus was much less pathogenic. During the first week of infection, virus replication and inflammation in the lungs were similar for wild-type and F-L179V viruses. After approximately 1 week of infection, the clearance of F-L179V virus was delayed, and more extensive interstitial inflammation and necrosis were observed in the lungs, affecting entire lobes of the lungs and having significantly greater numbers of syncytial cell masses in alveolar spaces on day 10. On the other hand, the slower-growing F-K180Q virus caused much less extensive inflammation than wild-type virus, presumably due to its reduced replication rate, and did not cause observable syncytium formation in the lungs. Overall, the results show that residues in the heptad repeat A region of the F protein modulate the virulence of Sendai virus in mice by influencing both the spread and clearance of the virus and the extent and severity of inflammation. An understanding of how the F protein contributes to infection and inflammation in vivo may assist in the development of antiviral therapies against respiratory paramyxoviruses.

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