scholarly journals Effects of Single α-to-β Residue Replacements on Recognition of an Extended Segment in a Viral Fusion Protein

2020 ◽  
Author(s):  
Victor Outlaw ◽  
Dale F. Kreitler ◽  
Debora Stelitano ◽  
Matteo Porotto ◽  
Anne Moscona ◽  
...  
Keyword(s):  
Amino Acid ◽  
Terminal Segment ◽  
Viral Envelope ◽  
Partial Replacement ◽  
Viral Fusion ◽  
Amino Acid Residues ◽  
Target Engagement ◽  
Viral Fusion Protein ◽  
Target Cell Membrane ◽  
The Impact

Partial replacement of α-amino acid residues with β-amino acid residues has been established as a strategy for preserving target-engagement by helix-forming polypeptides while suppressing susceptibility to proteolysis. The impact of β-residue incorporation within polypeptides that adopt less regular conformations, however, has received less attention. The HRC domains of fusion glycoproteins from pathogenic paramyxoviruses contain a segment that must adopt an extended conformation in order to engage the HRN domain in the post-fusion state and drive merger of the viral envelope with a target cell membrane. Here we examine the impact of single α-to-β substi-tutions within this extended N-terminal segment of the engineered HRC peptide VIQKI. Stabilities of helix-bundles formed with a native viral HRN have been evaluated, the structures of five helix-bundles have been determined, and antiviral efficacies have been measured. Many sites within the extended segment show functional tolerance of α-to-β substitution. These results offer a basis for future develop-ment of protease-resistant inhibitors of paramyxovirus infection.

scholarly journals Effects of Single α-to-β Residue Replacements on Recognition of an Extended Segment in a Viral Fusion Protein

2020 ◽  
Author(s):  
Victor Outlaw ◽  
Dale F. Kreitler ◽  
Debora Stelitano ◽  
Matteo Porotto ◽  
Anne Moscona ◽  
...  
Keyword(s):  
Amino Acid ◽  
Terminal Segment ◽  
Viral Envelope ◽  
Partial Replacement ◽  
Viral Fusion ◽  
Amino Acid Residues ◽  
Target Engagement ◽  
Viral Fusion Protein ◽  
Target Cell Membrane ◽  
The Impact

Partial replacement of α-amino acid residues with β-amino acid residues has been established as a strategy for preserving target-engagement by helix-forming polypeptides while suppressing susceptibility to proteolysis. The impact of β-residue incorporation within polypeptides that adopt less regular conformations, however, has received less attention. The HRC domains of fusion glycoproteins from pathogenic paramyxoviruses contain a segment that must adopt an extended conformation in order to engage the HRN domain in the post-fusion state and drive merger of the viral envelope with a target cell membrane. Here we examine the impact of single α-to-β substi-tutions within this extended N-terminal segment of the engineered HRC peptide VIQKI. Stabilities of helix-bundles formed with a native viral HRN have been evaluated, the structures of five helix-bundles have been determined, and antiviral efficacies have been measured. Many sites within the extended segment show functional tolerance of α-to-β substitution. These results offer a basis for future develop-ment of protease-resistant inhibitors of paramyxovirus infection.

Hydrophobic-at-Interface Regions in Viral Fusion Protein Ectodomains

2000 ◽  
Vol 20 (6) ◽  
pp. 519-533 ◽  
Author(s):  
José L. Nieva ◽  
Tatiana Suárez
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Viral Envelope ◽  
Fusion Pore ◽  
Viral Fusion ◽  
New Approach ◽  
Viral Fusion Protein ◽  
Membrane Partitioning ◽  
Viral Glycoproteins ◽  
New Procedures

In this chapter we shall describe how to apply the hydrophobicity-at-interface scale, as proposed by Wimley and White [Wimley, W. C. and White, S. H. (1996) Nature Struct. Biol. 3:842–848], to the detection of amino acid sequences of viral envelope glycoproteins putatively engaged in interactions with the target membranes. In addition, a new approach will be briefly introduced to infer the bilayer location at equilibrium of membrane-partitioning sequences. The use of these new procedures may be important in describing the molecular mechanism leading to the formation of a fusion pore by viral glycoproteins.

scholarly journals Novel antibody binding determinants on the capsid surface of serotype O foot-and-mouth disease virus

2014 ◽  
Vol 95 (5) ◽  
pp. 1104-1116 ◽  
Author(s):  
Amin S. Asfor ◽  
Sasmita Upadhyaya ◽  
Nick J. Knowles ◽  
Donald P. King ◽  
David J. Paton ◽  
...  
Keyword(s):  
Amino Acid ◽  
Guinea Pig ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Amino Acid Residues ◽  
Serotype O ◽  
Antigenic Sites ◽  
Mouth Disease Virus ◽  
Foot And Mouth ◽  
The Impact

Five neutralizing antigenic sites have been described for serotype O foot-and-mouth disease viruses (FMDV) based on monoclonal antibody (mAb) escape mutant studies. However, a mutant virus selected to escape neutralization of mAb binding at all five sites was previously shown to confer complete cross-protection with the parental virus in guinea pig challenge studies, suggesting that amino acid residues outside the mAb binding sites contribute to antibody-mediated in vivo neutralization of FMDV. Comparison of the ability of bovine antisera to neutralize a panel of serotype O FMDV identified three novel putative sites at VP2-74, VP2-191 and VP3-85, where amino acid substitutions correlated with changes in sero-reactivity. The impact of these positions was tested using site-directed mutagenesis to effect substitutions at critical amino acid residues within an infectious copy of FMDV O1 Kaufbeuren (O1K). Recovered viruses containing additional mutations at VP2-74 and VP2-191 exhibited greater resistance to neutralization with both O1K guinea pig and O BFS bovine antisera than a virus that was engineered to include only mutations at the five known antigenic sites. The changes at VP2-74 and VP3-85 are adjacent to critical amino acids that define antigenic sites 2 and 4, respectively. However VP2-191 (17 Å away from VP2-72), located at the threefold axis and more distant from previously identified antigenic sites, exhibited the most profound effect. These findings extend our knowledge of the surface features of the FMDV capsid known to elicit neutralizing antibodies, and will improve our strategies for vaccine strain selection and rational vaccine design.

scholarly journals Regulation of N-linked core glycosylation: use of a site-directed mutagenesis approach to identify Asn-Xaa-Ser/Thr sequons that are poor oligosaccharide acceptors

1997 ◽  
Vol 323 (2) ◽  
pp. 415-419 ◽  
Author(s):  
Lakshmi KASTURI ◽  
Hegang CHEN ◽  
Susan H. SHAKIN-ESHLEMAN
Keyword(s):  
Amino Acid ◽  
Amino Acid Residue ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Free Translation ◽  
A Cell ◽  
And Function ◽  
A Site ◽  
The Impact

N-linked glycosylation can profoundly affect protein expression and function. N-linked glycosylation usually occurs at the sequon Asn-Xaa-Ser/Thr, where Xaa is any amino acid residue except Pro. However, many Asn-Xaa-Ser/Thr sequons are glycosylated inefficiently or not at all for reasons that are poorly understood. We have used a site-directed mutagenesis approach to examine how the Xaa and hydroxy (Ser/Thr) amino acid residues in sequons influence core-glycosylation efficiency. We recently demonstrated that certain Xaa amino acids inhibit core glycosylation of the sequon, Asn37-Xaa-Ser, in rabies virus glycoprotein (RGP). Here we examine the impact of different Xaa residues on core-glycosylation efficiency when the Ser residue in this sequon is replaced with Thr. The core-glycosylation efficiencies of RGP variants with different Asn37-Xaa-Ser/Thr sequons were compared by using a cell-free translation/glycosylation system. Using this approach we confirm that four Asn-Xaa-Ser sequons are poor oligosaccharide acceptors: Asn-Trp-Ser, Asn-Asp-Ser, Asn-Glu-Ser and Asn-Leu-Ser. In contrast, Asn-Xaa-Thr sequons are efficiently glycosylated, even when Xaa = Trp, Asp, Glu or Leu. A comparison of the glycosylation status of Asn-Xaa-Ser and Asn-Xaa-Thr sequons in other glycoproteins confirms that sequons with Xaa = Trp, Asp, Glu or Leu are rarely glycosylated when Ser is the hydroxy amino acid residue, and that these sequons are unlikely to serve as glycosylation sites when introduced into proteins by site-directed mutagenesis.

Modulation of procaspase-7 self-activation by PEST amino acid residues of the N-terminal prodomain and intersubunit linker

2017 ◽  
Vol 95 (6) ◽  
pp. 634-643
Author(s):  
Juliano Alves ◽  
Miguel Garay-Malpartida ◽  
João M. Occhiucci ◽  
José E. Belizário
Keyword(s):  
Amino Acid ◽  
Large Subunit ◽  
Small Subunit ◽  
Cleavage Sites ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Caspase 7 ◽  
Caspase Family ◽  
The Impact

Procaspase-7 zymogen polypeptide is composed of a short prodomain, a large subunit (p20), and a small subunit (p10) connected to an intersubunit linker. Caspase-7 is activated by an initiator caspase-8 and -9, or by autocatalysis after specific cleavage at IQAD198↓S located at the intersubunit linker. Previously, we identified that PEST regions made of amino acid residues Pro (P), Glu (E), Asp (D), Ser (S), Thr (T), Asn (N), and Gln (Q) are conserved flanking amino acid residues in the cleavage sites within a prodomain and intersubunit linker of all caspase family members. Here we tested the impact of alanine substitution of PEST amino acid residues on procaspase-7 proteolytic self-activation directly in Escherichia coli. The p20 and p10 subunit cleavage were significantly delayed in double caspase-7 mutants in the prodomain (N18A/P26A) and intersubunit linker (S199A/P201A), compared with the wild-type caspase-7. The S199A/P201A mutants effectively inhibited the p10 small subunit cleavage. However, the mutations did not change the kinetic parameters (kcat/KM) and optimal tetrapeptide specificity (DEVD) of the purified mutant enzymes. The results suggest a role of PEST-amino acid residues in the molecular mechanism for prodomain and intersubunit cleavage and caspase-7 self-activation.

scholarly journals Identification of Amino Acid Residues in CD81 Critical for Interaction with Hepatitis C Virus Envelope Glycoprotein E2

2000 ◽  
Vol 74 (8) ◽  
pp. 3642-3649 ◽  
Author(s):  
Adrian Higginbottom ◽  
Elizabeth R. Quinn ◽  
Chiung-Chi Kuo ◽  
Mike Flint ◽  
Louise H. Wilson ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Envelope Glycoprotein ◽  
Tertiary Structure ◽  
Antibody Binding ◽  
Viral Envelope ◽  
Amino Acid Residues ◽  
Virus Envelope ◽  
Glycoprotein E2

ABSTRACT Human CD81 has been previously identified as the putative receptor for the hepatitis C virus envelope glycoprotein E2. The large extracellular loop (LEL) of human CD81 differs in four amino acid residues from that of the African green monkey (AGM), which does not bind E2. We mutated each of the four positions in human CD81 to the corresponding AGM residues and expressed them as soluble fusion LEL proteins in bacteria or as complete membrane proteins in mammalian cells. We found human amino acid 186 to be critical for the interaction with the viral envelope glycoprotein. This residue was also important for binding of certain anti-CD81 monoclonal antibodies. Mutating residues 188 and 196 did not affect E2 or antibody binding. Interestingly, mutation of residue 163 increased both E2 and antibody binding, suggesting that this amino acid contributes to the tertiary structure of CD81 and its ligand-binding ability. These observations have implications for the design of soluble high-affinity molecules that could target the CD81-E2 interaction site(s).

scholarly journals The N-terminal amino acid sequence of pig kidney endopeptidase-24.11 shows homology with pro-sucrase-isomaltase

1986 ◽  
Vol 240 (1) ◽  
pp. 305-308 ◽  
Author(s):  
I S Fulcher ◽  
D J Pappin ◽  
A J Kenny
Keyword(s):  
Amino Acid ◽  
Solid Phase ◽  
Consensus Sequence ◽  
Terminal Segment ◽  
Amino Acid Residues ◽  
Endopeptidase 24.11 ◽  
Cytoplasmic Face ◽  
Arginine Residues ◽  
Terminal Amino ◽  
Hydrophobic Anchor

Endopeptidase-24.11 (EC 3.4.24.11), a widely distributed ectoenzyme, was isolated from pig kidneys by detergent solubilization of membranes and immuno-affinity chromatography. In all, 12 preparations of the enzyme were submitted to solid-phase sequencing, yielding a consensus sequence of 25 amino acid residues of the N-terminal segment. Some samples were treated with either trypsin or Staphylococcus aureus V8 proteinase before sequencing. There were four lysine and one arginine residues in the first nine positions. This segment was susceptible to hydrolysis by trypsin and, in some samples, to endogenous proteinases. From residue 19 onwards, the sequence became intensely hydrophobic. There was a striking homology with the N-terminal sequence of pro-sucrase-isomaltase. From Lys7 to Leu20 there were seven identical amino acid residues and four conservative substitutions. We suggest that endopeptidase-24.11 is topologically similar to this glycosidase, the N-terminus at the cytoplasmic face and hydrophobic segment serving the roles of both signal peptide and hydrophobic anchor.

scholarly journals Characterization of X- box binding protein 1(XBP1) and BiP functions and investigation their roles in YHV replication in P. monodon

2021 ◽  
Vol 20 (4) ◽  
Author(s):  
Piyawadee Rugsanit ◽  
◽  
Pongsopee Attasart ◽  
Apinunt Udomkit ◽  
Witoon Tirasophon ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Amino Acid ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Amino Acid Residues ◽  
Total Change ◽  
Yellow Head Virus ◽  
Black Tiger Shrimp ◽  
Tiger Shrimp ◽  
The Impact

Abstract Eukaryotic cells have mechanisms to cope with stress in endoplasmic reticulum (ER) called unfolded protein response (UPR). In this study, we characterized putative X-box DNA binding protein1 (XBP1) and Binding protein (BiP) cDNAs from black tiger shrimp (Peneaus monodon). When the shrimp were infected with Yellow head virus (YHV), the levels of XBP1 and BiP mRNA transcripts were elevated approximately 8 and 55 folds, respectively. In normal shrimp, the putative XBP1 (XBP1u) was predicted to encode a protein with 283 amino acid residues. When shrimp were infected with YHV, portion of cDNA with 17 nucleotides intron elimination (XBP1s) was observed. The elimination caused alteration of its translational frame. The predicted protein from this is 469 amino acids in length with total change in its amino acid sequence downstream of the intron. Functional analysis of these XBP1 proteins in mammalian cells clearly showed that overexpression of P. monodon XBP1s was capable of activating the transcription rate of mammalian UPR responsive genes (BiP, ERdj4 and P58IPK). Finally, the impact of XBP1 and BiP on YHV multiplication in black tiger shrimp was investigated by RNAi approach. Knocking down either XBP1 or BiP expression efficiently inhibited YHV replication. Therefore, these two components in the UPR pathway may be an interesting target for anti YHV development in the future. Keywords: Endoplasmic reticulum, siRNA, Stress-inducible genes, Transcriptional regulation

scholarly journals Ultrashort Cationic Lipopeptides–Effect of N-Terminal Amino Acid and Fatty Acid Type on Antimicrobial Activity and Hemolysis

Molecules ◽  
2020 ◽  
Vol 25 (2) ◽  
pp. 257 ◽  
Author(s):  
Damian Neubauer ◽  
Maciej Jaśkiewicz ◽  
Marta Bauer ◽  
Krzysztof Gołacki ◽  
Wojciech Kamysz
Keyword(s):  
Fatty Acid ◽  
Amino Acid ◽  
Antimicrobial Agents ◽  
Basic Amino Acid ◽  
Amino Acid Residues ◽  
Bacterial Strains ◽  
Fatty Acid Chain ◽  
N Terminus ◽  
Arginine Residues ◽  
The Impact

Ultrashort cationic lipopeptides (USCLs) are promising antimicrobial agents that hypothetically may be alternatively used to combat pathogens such as bacteria and fungi. In general, USCLs consist of fatty acid chains and a few basic amino acid residues. The main shortcoming of USCLs is their relatively high cytotoxicity and hemolytic activity. This study focuses on the impact of the hydrophobic fatty acid chain, on both antimicrobial and hemolytic activities. To learn more about this region, a series of USCLs with different straight-chain fatty acids (C8, C10, C12, C14) attached to the tripeptide with two arginine residues were synthesized. The amino acid at the N-terminal position was exchanged for proteinogenic and non-proteinogenic amino acid residues (24 in total). Moreover, the branched fatty acid residues were conjugated to N-terminus of a dipeptide with two arginine residues. All USCLs had C-terminal amides. USCLs were tested against reference bacterial strains (including ESKAPE group) and Candida albicans. The hemolytic potential was tested on human erythrocytes. Hydrophobicity of the compounds was evaluated by RP-HPLC. Shortening of the fatty acid chain and simultaneous addition of amino acid residue at N-terminus were expected to result in more selective and active compounds than those of the reference lipopeptides with similar lipophilicity. Hypothetically, this approach would also be beneficial to other antimicrobial peptides where N-lipidation strategy was used to improve their biological characteristics.

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

2016 ◽  
Vol 90 (12) ◽  
pp. 5586-5600 ◽  
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.

Sign in / Sign up

Export Citation Format

Share Document