scholarly journals Identification of the Murine Coronavirus MP1 Cleavage Site Recognized by Papain-Like Proteinase 2

2003 ◽  
Vol 77 (13) ◽  
pp. 7376-7382 ◽  
Author(s):  
Amornrat Kanjanahaluethai ◽  
Dalia Jukneliene ◽  
Susan C. Baker
Keyword(s):  
Cleavage Site ◽  
Edman Degradation ◽  
Putative Cleavage Site ◽  
Amino Terminal ◽  
Cleavage Assay ◽  
Degradation Reaction ◽  
Methionine Residues ◽  
Identification And Characterization ◽  
Murine Coronavirus

ABSTRACT The replicase polyprotein of murine coronavirus is extensively processed by three proteinases, two papain-like proteinases (PLPs), termed PLP1 and PLP2, and a picornavirus 3C-like proteinase (3CLpro). Previously, we established a trans-cleavage assay and showed that PLP2 cleaves the replicase polyprotein between p210 and membrane protein 1 (MP1) (A. Kanjanahaluethai and S. C. Baker, J. Virol. 74:7911-7921, 2000). Here, we report the results of our studies identifying and characterizing this cleavage site. To determine the approximate position of the cleavage site, we expressed constructs that extended various distances upstream from the previously defined C-terminal end of MP1. We found that the construct extending from the putative PLP2 cleavage site at glycine 2840-alanine 2841 was most similar in size to the processed MP1 replicase product generated in a trans-cleavage assay. To determine which amino acids are critical for PLP2 recognition and processing, we generated 14 constructs with amino acid substitutions upstream and downstream of the putative cleavage site and assessed the effects of the mutations in the PLP2 trans-cleavage assay. We found that substitutions at phenylalanine 2835, glycine 2839, or glycine 2840 resulted in a reduction in cleavage of MP1. Finally, to unequivocally identify this cleavage site, we isolated radiolabeled MP1 protein and determined the position of [35S]methionine residues released by Edman degradation reaction. We found that the amino-terminal residue of MP1 corresponds to alanine 2841. Therefore, murine coronavirus PLP2 cleaves the replicase polyprotein between glycine 2840 and alanine 2841, and the critical determinants for PLP2 recognition and processing occupy the P6, P2, and P1 positions of the cleavage site. This study is the first report of the identification and characterization of a cleavage site recognized by murine coronavirus PLP2 activity.

scholarly journals Identification and Characterization of a Cleavage Site in the Proteolysis of Orf Virus 086 Protein

2016 ◽  
Vol 7 ◽  
Author(s):  
Xiaoping Wang ◽  
Bin Xiao ◽  
Jiafeng Zhang ◽  
Daxiang Chen ◽  
Wei Li ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Orf Virus ◽  
Identification And Characterization

scholarly journals Identification and characterization of a serine-like proteinase of the murine coronavirus MHV-A59.

1995 ◽  
Vol 69 (6) ◽  
pp. 3554-3559 ◽  
Author(s):  
Y Lu ◽  
X Lu ◽  
M R Denison
Keyword(s):  
Identification And Characterization ◽  
Murine Coronavirus

Identification and Characterization of a Divalent Metal Ion-Dependent Cleavage Site in the Hammerhead Ribozyme†

Biochemistry ◽  
2001 ◽  
Vol 40 (46) ◽  
pp. 13849-13856 ◽  
Author(s):  
John C. Markley ◽  
Frédéric Godde ◽  
Snorri Th. Sigurdsson
Keyword(s):  
Cleavage Site ◽  
Metal Ion ◽  
Hammerhead Ribozyme ◽  
Divalent Metal ◽  
Divalent Metal Ion ◽  
Identification And Characterization

Proteolytic Processing at the Amino Terminus of Human Coronavirus 229E Gene 1-Encoded Polyproteins: Identification of a Papain-Like Proteinase and Its Substrate

1998 ◽  
Vol 72 (2) ◽  
pp. 910-918 ◽  
Author(s):  
Jens Herold ◽  
Alexander E. Gorbalenya ◽  
Volker Thiel ◽  
Barbara Schelle ◽  
Stuart G. Siddell
Keyword(s):  
Amino Acids ◽  
Cleavage Site ◽  
Cysteine Proteinase ◽  
Proteolytic Processing ◽  
Amino Terminus ◽  
Human Coronavirus ◽  
Amino Terminal ◽  
Cleavage Assay ◽  
Human Coronavirus 229E

ABSTRACT Expression of the coronavirus gene 1-encoded polyproteins, pp1a and pp1ab, is linked to a series of proteolytic events involving virus-encoded proteinases. In this study, we used transfection and immunoprecipitation assays to show that the human coronavirus 229E-encoded papain-like cysteine proteinase, PCP1, is responsible for the release of an amino-terminal protein, p9, from the gene 1-encoded polyproteins. The same protein, p9, has also been identified in virus-infected cells. Furthermore, using an in vitrotrans-cleavage assay, we defined the proteolytic cleavage site at the carboxyl terminus of p9 as pp1a-pp1ab amino acids Gly-111 and Asn-112. These results and a comparative sequence analysis suggest that substrate positions P1 and P5 seem to be the major determinants of the PCP1 cleavage site and that the latter can occupy a variable position at the amino terminus of the coronavirus pp1a and pp1ab polyproteins. By combining the trans-cleavage assay with deletion mutagenesis, we were also able to locate the boundaries of the active PCP1 domain between pp1a-pp1ab amino acids Gly-861–Glu-975 and Asn-1209–Gln-1285. Finally, codon mutagenesis was used to show that Cys-1054 and His-1205 are essential for PCP1 proteolytic activity, suggesting that these amino acids most likely have a catalytic function.

scholarly journals Identification and Characterization of a 65-kDa Protein Processed from the Gene 1 Polyprotein of the Murine Coronavirus MHV-A59

Virology ◽  
1995 ◽  
Vol 207 (1) ◽  
pp. 316-320 ◽  
Author(s):  
Mark R. Denison ◽  
Scott A. Hughes ◽  
Susan R. Weiss
Keyword(s):  
Identification And Characterization ◽  
Murine Coronavirus

scholarly journals Identification and Characterization of a Proteolytically Primed Form of the Murine Coronavirus Spike Proteins after Fusion with the Target Cell

2014 ◽  
Vol 88 (9) ◽  
pp. 4943-4952 ◽  
Author(s):  
O. Wicht ◽  
C. Burkard ◽  
C. A. M. de Haan ◽  
F. J. M. van Kuppeveld ◽  
P. J. M. Rottier ◽  
...  
Keyword(s):  
Target Cell ◽  
Identification And Characterization ◽  
Murine Coronavirus ◽  
Spike Proteins

Characterization of a Mode of Specific Binding of Fibrin Monomer through its Amino-Terminal Domain by Macrophages and Macrophage Cell-Lines

1990 ◽  
Vol 63 (02) ◽  
pp. 193-203 ◽  
Author(s):  
John R Shainoff ◽  
Deborah J Stearns ◽  
Patricia M DiBello ◽  
Youko Hishikawa-Itoh
Keyword(s):  
Peritoneal Macrophages ◽  
Affinity Binding ◽  
Macrophage Cell ◽  
High Affinity Binding ◽  
Amino Terminal ◽  
High Affinity ◽  
Terminal Domain ◽  
Weak Binding ◽  
Amino Terminal Domain

SummaryThe studies reported here probe the existence of a receptor-mediated mode of fibrin-binding by macrophages that is associated with the chemical change underlying the fibrinogen-fibrin conversion (the release of fibrinopeptides from the amino-terminal domain) without depending on fibrin-aggregation. The question is pursued by 1) characterization of binding in relation to fibrinopeptide content of both the intact protein and the CNBr-fragment comprising the amino-terminal domain known as the NDSK of the protein, 2) tests of competition for binding sites, and 3) photo-affinity labeling of macrophage surface proteins. The binding of intact monomers of types lacking either fibrinopeptide A alone (α-fibrin) or both fibrinopeptides A and B (αβ-fibrin) by peritoneal macrophages is characterized as proceeding through both a fibrin-specific low density/high affinity (BMAX ≃ 200–800 molecules/cell, KD ≃ 10−12 M) interaction that is not duplicated with fibrinogen, and a non-specific high density/low affinity (BMAX ≥ 105 molecules/cell, KD ≥ 10−6 M) interaction equivalent to the weak binding of fibrinogen. Similar binding characteristics are displayed by monocyte/macrophage cell lines (J774A.1 and U937) as well as peritoneal macrophages towards the NDSK preparations of these proteins, except for a slightly weaker (KD ≃ 10−10 M) high-affinity binding. The high affinity binding of intact monomer is inhibitable by fibrin-NDSK, but not fibrinogen-NDSK. This binding appears principally dependent on release of fibrinopeptide-A, because a species of fibrin (β-fibrin) lacking fibrinopeptide-B alone undergoes only weak binding similar to that of fibrinogen. Synthetic Gly-Pro-Arg and Gly-His-Arg-Pro corresponding to the N-termini of to the α- and the β-chains of fibrin both inhibit the high affinity binding of the fibrin-NDSKs, and the cell-adhesion peptide Arg-Gly-Asp does not. Photoaffinity-labeling experiments indicate that polypeptides with elec-trophoretically estimated masses of 124 and 187 kDa are the principal membrane components associated with specifically bound fibrin-NDSK. The binding could not be up-regulated with either phorbol myristyl acetate, interferon gamma or ADP, but was abolished by EDTA and by lipopolysaccharide. Because of the low BMAX, it is suggested that the high-affinity mode of binding characterized here would be too limited to function by itself in scavenging much fibrin, but may act cooperatively with other, less limited modes of fibrin binding.

Identification and characterization of peptidases secreted by quahog parasite unknown (QPX), the protistan parasite of hard clams

2016 ◽  
Vol 122 (1) ◽  
pp. 21-33 ◽  
Author(s):  
E Rubin ◽  
GT Werneburg ◽  
E Pales Espinosa ◽  
DG Thanassi ◽  
B Allam
Keyword(s):  
Quahog Parasite Unknown ◽  
Hard Clams ◽  
Identification And Characterization
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