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 Conformational States of the Severe Acute Respiratory Syndrome Coronavirus Spike Protein Ectodomain

2006 ◽  
Vol 80 (14) ◽  
pp. 6794-6800 ◽  
Author(s):  
Fang Li ◽  
Marcelo Berardi ◽  
Wenhui Li ◽  
Michael Farzan ◽  
Philip R. Dormitzer ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Membrane Fusion ◽  
Receptor Binding ◽  
Conformational Changes ◽  
Viral Entry ◽  
Protein S ◽  
Spike Protein ◽  
Fusion Peptides ◽  
Conformational States ◽  
Protease Cleavage

ABSTRACT The severe acute respiratory syndrome coronavirus enters cells through the activities of a spike protein (S) which has receptor-binding (S1) and membrane fusion (S2) regions. We have characterized four sequential states of a purified recombinant S ectodomain (S-e) comprising S1 and the ectodomain of S2. They are S-e monomers, uncleaved S-e trimers, cleaved S-e trimers, and dissociated S1 monomers and S2 trimer rosettes. Lowered pH induces an irreversible transition from flexible, L-shaped S-e monomers to clove-shaped trimers. Protease cleavage of the trimer occurs at the S1-S2 boundary; an ensuing S1 dissociation leads to a major rearrangement of the trimeric S2 and to formation of rosettes likely to represent clusters of elongated, postfusion trimers of S2 associated through their fusion peptides. The states and transitions of S suggest conformational changes that mediate viral entry into cells.

Headless henipaviral receptor binding glycoproteins reveal key post-receptor binding contributions of the globular head and head/stalk interface in fusion promotion

2021 ◽  
Author(s):  
Yao Yu Yeo ◽  
David W. Buchholz ◽  
Amandine Gamble ◽  
Mason Jager ◽  
Hector C. Aguilar
Keyword(s):  
Cell Fusion ◽  
Receptor Binding ◽  
Viral Entry ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Wild Type ◽  
Emerging Viruses ◽  
Multinucleated Cells ◽  
Head Domain ◽  
Cell Cell

Cedar virus (CedV) is a nonpathogenic member of the Henipavirus (HNV) genus of emerging viruses, which includes the deadly Nipah (NiV) and Hendra (HeV) viruses. CedV forms syncytia, a hallmark of henipaviral and paramyxoviral infections and pathogenicity. However, the intrinsic fusogenic capacity of CedV relative to NiV or HeV remains unquantified. HNV entry is mediated by concerted interactions between the attachment (G) and fusion (F) glycoproteins. Upon receptor binding by the HNV G head domain, a fusion-activating G stalk region is exposed and triggers F to undergo a conformational cascade that leads to viral entry or cell-cell fusion. Here, we first demonstrated quantitatively that CedV is inherently significantly less fusogenic than NiV at equivalent G and F cell surface expression levels. We then generated and tested six headless CedV G mutants of distinct stalk C-terminal lengths, surprisingly revealing highly hyperfusogenic cell-cell fusion phenotypes 3 to 4-fold greater than wild-type CedV levels. Additionally, similarly to NiV, a headless HeV G mutant yielded a less pronounced hyperfusogenic phenotype compared to wild-type HeV. Further, coimmunoprecipitation and cell-cell fusion assays revealed heterotypic NiV/CedV functional G/F bidentate interactions, as well as evidence of HNV G head domain involvement beyond receptor binding or G stalk exposure. All evidence points to the G head/stalk junction being key to modulating HNV fusogenicity, supporting the notion that head domains play several distinct and central roles in modulating stalk domain fusion promotion. Further, this study exemplifies how CedV may help elucidate important mechanistic underpinnings of HNV entry and pathogenicity. IMPORTANCE The Henipavirus genus in the Paramyxoviridae family includes the zoonotic Nipah (NiV) and Hendra (HeV) viruses. NiV and HeV infections often cause fatal encephalitis and pneumonia, but no vaccines or therapeutics are currently approved for human use. Upon viral entry, Henipavirus infections yield the formation of multinucleated cells (syncytia). Viral entry and cell-cell fusion are mediated by the attachment (G) and fusion (F) glycoproteins. Cedar virus (CedV), a nonpathogenic henipavirus, may be a useful tool to gain knowledge on henipaviral pathogenicity. Here, using homotypic and heterotypic full-length and headless CedV, NiV, and HeV G/F combinations, we discovered that CedV G/F are significantly less fusogenic than NiV or HeV G/F, and that the G head/stalk junction is key to modulating cell-cell fusion, refining the mechanism of henipaviral membrane fusion events. Our study exemplifies how CedV may be a useful tool to elucidate broader mechanistic understanding for the important henipaviruses.

Abstract 049: Functional Characterization of KCNQ1 Channel Subunit With an A590T Mutation

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Koshi Kinoshita ◽  
Katsuya Kimoto ◽  
Takuto Komatsu ◽  
Kohki Nishide ◽  
Toshihide Tabata ◽  
...  
Keyword(s):  
Cell Surface ◽  
Functional Characterization ◽  
Coiled Coil ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Delayed Rectifier ◽  
Wild Type ◽  
Cardiac Events ◽  
Channel Function ◽  
Similar Density

Background: KCNQ1 encodes the alpha subunit of the voltage-gated K + channel that mediates the cardiac slow delayed rectifier K + current (I Ks ). A mutation, A590T, in KCNQ1 was incidentally identified in a 40 years old female. She had a mild QTc prolongation in electrocardiogram but has never experienced any cardiac events. A590 is located in the C-terminal domain forming a coiled-coil structure, which has been suggested as a pivotal component for subunit tetramerization and channel trafficking to the cell surface. The previously reported mutations around A590 result in markedly reduced cell surface expression and loss of functional channel. We, for the first time, examined whether and how the A590T mutation affects the I Ks channel function. Methods: To assess the trafficking and channel function of KCNQ1(A590T) mutant subunit, we performed immunostaining, immunoblotting, and voltage-clamp measurements in HEK-293T cells transfected with wild-type or the A590T mutant KCNQ1 or their mixture (WT, A590T, and A590T/WT cells, respectively). Results: The density of a depolarization-activated current in the A590T cells was smaller than that in the WT cells. The threshold, half-maximal activation, and saturating voltages of the depolarization-activated current in the A590T cells were more positive than those in the WT cells. The immunoreactivity against KCNQ1 subunit on the cell surface in the A590T cells is lower than in WT cells. The A590T/WT cells had a similar density of the depolarization-activated current and a similar level of immunoreactivity against the channel subunit to the WT cells. Furthermore, the immunoblotting detected subunit oligomers in the membrane fraction of the A590T cells while their densities were lower than those of the WT cells. Conclusion: Although the A590T mutant subunit can form oligomers for itself, this subunit is not efficiently trafficked to the cell surface without the aid of the WT subunit. Thus, homozygous inheritance of the mutant KCNQ1 might be pathogenic. By contrast, the cells expressing both the mutant and wild-type KCNQ1 subunit had normal I Ks and cell surface expression, indicating that the heterozygous inheritance of the mutant KCNQ1 might not cause severe cardiac diseases.

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 Structural impact on SARS-CoV-2 spike protein by D614G substitution

Science ◽  
2021 ◽  
pp. eabf2303
Author(s):  
Jun Zhang ◽  
Yongfei Cai ◽  
Tianshu Xiao ◽  
Jianming Lu ◽  
Hanqin Peng ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Membrane Fusion ◽  
Receptor Binding ◽  
Viral Entry ◽  
Vaccine Development ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Structural Rearrangements ◽  
Viral Spread ◽  
Structural Impact

Substitution for aspartic acid by glycine at position 614 in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 appears to facilitate rapid viral spread. The G614 strain and its recent variants are now the dominant circulating forms. We report here cryo-EM structures of a full-length G614 S trimer, which adopts three distinct prefusion conformations differing primarily by the position of one receptor-binding domain. A loop disordered in the D614 S trimer wedges between domains within a protomer in the G614 spike. This added interaction appears to prevent premature dissociation of the G614 trimer, effectively increasing the number of functional spikes and enhancing infectivity, and to modulate structural rearrangements for membrane fusion. These findings extend our understanding of viral entry and suggest an improved immunogen for vaccine development.

scholarly journals Effects of Mutations in the Rubella Virus E1 Glycoprotein on E1-E2 Interaction and Membrane Fusion Activity

1998 ◽  
Vol 72 (11) ◽  
pp. 8747-8755 ◽  
Author(s):  
Decheng Yang ◽  
Dorothy Hwang ◽  
Zhiyong Qiu ◽  
Shirley Gillam
Keyword(s):  
Cell Surface ◽  
Membrane Fusion ◽  
Rubella Virus ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Fusion Activity ◽  
Wild Type ◽  
Hydrophobic Region ◽  
Hydrophobic Domain ◽  
Infected Cells

ABSTRACT Rubella virus (RV) virions contain two glycosylated membrane proteins, E1 and E2, that exist as a heterodimer and form the viral spike complexes on the virion surface. Formation of an E1-E2 heterodimer is required for transport of E1 out of the endoplasmic reticulum lumen to the Golgi apparatus and plasma membrane. To investigate the nature of the E1-E2 interaction, we have introduced mutations in the internal hydrophobic region (residues 81 to 109) of E1. Substitution of serine at Cys82 (mutant C82S) or deletion of this hydrophobic domain (mutant dt) of E1 resulted in a disruption of the E1 conformation that ultimately affected E1-E2 heterodimer formation and cell surface expression of both E1 and E2. Substitution of either aspartic acid at Gly93 (G93D) or glycine at Pro104 (P104G) was found to impair neither E1-E2 heterodimer formation nor the transport of E1 and E2 to the cell surface. Fusion of RV-infected cells is induced by a brief treatment at a pH below 6.0. To test whether this internal hydrophobic domain is involved in the membrane fusion activity of RV, transformed BHK cell lines expressing either wild-type or mutant spike proteins were exposed to an acidic pH and polykaryon formation was measured. No fusion activity was observed in the C82S, dt, and G93D mutants; however, the wild type and the P104G mutant exhibited fusogenic activities, with greater than 60% and 20 to 40% of the cells being fused, respectively, at pH 4.8. These results suggest that it is likely that the region of E1 between amino acids 81 and 109 is involved in the membrane fusion activity of RV and that it may be important for the interaction of that protein with E2 to form the E1-E2 heterodimer.

scholarly journals Structural impact on SARS-CoV-2 spike protein by D614G substitution

2020 ◽  
Author(s):  
Jun Zhang ◽  
Yongfei Cai ◽  
Tianshu Xiao ◽  
Jianming Lu ◽  
Hanqin Peng ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Membrane Fusion ◽  
Viral Entry ◽  
Vaccine Development ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Structural Rearrangements ◽  
S Protein ◽  
Viral Spread ◽  
Structural Impact

AbstractSubstitution for aspartic acid by glycine at position 614 in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the ongoing pandemic, appears to facilitate rapid viral spread. The G614 variant has now replaced the D614-carrying virus as the dominant circulating strain. We report here cryo-EM structures of a full-length S trimer carrying G614, which adopts three distinct prefusion conformations differing primarily by the position of one receptor-binding domain (RBD). A loop disordered in the D614 S trimer wedges between domains within a protomer in the G614 spike. This added interaction appears to prevent premature dissociation of the G614 trimer, effectively increasing the number of functional spikes and enhancing infectivity. The loop transition may also modulate structural rearrangements of S protein required for membrane fusion. These findings extend our understanding of viral entry and suggest an improved immunogen for vaccine development.

scholarly journals Amino Acids 1055 to 1192 in the S2 Region of Severe Acute Respiratory Syndrome Coronavirus S Protein Induce Neutralizing Antibodies: Implications for the Development of Vaccines and Antiviral Agents

2005 ◽  
Vol 79 (6) ◽  
pp. 3289-3296 ◽  
Author(s):  
Choong-Tat Keng ◽  
Aihua Zhang ◽  
Shuo Shen ◽  
Kuo-Ming Lip ◽  
Burtram C. Fielding ◽  
...  
Keyword(s):  
Amino Acids ◽  
Severe Acute Respiratory Syndrome ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Antiviral Agents ◽  
Host Cells ◽  
Heptad Repeat ◽  
Antigenic Determinants ◽  
Amino Acid Residues ◽  
S Protein

ABSTRACT The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-SΔ10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.

scholarly journals Engineered ACE2 receptor traps potently neutralize SARS-CoV-2

2020 ◽  
Vol 117 (45) ◽  
pp. 28046-28055 ◽  
Author(s):  
Anum Glasgow ◽  
Jeff Glasgow ◽  
Daniel Limonta ◽  
Paige Solomon ◽  
Irene Lui ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Viral Entry ◽  
Neutralizing Antibodies ◽  
Surface Display ◽  
Random Mutagenesis ◽  
Yeast Surface Display ◽  
Host Cells ◽  
Spike Protein ◽  
Receptor Protein ◽  
Yeast Surface

An essential mechanism for severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here, we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2–RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest-affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human immunoglobulin crystallizable fragment (Fc) domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2–pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50s) in the 10- to 100-ng/mL range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-using coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be predesigned for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated from convalescent patients.

scholarly journals Preventing SARS-CoV-2 infection by blocking a tissue serine protease

2020 ◽  
Vol 7 ◽  
pp. 204993612093307
Author(s):  
Katherine C. Jankousky ◽  
Jonathan Schultz ◽  
Samuel Windham ◽  
Andrés F. Henao-Martínez ◽  
Carlos Franco-Paredes ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Severe Acute Respiratory Syndrome ◽  
Serine Protease ◽  
Viral Entry ◽  
Clinical Impact ◽  
Spike Protein ◽  
Viral Pathogens ◽  
Pharmacological Targets ◽  
Alternative Approach ◽  
Viral Surface

Currently, there are no proven pharmacologic interventions to reduce the clinical impact and prevent complications of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection, the cause of the ongoing Coronavirus Disease of 2019 (COVID-19) pandemic. Selecting specific pharmacological targets for the treatment of viral pathogens has traditionally relied in blockage of specific steps in their replicative lifecycle in human cells. However, an alternative approach is reducing the molecular cleavage of the viral surface spike protein of SARS-CoV-2 to prevent viral entry into epithelial cells.

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