Enhanced Cholesterol-dependent Hemifusion by Internal Fusion Peptide 1 of SARS Coronavirus-2 Compared to its N-terminal Counterpart

ABSTRACTMembrane fusion is an important step for the entry of the lipid-sheathed viruses into the host cells. The fusion process is being carried out by fusion proteins present in the viral envelope. The class I viruses contains a 20-25 amino acid sequence at its N-terminal of the fusion domain, which is instrumental in fusion, and is termed as ‘fusion peptide’. However, Severe Acute Respiratory Syndrome Coronavirus (SARS) coronaviruses contain more than one fusion peptide sequences. We have shown that the internal fusion peptide 1 (IFP1) of SARS-CoV is far more efficient than its N-terminal counterpart (FP) to induce hemifusion between small unilamellar vesicles. Moreover, the ability of IFP1 to induce hemifusion formation increases dramatically with growing cholesterol content in the membrane. Interestingly, IFP1 is capable of inducing hemifusion, but fails to open pore.

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Identification of the Fusion Peptide-Containing Region in Betacoronavirus Spike Glycoproteins

Journal of Virology ◽

10.1128/jvi.00015-16 ◽

2016 ◽

Vol 90 (12) ◽

pp. 5586-5600 ◽

Cited By ~ 30

Author(s):

Xiuyuan Ou ◽

Wangliang Zheng ◽

Yiwei Shan ◽

Zhixia Mu ◽

Samuel R. Dominguez ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Cell Fusion ◽

Fusion Proteins ◽

Virus Entry ◽

Amino Acid Sequences ◽

Viral Envelope ◽

Class I ◽

Viral Fusion ◽

Viral Fusion Proteins

ABSTRACTThe fusion peptides (FP) play an essential role in fusion of viral envelope with cellular membranes. The location and properties of the FPs in the spike (S) glycoproteins of different coronaviruses (CoV) have not yet been determined. Through amino acid sequence analysis of S proteins of representative CoVs, we identified a common region as a possible FP (pFP) that shares the characteristics of FPs of class I viral fusion proteins, including high Ala/Gly content, intermediate hydrophobicity, and few charged residues. To test the hypothesis that this region contains the CoV FP, we systemically mutated every residue in the pFP of Middle East respiratory syndrome betacoronavirus (MERS-CoV) and found that 11 of the 22 residues in the pFP (from G953 to L964, except for A956) were essential for S protein-mediated cell-cell fusion and virus entry. The synthetic MERS-CoV pFP core peptide (955IAGVGWTAGL964) induced extensive fusion of liposome membranes, while mutant peptide failed to induce any lipid mixing. We also selectively mutated residues in pFPs of two other β-CoVs, severe acute respiratory syndrome coronavirus (SARS-CoV) and mouse hepatitis virus (MHV). Although the amino acid sequences of these two pFPs differed significantly from that of MERS-CoV and each other, most of the pFP mutants of SARS-CoV and MHV also failed to mediate membrane fusion, suggesting that these pFPs are also the functional FPs. Thus, the FPs of 3 different lineages of β-CoVs are conserved in location within the S glycoproteins and in their functions, although their amino acid sequences have diverged significantly during CoV evolution.IMPORTANCEWithin the class I viral fusion proteins of many enveloped viruses, the FP is the critical mediator of fusion of the viral envelope with host cell membranes leading to virus infection. FPs from within a virus family, like influenza viruses or human immunodeficiency viruses (HIV), tend to share high amino acid sequence identity. In this study, we determined the location and amino acid sequences of the FPs of S glycoproteins of 3 β-CoVs, MERS-CoV, SARS-CoV, and MHV, and demonstrated that they were essential for mediating cell-cell fusion and virus entry. Interestingly, in marked contrast to the FPs of influenza and HIV, the primary amino acid sequences of the FPs of β-CoVs in 3 different lineages differed significantly. Thus, during evolution the FPs of β-CoVs have diverged significantly in their primary sequences while maintaining the same essential biological functions. Our findings identify a potential new target for development of drugs against CoVs.

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Antibodies that inhibit fusion of human immunodeficiency virus-infected cells bind a 24-amino acid sequence of the viral envelope, gp120.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.85.9.3198 ◽

1988 ◽

Vol 85 (9) ◽

pp. 3198-3202 ◽

Cited By ~ 325

Author(s):

J. R. Rusche ◽

K. Javaherian ◽

C. McDanal ◽

J. Petro ◽

D. L. Lynn ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Amino Acid ◽

Amino Acid Sequence ◽

Viral Envelope ◽

Immunodeficiency Virus ◽

Infected Cells ◽

Envelope Gp120

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Cloning and Characterization of the Ferulic Acid Catabolic Genes of Sphingomonas paucimobilis SYK-6

Applied and Environmental Microbiology ◽

10.1128/aem.68.9.4416-4424.2002 ◽

2002 ◽

Vol 68 (9) ◽

pp. 4416-4424 ◽

Cited By ~ 62

Author(s):

Eiji Masai ◽

Kyo Harada ◽

Xue Peng ◽

Hirotaka Kitayama ◽

Yoshihiro Katayama ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Ferulic Acid ◽

Sinapinic Acid ◽

Host Cells ◽

Acid Decarboxylase ◽

Sphingomonas Paucimobilis ◽

Sequencing Analysis ◽

Catabolic Genes ◽

Insertional Inactivation

ABSTRACT Sphingomonas paucimobilis SYK-6 degrades ferulic acid to vanillin, and it is further metabolized through the protocatechuate 4,5-cleavage pathway. We obtained a Tn5 mutant of SYK-6, FA2, which was able to grow on vanillic acid but not on ferulic acid. A cosmid which complemented the growth deficiency of FA2 on ferulic acid was isolated. The 5.2-kb BamHI-EcoRI fragment in this cosmid conferred the transformation activity of ferulic acid to vanillin on Escherichia coli host cells. A sequencing analysis revealed the genes ferB and ferA in this fragment; these genes consist of 852- and 2,127-bp open reading frames, respectively. The deduced amino acid sequence of ferB showed 40 to 48% identity with that of the feruloyl-coenzyme A (CoA) hydratase/lyase genes of Pseudomonas and Amycolatopsis ferulic acid degraders. On the other hand, the deduced amino acid sequence of ferA showed no significant similarity to the feruloyl-CoA synthetase genes of other ferulic acid degraders. However, the deduced amino acid sequence of ferA did show 31% identity with pimeloyl-CoA synthetase of Pseudomonas mendocina 35, which has been classified as a new superfamily of acyl-CoA synthetase (ADP forming) with succinyl-CoA synthetase (L. B. Sánchez, M. Y. Galperin, and M. Müller, J. Biol. Chem. 275:5794-5803, 2000). On the basis of the enzyme activity of E. coli carrying each of these genes, ferA and ferB were shown to encode a feruloyl-CoA synthetase and feruloyl-CoA hydratase/lyase, respectively. p-coumaric acid, caffeic acid, and sinapinic acid were converted to their corresponding benzaldehyde derivatives by the cell extract containing FerA and FerB, thereby indicating their broad substrate specificities. We found a ferB homolog, ferB2, upstream of a 5-carboxyvanillic acid decarboxylase gene (ligW) involved in the degradation of 5,5′-dehydrodivanillic acid. The deduced amino acid sequence of ferB2 showed 49% identity with ferB, and its gene product showed feruloyl-CoA hydratase/lyase activity with a substrate specificity similar to that of FerB. Insertional inactivation of each fer gene in S. paucimobilis SYK-6 suggested that the ferA gene is essential and that ferB and ferB2 genes are involved in ferulic acid degradation.

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Identification of a F.VIII epitope recognized by a human hemophilic inhibitor

Blood ◽

10.1182/blood.v73.2.497.497 ◽

1989 ◽

Vol 73 (2) ◽

pp. 497-499 ◽

Cited By ~ 20

Author(s):

BC Lubahn ◽

J Ware ◽

DW Stafford ◽

HM Reisner

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Factor Viii ◽

Fusion Proteins ◽

Cleavage Site ◽

Hemophilia A ◽

Antibody Binding ◽

Bleeding Disorders ◽

Enzymatic Cleavage ◽

Coagulant Activity

Abstract Hemophilia A, one of the most common of the inherited bleeding disorders, results from a deficiency or abnormality of factor VIII (F.VIII). In approximately 15% of persons with hemophilia, treatment with exogenous F.VIII is complicated by the development of anti-F.VIII antibodies which block F.VIII coagulant activity. These antibodies have been termed inhibitors. To localize epitopes recognized by inhibitors, we used a lambda gt11 library which expresses small random fragments of F.VIII as fusion proteins. One epitope has been mapped to the 25-amino acid sequence lys-338 through asp-362 of F.VIII (E338–362). Immunoaffinity-purified antibodies that react with this epitope neutralize F.VIII:C activity. E338–362 is adjacent to an enzymatic cleavage site at arg-372 which is important in F.VIII activation. Hence, an antibody binding to E338–362 would probably block this cleavage and thereby block activation of F.VIII.

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Identification of a F.VIII epitope recognized by a human hemophilic inhibitor

Blood ◽

10.1182/blood.v73.2.497.bloodjournal732497 ◽

1989 ◽

Vol 73 (2) ◽

pp. 497-499

Author(s):

BC Lubahn ◽

J Ware ◽

DW Stafford ◽

HM Reisner

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Factor Viii ◽

Fusion Proteins ◽

Cleavage Site ◽

Hemophilia A ◽

Antibody Binding ◽

Bleeding Disorders ◽

Enzymatic Cleavage ◽

Coagulant Activity

Hemophilia A, one of the most common of the inherited bleeding disorders, results from a deficiency or abnormality of factor VIII (F.VIII). In approximately 15% of persons with hemophilia, treatment with exogenous F.VIII is complicated by the development of anti-F.VIII antibodies which block F.VIII coagulant activity. These antibodies have been termed inhibitors. To localize epitopes recognized by inhibitors, we used a lambda gt11 library which expresses small random fragments of F.VIII as fusion proteins. One epitope has been mapped to the 25-amino acid sequence lys-338 through asp-362 of F.VIII (E338–362). Immunoaffinity-purified antibodies that react with this epitope neutralize F.VIII:C activity. E338–362 is adjacent to an enzymatic cleavage site at arg-372 which is important in F.VIII activation. Hence, an antibody binding to E338–362 would probably block this cleavage and thereby block activation of F.VIII.

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Antibodies that inhibit fusion of human immunodeficiency virus-infected cells bind a 24-amino acid sequence of the viral envelope, gp120

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.85.22.8697 ◽

1988 ◽

Vol 85 (22) ◽

pp. 8697-8697 ◽

Cited By ~ 1

Keyword(s):

Human Immunodeficiency Virus ◽

Amino Acid ◽

Amino Acid Sequence ◽

Viral Envelope ◽

Immunodeficiency Virus ◽

Infected Cells ◽

Envelope Gp120

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Isolation and characterization of different C-terminal fragments of dystrophin expressed in Escherichia coli

Biochemical Journal ◽

10.1042/bj2881037 ◽

1992 ◽

Vol 288 (3) ◽

pp. 1037-1044 ◽

Cited By ~ 21

Author(s):

R E Milner ◽

J Busaan ◽

M Michalak

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Amino Acid Sequence ◽

Fusion Proteins ◽

Cytoskeletal Proteins ◽

Terminal Region ◽

Soluble Form ◽

E Coli ◽

Terminal Domain ◽

Isolation And Characterization

Dystrophin, the protein product of the Duchenne muscular dystrophy gene, is thought to belong to a family of membrane cytoskeletal proteins. Based on its deduced amino-acid sequence, it is postulated to have several distinct structural domains; an N-terminal region; a central, rod-shaped, domain; and a C-terminal domain [Koenig, Monaco & Kunkel (1988) Cell 53, 219-228]. The C-terminal domain is further divided into two regions; the first has some sequence similarity to slime mould alpha-actinin, and is rich in cysteine residues; this is followed by the C-terminal amino-acid sequence that is unique to dystrophin. Dystrophin is very difficult to purify in quantities sufficient for detailed studies of the structure/function relationships within the molecule. Therefore, in this study, we have expressed selected fragments of the C-terminal region of dystrophin, as fusion proteins, in Escherichia coli. Importantly, we describe the first successful purification, from E. coli lysates, of large quantities of fragments of dystrophin in a soluble form. The first fragment, termed CT-1, encodes the C-terminal 201 amino acids of the protein; the second, termed CT-2, spans the cysteine-rich region of the C-terminal domain. These fusion proteins were identified by their mobility in SDS/PAGE, by their interaction with appropriate affinity columns and by their reactivity with anti-dystrophin antibodies. The fragment CT-2, which spans a region containing putative EF-hand-like sequences, was found to bind Ca2+ in 45Ca2+ overlay experiments. In addition, we have discovered that the fragment CT-1, but not fragment CT-2, interacts specifically with the E. coli DnaK gene product [analogue of heat shock protein 70 (hsp70)]. This interaction is disrupted, in vitro, by the addition of ATP. Our results indicate that the two C-terminal fragments of dystrophin have differing biophysical properties, indicating that they may play distinct roles in the function of the protein.

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Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM

10.1101/2020.06.30.177097 ◽

2020 ◽

Cited By ~ 4

Author(s):

Cong Xu ◽

Yanxing Wang ◽

Caixuan Liu ◽

Chao Zhang ◽

Wenyu Han ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Receptor Binding ◽

Viral Entry ◽

Conformational Dynamics ◽

Conformational Transitions ◽

Fusion Peptide ◽

Host Cells ◽

Structural Basis ◽

Receptor Binding Domain ◽

Viral Determinants

AbstractThe recent outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its rapid international spread pose a global health emergency. The trimeric spike (S) glycoprotein interacts with its receptor human ACE2 to mediate viral entry into host-cells. Here we present cryo-EM structures of an uncharacterized tightly closed SARS-CoV-2 S-trimer and the ACE2-bound-S-trimer at 2.7-Å and 3.8-Å-resolution, respectively. The tightly closed S-trimer with inactivated fusion peptide may represent the ground prefusion state. ACE2 binding to the up receptor-binding domain (RBD) within S-trimer triggers continuous swing-motions of ACE2-RBD, resulting in conformational dynamics of S1 subunits. Noteworthy, SARS-CoV-2 S-trimer appears much more sensitive to ACE2-receptor than SARS-CoV S-trimer in terms of receptor-triggered transformation from the closed prefusion state to the fusion-prone open state, potentially contributing to the superior infectivity of SARS-CoV-2. We defined the RBD T470-T478 loop and residue Y505 as viral determinants for specific recognition of SARS-CoV-2 RBD by ACE2, and provided structural basis of the spike D614G-mutation induced enhanced infectivity. Our findings offer a thorough picture on the mechanism of ACE2-induced conformational transitions of S-trimer from ground prefusion state towards postfusion state, thereby providing important information for development of vaccines and therapeutics aimed to block receptor binding.

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