Transient lipid-bound states of spike protein heptad repeats provide insights into SARS-CoV-2 membrane fusion

ABSTRACT The prototype JHM strain of murine hepatitis virus (MHV) is an enveloped, RNA-containing coronavirus that has been selected in vivo for extreme neurovirulence. This virus encodes spike (S) glycoproteins that are extraordinarily effective mediators of intercellular membrane fusion, unique in their ability to initiate fusion even without prior interaction with the primary MHV receptor, a murine carcinoembryonic antigen-related cell adhesion molecule (CEACAM). In considering the possible role of this hyperactive membrane fusion activity in neurovirulence, we discovered that the growth of JHM in tissue culture selected for variants that had lost murine CEACAM-independent fusion activity. Among the collection of variants, mutations were identified in regions encoding both the receptor-binding (S1) and fusion-inducing (S2) subunits of the spike protein. Each mutation was separately introduced into cDNA encoding the prototype JHM spike, and the set of cDNAs was expressed using vaccinia virus vectors. The variant spikes were similar to that of JHM in their assembly into oligomers, their proteolysis into S1 and S2 cleavage products, their transport to cell surfaces, and their affinity for a soluble form of murine CEACAM. However, these tissue culture-adapted spikes were significantly stabilized as S1-S2 heteromers, and their entirely CEACAM-dependent fusion activity was delayed or reduced relative to prototype JHM spikes. The mutations that we have identified therefore point to regions of the S protein that specifically regulate the membrane fusion reaction. We suggest that cultured cells, unlike certain in vivo environments, select for S proteins with delayed, CEACAM-dependent fusion activities that may increase the likelihood of virus internalization prior to the irreversible uncoating process.

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Peptide-Based Membrane Fusion Inhibitors Targeting HCoV-229E Spike Protein HR1 and HR2 Domains

International Journal of Molecular Sciences ◽

10.3390/ijms19020487 ◽

2018 ◽

Vol 19 (2) ◽

pp. 487 ◽

Cited By ~ 20

Author(s):

Shuai Xia ◽

Wei Xu ◽

Qian Wang ◽

Cong Wang ◽

Chen Hua ◽

...

Keyword(s):

Membrane Fusion ◽

Spike Protein ◽

Fusion Inhibitors

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

Journal of Virology ◽

10.1128/jvi.02744-05 ◽

2006 ◽

Vol 80 (14) ◽

pp. 6794-6800 ◽

Cited By ~ 76

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.

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A Single Point Mutation Controls the Cholesterol Dependence of Semliki Forest Virus Entry and Exit

The Journal of Cell Biology ◽

10.1083/jcb.140.1.91 ◽

1998 ◽

Vol 140 (1) ◽

pp. 91-99 ◽

Cited By ~ 108

Author(s):

Malini Vashishtha ◽

Thomas Phalen ◽

Marianne T. Marquardt ◽

Jae S. Ryu ◽

Alice C. Ng ◽

...

Keyword(s):

Membrane Fusion ◽

Semliki Forest Virus ◽

Single Point ◽

Cellular Membrane ◽

Spike Protein ◽

Single Amino Acid ◽

Progeny Virus ◽

Single Point Mutation ◽

Protein Subunit ◽

Single Amino Acid Change

Membrane fusion and budding are key steps in the life cycle of all enveloped viruses. Semliki Forest virus (SFV) is an enveloped alphavirus that requires cellular membrane cholesterol for both membrane fusion and efficient exit of progeny virus from infected cells. We selected an SFV mutant, srf-3, that was strikingly independent of cholesterol for growth. This phenotype was conferred by a single amino acid change in the E1 spike protein subunit, proline 226 to serine, that increased the cholesterol independence of both srf-3 fusion and exit. The srf-3 mutant emphasizes the relationship between the role of cholesterol in membrane fusion and virus exit, and most significantly, identifies a novel spike protein region involved in the virus cholesterol requirement.

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A Conserved Region in the F2 Subunit of Paramyxovirus Fusion Proteins Is Involved In Fusion Regulation

Journal of Virology ◽

10.1128/jvi.00366-07 ◽

2007 ◽

Vol 81 (15) ◽

pp. 8303-8314 ◽

Cited By ~ 29

Author(s):

Amanda E. Gardner ◽

Rebecca E. Dutch

Keyword(s):

Membrane Fusion ◽

Cell Fusion ◽

Critical Role ◽

Simian Virus ◽

Hendra Virus ◽

Heptad Repeat ◽

F Protein ◽

Conserved Region ◽

Heptad Repeats ◽

Cell Cell

ABSTRACT Paramyxoviruses utilize both an attachment protein and a fusion (F) protein to drive virus-cell and cell-cell fusion. F exists functionally as a trimer of two disulfide-linked subunits: F1 and F2. Alignment and analysis of a set of paramyxovirus F protein sequences identified three conserved blocks (CB): one in the fusion peptide/heptad repeat A domain, known to play important roles in fusion promotion, one in the region between the heptad repeats of F1 (CBF1) (A. E. Gardner, K. L. Martin, and R. E. Dutch, Biochemistry 46:5094-5105, 2007), and one in the F2 subunit (CBF2). To analyze the functions of CBF2, alanine substitutions at conserved positions were created in both the simian virus 5 (SV5) and Hendra virus F proteins. A number of the CBF2 mutations resulted in folding and expression defects. However, the CBF2 mutants that were properly expressed and trafficked had altered fusion promotion activity. The Hendra virus CBF2 Y79A and P89A mutants showed significantly decreased levels of fusion, whereas the SV5 CBF2 I49A mutant exhibited greatly increased cell-cell fusion relative to that for wild-type F. Additional substitutions at SV5 F I49 suggest that both side chain volume and hydrophobicity at this position are important in the folding of the metastable, prefusion state and the subsequent triggering of membrane fusion. The recently published prefusogenic structure of parainfluenza virus 5/SV5 F (H. S. Yin et al., Nature 439:38-44, 2006) places CBF2 in direct contact with heptad repeat A. Our data therefore indicate that this conserved region plays a critical role in stabilizing the prefusion state, likely through interactions with heptad repeat A, and in triggering membrane fusion.

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Extracellular vesicles containing ACE2 efficiently prevent infection by SARS-CoV-2 Spike protein-containing virus

10.1101/2020.07.08.193672 ◽

2020 ◽

Cited By ~ 1

Author(s):

Federico Cocozza ◽

Ester Piovesana ◽

Nathalie Névo ◽

Xavier Lahaye ◽

Julian Buchrieser ◽

...

Keyword(s):

Membrane Fusion ◽

Extracellular Vesicles ◽

Protein S ◽

Spike Protein ◽

S Protein ◽

Surface Receptor

ABSTRACTSARS-CoV-2 entry is mediated by binding of the spike protein (S) to the surface receptor ACE2 and subsequent priming by TMPRRS2 allowing membrane fusion. Here, we produced extracellular vesicles (EVs) exposing ACE2 and demonstrate that ACE2-EVs are efficient decoys for SARS-CoV-2 S protein-containing lentivirus. Reduction of infectivity positively correlates with the level of ACE2, is 500 to 1500 times more efficient than with soluble ACE2 and further enhanced by the inclusion of TMPRSS2.

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An Epitope of the Semliki Forest Virus Fusion Protein Exposed during Virus-Membrane Fusion

Journal of Virology ◽

10.1128/jvi.73.12.10029-10039.1999 ◽

1999 ◽

Vol 73 (12) ◽

pp. 10029-10039 ◽

Cited By ~ 28

Author(s):

Anna Ahn ◽

Matthew R. Klimjack ◽

Prodyot K. Chatterjee ◽

Margaret Kielian

Keyword(s):

Membrane Fusion ◽

Binding Site ◽

Semliki Forest Virus ◽

Fusion Reaction ◽

Ph Dependence ◽

Fusion Peptide ◽

Endocytic Pathway ◽

Spike Protein ◽

Viral Fusion ◽

Wide Range

ABSTRACT Semliki Forest virus (SFV) is an enveloped alphavirus that infects cells via a membrane fusion reaction triggered by acidic pH in the endocytic pathway. Fusion is mediated by the spike protein E1 subunit, an integral membrane protein that contains the viral fusion peptide and forms a stable homotrimer during fusion. We have characterized four monoclonal antibodies (MAbs) specific for the acid conformation of E1. These MAbs did not inhibit fusion, suggesting that they bind to an E1 region different from the fusion peptide. Competition analyses demonstrated that all four MAbs bound to spatially related sites on acid-treated virions or isolated spike proteins. To map the binding site, we selected for virus mutants resistant to one of the MAbs, E1a-1. One virus isolate, SFV 4-2, showed reduced binding of three acid-specific MAbs including E1a-1, while its binding of one acid-specific MAb as well as non-acid-specific MAbs to E1 and E2 was unchanged. The SFV 4-2 mutant was fully infectious, formed the E1 homotrimer, and had the wild-type pH dependence of infection. Sequence analysis demonstrated that the relevant mutation in SFV 4-2 was a change of E1 glycine 157 to arginine (G157R). Decreased binding of MAb E1a-1 was observed under a wide range of assay conditions, strongly suggesting that the E1 G157R mutation directly affects the MAb binding site. These data thus localize an E1 region that is normally hidden in the neutral pH structure and becomes exposed as part of the reorganization of the spike protein to its fusion-active conformation.

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