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

ABSTRACT Wild-type measles virus (MV) isolated in B95a cells could be adapted to Vero cells after several blind passages. In this study, we have determined the complete nucleotide sequences of the genomes of the wild type (T11wild) and its Vero cell-adapted (T11Ve-23) MV strain and identified amino acid substitutions R516G, E271K, D439E and G464W (D439E/G464W), N481Y/H495R, and Y187H/L204F in the nucleocapsid, V, fusion (F), hemagglutinin (H), and large proteins, respectively. Expression of mutated H and F proteins from cDNA revealed that the H495R substitution, in addition to N481Y, in the H protein was necessary for the wild-type H protein to use CD46 efficiently as a receptor and that the G464W substitution in the F protein was important for enhanced cell-cell fusion. Recombinant wild-type MV strains harboring the F protein with the mutations D439E/G464W [F(D439E/G464W)] and/or H(N481Y/H495R) protein revealed that both mutated F and H proteins were required for efficient syncytium formation and virus growth in Vero cells. Interestingly, a recombinant wild-type MV strain harboring the H(N481Y/H495R) protein penetrated slowly into Vero cells, while a recombinant wild-type MV strain harboring both the F(D439E/G464W) and H(N481Y/H495R) proteins penetrated efficiently into Vero cells, indicating that the F(D439E/G464W) protein compensates for the inefficient penetration of a wild-type MV strain harboring the H(N481Y/H495R) protein. Thus, the F and H proteins synergistically function to ensure efficient wild-type MV growth in Vero cells.

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

Journal of Virology ◽

10.1128/jvi.80.3.1302-1310.2006 ◽

2006 ◽

Vol 80 (3) ◽

pp. 1302-1310 ◽

Cited By ~ 38

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.

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

Journal of Virology ◽

10.1128/jvi.76.21.10708-10716.2002 ◽

2002 ◽

Vol 76 (21) ◽

pp. 10708-10716 ◽

Cited By ~ 53

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.

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A 12-Amino Acid Stretch in the Hypervariable Region of the Spike Protein S1 Subunit Is Critical for Cell Fusion Activity of Mouse Hepatitis Virus

Journal of Biological Chemistry ◽

10.1074/jbc.274.37.26085 ◽

1999 ◽

Vol 274 (37) ◽

pp. 26085-26090 ◽

Cited By ~ 14

Author(s):

Chang-Wu Tsai ◽

Shin C. Chang ◽

Ming-Fu Chang

Keyword(s):

Amino Acid ◽

Cell Fusion ◽

Hepatitis Virus ◽

Mouse Hepatitis Virus ◽

Hypervariable Region ◽

Spike Protein ◽

Fusion Activity ◽

Amino Acid Stretch

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In Silico Investigation of the New UK (B.1.1.7) and South African (501Y.V2) SARS-CoV-2 Variants with a Focus at the ACE2–Spike RBD Interface

International Journal of Molecular Sciences ◽

10.3390/ijms22041695 ◽

2021 ◽

Vol 22 (4) ◽

pp. 1695

Author(s):

Bruno O. Villoutreix ◽

Vincent Calvez ◽

Anne-Geneviève Marcelin ◽

Abdel-Majid Khatib

Keyword(s):

Amino Acid ◽

South African ◽

In Silico ◽

Neutralizing Antibodies ◽

Viral Escape ◽

Spike Protein ◽

Amino Acid Substitutions ◽

The South ◽

African Strain ◽

The Uk

SARS-CoV-2 exploits angiotensin-converting enzyme 2 (ACE2) as a receptor to invade cells. It has been reported that the UK and South African strains may have higher transmission capabilities, eventually in part due to amino acid substitutions on the SARS-CoV-2 Spike protein. The pathogenicity seems modified but is still under investigation. Here we used the experimental structure of the Spike RBD domain co-crystallized with part of the ACE2 receptor, several in silico methods and numerous experimental data reported recently to analyze the possible impacts of three amino acid replacements (Spike K417N, E484K, N501Y) with regard to ACE2 binding. We found that the N501Y replacement in this region of the interface (present in both the UK and South African strains) should be favorable for the interaction with ACE2, while the K417N and E484K substitutions (South African strain) would seem neutral or even unfavorable. It is unclear if the N501Y substitution in the South African strain could counterbalance the K417N and E484K Spike replacements with regard to ACE2 binding. Our finding suggests that the UK strain should have higher affinity toward ACE2 and therefore likely increased transmissibility and possibly pathogenicity. If indeed the South African strain has a high transmission level, this could be due to the N501Y replacement and/or to substitutions in regions located outside the direct Spike–ACE2 interface but not so much to the K417N and E484K replacements. Yet, it should be noted that amino acid changes at Spike position 484 can lead to viral escape from neutralizing antibodies. Further, these amino acid substitutions do not seem to induce major structural changes in this region of the Spike protein. This structure–function study allows us to rationalize some observations made for the UK strain but raises questions for the South African strain.

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Variations in Disparate Regions of the Murine Coronavirus Spike Protein Impact the Initiation of Membrane Fusion

Journal of Virology ◽

10.1128/jvi.75.6.2792-2802.2001 ◽

2001 ◽

Vol 75 (6) ◽

pp. 2792-2802 ◽

Cited By ~ 65

Author(s):

Dawn K. Krueger ◽

Sean M. Kelly ◽

Daniel N. Lewicki ◽

Rosanna Ruffolo ◽

Thomas M. Gallagher

Keyword(s):

Tissue Culture ◽

Membrane Fusion ◽

Cultured Cells ◽

Hepatitis Virus ◽

Fusion Reaction ◽

Spike Protein ◽

Soluble Form ◽

Fusion Activity ◽

Murine Hepatitis Virus

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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SARS-CoV-2 spike P681R mutation enhances and accelerates viral fusion

10.1101/2021.06.17.448820 ◽

2021 ◽

Author(s):

Akatsuki Saito ◽

Hesham Nasser ◽

Keiya Uriu ◽

Yusuke Kosugi ◽

Takashi Irie ◽

...

Keyword(s):

Cell Fusion ◽

Viral Genome ◽

Neutralizing Antibody ◽

Spike Protein ◽

Human Society ◽

Viral Infectivity ◽

Viral Fusion ◽

Antibody Resistance ◽

Cell Cell

During the current SARS-CoV-2 pandemic, a variety of mutations have been accumulated in the viral genome, and at least five variants of concerns (VOCs) have been considered as the hazardous SARS-CoV-2 variants to the human society. The newly emerging VOC, the B.1.617.2 lineage (delta variant), closely associates with a huge COVID-19 surge in India in Spring 2021. However, its virological property remains unclear. Here, we show that the B.1.617 variants are highly fusogenic and form prominent syncytia. Bioinformatic analyses reveal that the P681R mutation in the spike protein is highly conserved in this lineage. Although the P681R mutation decreases viral infectivity, this mutation confers the neutralizing antibody resistance. Notably, we demonstrate that the P681R mutation facilitates the furin-mediated spike cleavage and enhances and accelerates cell-cell fusion. Our data suggest that the P681R mutation is a hallmark characterizing the virological phenotype of this newest VOC, which may associate with viral pathogenicity.

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The genetic variant analyses of SARS-CoV-2 strains; circulating in Bangladesh

10.1101/2020.07.29.226555 ◽

2020 ◽

Author(s):

Abu Sayeed Mohmmad Mahmud ◽

Tarannum Taznin ◽

Md. Murshed Hasan Sarkar ◽

Mohammad Samir Uzzaman ◽

Eshrar Osman ◽

...

Keyword(s):

Amino Acid ◽

Mutation Rate ◽

Local Environment ◽

Spike Protein ◽

Amino Acid Substitutions ◽

List Type ◽

Nucleotide Substitutions ◽

Disease Manifestation ◽

Entire Genome ◽

Nucleotide Mutation

AbstractGenomic mutation of the virus may impact the viral adaptation to the local environment, their transmission, disease manifestation, and the effectiveness of existing treatment and vaccination. The objectives of this study were to characterize genomic variations, non-synonymous amino acid substitutions, especially in target proteins, mutation events per samples, mutation rate, and overall scenario of coronaviruses across the country. To investigate the genetic diversity, a total of 184 genomes of virus strains sampled from different divisions of Bangladesh with sampling dates between the 10th of May 2020 and the 27th of June 2020 were analyzed. To date, a total of 634 mutations located along the entire genome resulting in non-synonymous 274 amino acid substitutions in 22 different proteins were detected with nucleotide mutation rate estimated to be 23.715 substitutions per year. The highest non-synonymous amino acid substitutions were observed at 48 different positions of the papain-like protease (nsp3). Although no mutations were found in nsp7, nsp9, nsp10, and nsp11, yet orf1ab accounts for 56% of total mutations. Among the structural proteins, the highest non-synonymous amino acid substitution (at 36 positions) observed in spike proteins, in which 9 unique locations were detected relative to the global strains, including 516E>Q in the boundary of the ACE2 binding region. The most dominated variant G614 (95%) based in spike protein is circulating across the country with co-evolving other variants including L323 (94%) in RNA dependent RNA polymerase (RdRp), K203 (82%) and R204 (82%) in nucleocapsid, and F120 (78%) in NSP2. These variants are mostly seen as linked mutations and are part of a haplotype observed in Europe. Data suggest effective containment of clade G strains (4.8%) with sub-clusters GR 82.4%, and GH clade 6.4%.HighlightsWe have sequenced 137 and analyzed 184 whole-genomes sequences of SARS-CoV-2 strains from different divisions of Bangladesh.A total of 634 mutation sites across the SARS-CoV-2 genome and 274 non-synonymous amino acid substitutions were detected.The mutation rate of SARS-CoV-2 estimated to be 23.715 nucleotide substitutions per year.Nine unique variants were detected based on non-anonymous amino acid substitutions in spike protein relative to the global SARS-CoV-2 strains.

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Cleavage Inhibition of the Murine Coronavirus Spike Protein by a Furin-Like Enzyme Affects Cell-Cell but Not Virus-Cell Fusion

Journal of Virology ◽

10.1128/jvi.78.11.6048-6054.2004 ◽

2004 ◽

Vol 78 (11) ◽

pp. 6048-6054 ◽

Cited By ~ 100

Author(s):

Cornelis A. M. de Haan ◽

Konrad Stadler ◽

Gert-Jan Godeke ◽

Berend Jan Bosch ◽

Peter J. M. Rottier

Keyword(s):

Cell Fusion ◽

Spike Protein ◽

Dependent Manner ◽

Protein Cleavage ◽

Concentration Dependent Manner ◽

Cell Cell ◽

Murine Coronavirus

ABSTRACT Cleavage of the mouse hepatitis coronavirus strain A59 spike protein was blocked in a concentration-dependent manner by a peptide furin inhibitor, indicating that furin or a furin-like enzyme is responsible for this process. While cell-cell fusion was clearly affected by preventing spike protein cleavage, virus-cell fusion was not, indicating that these events have different requirements.

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