scholarly journals Important Role for the Transmembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Protein during Entry

2006 ◽  
Vol 80 (3) ◽  
pp. 1302-1310 ◽  
Author(s):  
Rene Broer ◽  
Bertrand Boson ◽  
Willy Spaan ◽  
François-Loïc Cosset ◽  
Jeroen Corver
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Membrane Fusion ◽  
Cell Fusion ◽  
Transmembrane Domain ◽  
Cytoplasmic Tail ◽  
Transmembrane Domains ◽  
Spike Protein ◽  
Fusion Activity ◽  
Wild Type ◽  
Cell Cell

ABSTRACT The spike protein (S) of severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for receptor binding and membrane fusion. It contains a highly conserved transmembrane domain that consists of three parts: an N-terminal tryptophan-rich domain, a central domain, and a cysteine-rich C-terminal domain. The cytoplasmic tail of S has previously been shown to be required for assembly. Here, the roles of the transmembrane and cytoplasmic domains of S in the infectivity and membrane fusion activity of SARS-CoV have been studied. SARS-CoV S-pseudotyped retrovirus (SARSpp) was used to measure S-mediated infectivity. In addition, the cell-cell fusion activity of S was monitored by a Renilla luciferase-based cell-cell fusion assay. Svsv-cyt, an S chimera with a cytoplasmic tail derived from vesicular stomatitis virus G protein (VSV-G), and Smhv-tmdcyt, an S chimera with the cytoplasmic and transmembrane domains of mouse hepatitis virus, displayed wild-type-like activity in both assays. Svsv-tmdcyt, a chimera with the cytoplasmic and transmembrane domains of VSV-G, was impaired in the SARSpp and cell-cell fusion assays, showing 3 to 25% activity compared to the wild type, depending on the assay and the cells used. Examination of the oligomeric state of the chimeric S proteins in SARSpp revealed that Svsv-tmdcyt trimers were less stable than wild-type S trimers, possibly explaining the lowered fusogenicity and infectivity.

scholarly journals The Transmembrane Domain and Cytoplasmic Tail of Herpes Simplex Virus Type 1 Glycoprotein H Play a Role in Membrane Fusion

2002 ◽  
Vol 76 (21) ◽  
pp. 10708-10716 ◽  
Author(s):  
Andrew Harman ◽  
Helena Browne ◽  
Tony Minson
Keyword(s):  
Amino Acid ◽  
Cell Fusion ◽  
Transmembrane Domain ◽  
Cytoplasmic Tail ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Glycoprotein H ◽  
Membrane Spanning ◽  
Simplex Virus ◽  
Cell Cell

ABSTRACT Herpes simplex virus glycoprotein H (gH) is one of the four virion envelope proteins which are required for virus entry and for cell-cell fusion in a transient system. In this report, the role of the transmembrane and cytoplasmic tail domains of gH in membrane fusion was investigated by generating chimeric constructs in which these regions were replaced with analogous domains from other molecules and by introducing amino acid substitutions within the membrane-spanning sequence. gH molecules which lack the authentic transmembrane domain or cytoplasmic tail were unable to mediate cell-cell fusion when coexpressed with gB, gD, and gL and were unable to rescue the infectivity of a gH-null virus as efficiently as a wild-type gH molecule. Many amino acid substitutions of specific amino acid residues within the transmembrane domain also affected cell-cell fusion, in particular, those introduced at a conserved glycine residue. Some gH mutants that were impaired in cell-cell fusion were nevertheless able to rescue the infectivity of a gH-negative virus, but these pseudotyped virions entered cells more slowly than wild-type virions. These results indicate that the fusion event mediated by the coexpression of gHL, gB, and gD in cells shares common features with the fusion of the virus envelope with the plasma membrane, they point to a likely role for the membrane-spanning and cytoplasmic tail domains of gH in both processes, and they suggest that a conserved glycine residue in the membrane-spanning sequence is crucial for efficient fusion.

scholarly journals Acquisition of Cell–Cell Fusion Activity by Amino Acid Substitutions in Spike Protein Determines the Infectivity of a Coronavirus in Cultured Cells

PLoS ONE ◽  
2009 ◽  
Vol 4 (7) ◽  
pp. e6130 ◽  
Author(s):  
Yoshiyuki Yamada ◽  
Xiao Bo Liu ◽  
Shou Guo Fang ◽  
Felicia P. L. Tay ◽  
Ding Xiang Liu
Keyword(s):  
Amino Acid ◽  
Cell Fusion ◽  
Cultured Cells ◽  
Spike Protein ◽  
Amino Acid Substitutions ◽  
Fusion Activity ◽  
Cell Cell

scholarly journals Polybasic KKR Motif in the Cytoplasmic Tail of Nipah Virus Fusion Protein Modulates Membrane Fusion by Inside-Out Signaling

2007 ◽  
Vol 81 (9) ◽  
pp. 4520-4532 ◽  
Author(s):  
Hector C. Aguilar ◽  
Kenneth A. Matreyek ◽  
Daniel Y. Choi ◽  
Claire Marie Filone ◽  
Sophia Young ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Cell Fusion ◽  
Viral Entry ◽  
Nipah Virus ◽  
Cytoplasmic Tail ◽  
Wild Type ◽  
Strong Negative Correlation ◽  
Virus Fusion ◽  
Inside Out ◽  
Cell Cell

ABSTRACT The cytoplasmic tails of the envelope proteins from multiple viruses are known to contain determinants that affect their fusogenic capacities. Here we report that specific residues in the cytoplasmic tail of the Nipah virus fusion protein (NiV-F) modulate its fusogenic activity. Truncation of the cytoplasmic tail of NiV-F greatly inhibited cell-cell fusion. Deletion and alanine scan analysis identified a tribasic KKR motif in the membrane-adjacent region as important for modulating cell-cell fusion. The K1A mutation increased fusion 5.5-fold, while the K2A and R3A mutations decreased fusion 3- to 5-fold. These results were corroborated in a reverse-pseudotyped viral entry assay, where receptor-pseudotyped reporter virus was used to infect cells expressing wild-type or mutant NiV envelope glycoproteins. Differential monoclonal antibody binding data indicated that hyper- or hypofusogenic mutations in the KKR motif affected the ectodomain conformation of NiV-F, which in turn resulted in faster or slower six-helix bundle formation, respectively. However, we also present evidence that the hypofusogenic phenotypes of the K2A and R3A mutants were effected via distinct mechanisms. Interestingly, the K2A mutant was also markedly excluded from lipid rafts, where ∼20% of wild-type F and the other mutants can be found. Finally, we found a strong negative correlation between the relative fusogenic capacities of these cytoplasmic-tail mutants and the avidities of NiV-F and NiV-G interactions (P = 0.007, r 2 = 0.82). In toto, our data suggest that inside-out signaling by specific residues in the cytoplasmic tail of NiV-F can modulate its fusogenicity by multiple distinct mechanisms.

scholarly journals N- (4-Hydroxyphenyl) retinamide suppresses SARS-CoV-2 spike protein-mediated cell-cell fusion by a dihydroceramide Δ4-desaturase 1-independent mechanism

2021 ◽  
Author(s):  
Yasuhiro Hayashi ◽  
Kiyoto Tsuchiya ◽  
Mizuki Yamamoto ◽  
Yoko Nemoto-Sasaki ◽  
Kazunari Tanigawa ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Viral Infection ◽  
Membrane Fusion ◽  
Membrane Fluidity ◽  
Cell Fusion ◽  
Host Cells ◽  
Spike Protein ◽  
Sphingolipid Metabolism ◽  
Independent Mechanism ◽  
Cell Cell

The membrane fusion between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host cells is essential for the initial step of infection; therefore, the host cell membrane components, including sphingolipids, influence the viral infection. We assessed several inhibitors of the enzymes pertaining to sphingolipid metabolism, against SARS-CoV-2 spike protein (S)-mediated cell-cell fusion and viral infection. N -(4-hydroxyphenyl) retinamide (4-HPR), an inhibitor of dihydroceramide Δ4-desaturase 1 (DES1), suppressed cell-cell fusion, and viral infection. The analysis of sphingolipid levels revealed that the inhibition efficiencies of cell-cell fusion and viral infection in 4-HPR-treated cells were consistent with an increased ratio of saturated sphinganine-based lipids to total sphingolipids. We investigated the relationship of DES1 with the inhibition efficiencies of cell-cell fusion. The changes in the sphingolipid profile induced by 4-HPR were mitigated by the supplementation with exogenous cell-permeable ceramide; however, the reduced cell-cell fusion could not be reversed. The efficiency of cell-cell fusion in DES1 knockout (KO) cells was at a level comparable to that in wild-type (WT) cells; however, the ratio of saturated sphinganine-based lipids to the total sphingolipids was higher in DES1 KO cells, compared to that in WT cells. 4-HPR reduced cell membrane fluidity without any significant effects on the expression or localization of angiotensin-converting enzyme 2, the SARS-CoV-2 receptor. Therefore, 4-HPR suppresses SARS-CoV-2 S-mediated membrane fusion through a DES1-independent mechanism, and this decrease in membrane fluidity induced by 4-HPR could be the major cause for the inhibition of SARS-CoV-2 infection. Importance Sphingolipids could play an important role in SARS-CoV-2 S-meditated membrane fusion with host cells. We studied the cell-cell fusion using SARS-CoV-2 S expressing cells and sphingolipid-manipulated target cells, with an inhibitor of the sphingolipid metabolism. 4-HPR (also known as fenretinide) is an inhibitor of DES1 and it exhibits antitumor activity and suppresses cell-cell fusion and viral infection. 4-HPR suppresses membrane fusion through a decrease in membrane fluidity, which could possibly be the cause for the inhibition of SARS-CoV-2 infection. There is accumulating clinical data on the safety of 4-HPR. Therefore, it could be a potential candidate drug against COVID-19.

scholarly journals Mechanisms of mutations inhibiting fusion and infection by Semliki Forest virus.

1996 ◽  
Vol 134 (4) ◽  
pp. 863-872 ◽  
Author(s):  
M Kielian ◽  
M R Klimjack ◽  
S Ghosh ◽  
W A Duffus
Keyword(s):  
Membrane Fusion ◽  
Cell Fusion ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Infectious Clone ◽  
Low Ph ◽  
Spike Protein ◽  
Initial Reaction ◽  
Target Membrane ◽  
Cell Cell

Semliki Forest virus (SFV) infects cells by an acid-dependent membrane fusion reaction catalyzed by the virus spike protein, a complex containing E1 and E2 transmembrane subunits. E1 carries the putative virus fusion peptide, and mutations in this domain of the spike protein were previously shown to shift the pH threshold of cell-cell fusion (G91A), or block cell-cell fusion (G91D). We have used an SFV infectious clone to characterize virus particles containing these mutations. In keeping with the previous spike protein results, G91A virus showed limited secondary infection and an acid-shifted fusion threshold, while G91D virus was noninfectious and inactive in both cell-cell and virus-liposome fusion assays. During the low pH- induced SFV fusion reaction, the E1 subunit exposes new epitopes for monoclonal antibody (mAb) binding and forms an SDS-resistant homotrimer, the virus associates hydrophobically with the target membrane, and fusion of the virus and target membranes occurs. After low pH treatment, G91A spike proteins were shown to bind conformation-specific mAbs, associate with target liposome membranes, and form the E1 homotrimer. However, both G91A membrane association and homotrimer formation had an acid-shifted pH threshold and reduced efficiency compared to wt virus. In contrast, studies of the fusion-defective G91D mutant showed that the virus efficiently reacted with low pH as assayed by mAb binding and liposome association, but was essentially inactive in homotrimer formation. These results suggest that the G91D mutant is noninfectious due to a block in a late step in membrane fusion, separate from the initial reaction to low pH and interaction with the target membrane, and involving the lack of efficient formation of the E1 homotrimer.

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 Modification of the Cytoplasmic Domain of Influenza Virus Hemagglutinin Affects Enlargement of the Fusion Pore

2000 ◽  
Vol 74 (16) ◽  
pp. 7529-7537 ◽  
Author(s):  
Christine Kozerski ◽  
Evgeni Ponimaskin ◽  
Britta Schroth-Diez ◽  
Michael F. G. Schmidt ◽  
Andreas Herrmann
Keyword(s):  
Influenza Virus ◽  
Membrane Fusion ◽  
Transmembrane Domain ◽  
Expression System ◽  
Fusion Pore ◽  
Target Cells ◽  
Fusion Activity ◽  
Wild Type ◽  
Influenza Virus Hemagglutinin ◽  
Pore Enlargement

ABSTRACT The fusion activity of chimeras of influenza virus hemagglutinin (HA) (from A/fpv/Rostock/34; subtype H7) with the transmembrane domain (TM) and/or cytoplasmic tail (CT) either from the nonviral, nonfusogenic T-cell surface protein CD4 or from the fusogenic Sendai virus F-protein was studied. Wild-type or chimeric HA was expressed in CV-1 cells by the transient T7-RNA-polymerase vaccinia virus expression system. Subsequently, the fusion activity of the expression products was monitored with red blood cells or ghosts as target cells. To assess the different steps of fusion, target cells were labeled with the fluorescent membrane label octadecyl rhodamine B-chloride (R18) (membrane fusion) and with the cytoplasmic fluorophores calcein (molecular weight [MW], 623; formation of small aqueous fusion pore) and tetramethylrhodamine-dextran (MW, 10,000; enlargement of fusion pore). All chimeric HA/F-proteins, as well as the chimera with the TM of CD4 and the CT of HA, were able to mediate the different steps of fusion very similarly to wild-type HA. Quite differently, chimeric proteins with the CT of CD4 were strongly impaired in mediating pore enlargement. However, membrane fusion and formation of small pores were similar to those of wild-type HA, indicating that the conformational change of the ectodomain and earlier fusion steps were not inhibited. Various properties of the CT which may affect pore enlargement are considered. We surmise that the hydrophobicity of the sequence adjacent to the transmembrane domain is important for pore dilation.

scholarly journals Aromatic Amino Acids in the Juxtamembrane Domain of Severe Acute Respiratory Syndrome Coronavirus Spike Glycoprotein Are Important for Receptor-Dependent Virus Entry and Cell-Cell Fusion

2008 ◽  
Vol 82 (6) ◽  
pp. 2883-2894 ◽  
Author(s):  
Megan W. Howard ◽  
Emily A. Travanty ◽  
Scott A. Jeffers ◽  
M. K. Smith ◽  
Sonia T. Wennier ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Fusion ◽  
Transmembrane Domain ◽  
Virus Entry ◽  
Aromatic Amino Acids ◽  
Wild Type ◽  
Aromatic Residues ◽  
Wild Type Level ◽  
S Proteins ◽  
Cell Cell

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) spike glycoprotein (S) is a class I viral fusion protein that binds to its receptor glycoprotein, human angiotensin converting enzyme 2 (hACE2), and mediates virus entry and cell-cell fusion. The juxtamembrane domain (JMD) of S is an aromatic amino acid-rich region proximal to the transmembrane domain that is highly conserved in all coronaviruses. Alanine substitutions for one or two of the six aromatic residues in the JMD did not alter the surface expression of the SARS-CoV S proteins with a deletion of the C-terminal 19 amino acids (S Δ19) or reduce binding to soluble human ACE2 (hACE2). However, hACE2-dependent entry of trypsin-treated retrovirus pseudotyped viruses expressing JMD mutant S Δ19 proteins was greatly reduced. Single alanine substitutions for aromatic residues reduced entry to 10 to 60% of the wild-type level. The greatest reduction was caused by residues nearest the transmembrane domain. Four double alanine substitutions reduced entry to 5 to 10% of the wild-type level. Rapid hACE2-dependent S-mediated cell-cell fusion was reduced to 60 to 70% of the wild-type level for all single alanine substitutions and the Y1188A/Y1191A protein. S Δ19 proteins with other double alanine substitutions reduced cell-cell fusion further, from 40% to less than 20% of wild-type levels. The aromatic amino acids in the JMD of the SARS-CoV S glycoprotein play critical roles in receptor-dependent virus-cell and cell-cell fusion. Because the JMD is so highly conserved in all coronavirus S proteins, it is a potential target for development of drugs that may inhibit virus entry and/or cell-cell fusion mediated by S proteins of all coronaviruses.

scholarly journals Functional Characterization of Heptad Repeat 1 and 2 Mutants of the Spike Protein of Severe Acute Respiratory Syndrome Coronavirus

2006 ◽  
Vol 80 (7) ◽  
pp. 3225-3237 ◽  
Author(s):  
Woan-Eng Chan ◽  
Chin-Kai Chuang ◽  
Shiou-Hwei Yeh ◽  
Mau-Sun Chang ◽  
Steve S.-L. Chen
Keyword(s):  
Molecular Weight ◽  
Severe Acute Respiratory Syndrome ◽  
Membrane Fusion ◽  
Viral Entry ◽  
Functional Characterization ◽  
Surface Expression ◽  
Spike Protein ◽  
Heptad Repeat ◽  
Cell Surface Expression ◽  
Wild Type

ABSTRACT To understand the roles of heptad repeat 1(HR1) and HR2 of the spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) in virus-cell interactions, the conserved Leu or Ile residues located at positions 913, 927, 941, and 955 in HR1 and 1151, 1165, and 1179 in HR2 were individually replaced with an α-helix-breaker Pro residue. The 913P mutant was expressed mainly as a faster-migrating, lower-molecular-weight SL form, while the wild type and all other mutants produced similar levels of both the SL form and the slower-migrating, higher-molecular-weight SH form. The wild-type SL form was processed to the SH form, whereas the SL form of the 913P mutant was inefficiently converted to the SH form after biosynthesis. None of these mutations affected cell surface expression or binding to its cognate ACE2 receptor. In a human immunodeficiency virus type 1/SARS S coexpression study, all mutants except the 913P mutant incorporated the SH form into the virions as effectively as did the wild-type SH form. The mutation at Ile-1151 did not affect membrane fusion or viral entry. The impaired viral entry of the 927P, 941P, 955P, and 1165P mutants was due to their inability to mediate membrane fusion, whereas the defect in viral entry of the 1179P mutant occurred not at the stage of membrane fusion but rather at a postfusion stage. Our study demonstrates the functional importance of HR1 and HR2 of the SARS-CoV spike protein in membrane fusion and viral entry.

scholarly journals Fusion Activity of Transmembrane and Cytoplasmic Domain Chimeras of the Influenza Virus Glycoprotein Hemagglutinin

1998 ◽  
Vol 72 (1) ◽  
pp. 133-141 ◽  
Author(s):  
Britta Schroth-Diez ◽  
Evgeni Ponimaskin ◽  
Helmut Reverey ◽  
Michael F. G. Schmidt ◽  
Andreas Herrmann
Keyword(s):  
Influenza Virus ◽  
Membrane Fusion ◽  
Lipid Bilayers ◽  
Transmembrane Domain ◽  
Sendai Virus ◽  
Expression System ◽  
Cytoplasmic Tail ◽  
Fusion Pore ◽  
Fusion Activity ◽  
Influenza Virus Hemagglutinin

ABSTRACT The role of the sequence of transmembrane and cytoplasmic/intraviral domains of influenza virus hemagglutinin (HA, subtype H7) for HA-mediated membrane fusion was explored. To analyze the influence of the two domains on the fusogenic properties of HA, we designed HA-chimeras in which the cytoplasmic tail and/or transmembrane domain of HA was replaced with the corresponding domains of the fusogenic glycoprotein F of Sendai virus. These chimeras, as well as constructs of HA in which the cytoplasmic tail was replaced by peptides of human neurofibromin type1 (NF1) or c-Raf-1, NF78 (residues 1441 to 1518), and Raf81 (residues 51 to 131), respectively, were expressed in CV-1 cells by using the vaccinia virus-T7 polymerase transient-expression system. Wild-type and chimeric HA were cleaved properly into two subunits and expressed as trimers. Membrane fusion between CV-1 cells and bound human erythrocytes (RBCs) mediated by parental or chimeric HA proteins was studied by a lipid-mixing assay with the lipid-like fluorophore octadecyl rhodamine B chloride (R18). No profound differences in either extent or kinetics could be observed. After the pH was lowered, the above proteins also induced a flow of the aqueous fluorophore calcein from preloaded RBCs into the cytoplasm of the protein-expressing CV-1 cells, indicating that membrane fusion involves both leaflets of the lipid bilayers and leads to formation of an aqueous fusion pore. We conclude that neither HA-specific sequences in the transmembrane and cytoplasmic domains nor their length is crucial for HA-induced membrane fusion activity.

Sign in / Sign up

Export Citation Format

Share Document