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 Human Coronavirus 229E Papain-Like Proteases Have Overlapping Specificities but Distinct Functions in Viral Replication

2007 ◽  
Vol 81 (8) ◽  
pp. 3922-3932 ◽  
Author(s):  
John Ziebuhr ◽  
Barbara Schelle ◽  
Nadja Karl ◽  
Ekaterina Minskaia ◽  
Sonja Bayer ◽  
...  
Keyword(s):  
Cell Culture ◽  
Viral Replication ◽  
Active Site ◽  
Reverse Genetics ◽  
Cysteine Proteases ◽  
Proteolytic Processing ◽  
Human Coronavirus ◽  
Mutant Genotype ◽  
Amino Terminal ◽  
Human Coronavirus 229E

ABSTRACT Expression of the exceptionally large RNA genomes of CoVs involves multiple regulatory mechanisms, including extensive proteolytic processing of the large replicase polyproteins, pp1a and pp1ab, by two types of cysteine proteases: the chymotrypsin-like main protease and papain-like accessory proteases (PLpros). Here, we characterized the proteolytic processing of the human coronavirus 229E (HCoV-229E) amino-proximal pp1a/pp1ab region by two paralogous PLpro activities. Reverse-genetics data revealed that replacement of the PL2pro active-site cysteine was lethal. By contrast, the PL1pro activity proved to be dispensable for HCoV-229E virus replication, although reversion of the PL1pro active-site substitution to the wild-type sequence after several passages in cell culture indicated that there was selection pressure to restore the PL1pro activity. Further experiments showed that both PL1pro and PL2pro were able to cleave the nsp1-nsp2 cleavage site, with PL2pro cleaving the site less efficiently. The PL1pro-negative mutant genotype could be stably maintained in cell culture when the nsp1-nsp2 site was replaced by a short autoproteolytic sequence, suggesting that the major driving force for the observed reversion of the PL1pro mutation was the requirement for efficient nsp1-nsp2 cleavage. The data suggest that the two HCoV-229E PLpro paralogs have overlapping substrate specificities but different functions in viral replication. Within the tightly controlled interplay of the two protease activities, PL2pro plays a universal and essential proteolytic role that appears to be assisted by the PL1pro paralog at specific sites. Functional and evolutionary implications of the differential amino-terminal polyprotein-processing pathways among the main CoV lineages are discussed.

scholarly journals Import of rat ornithine transcarbamylase precursor into mitochondria: two-step processing of the leader peptide.

1987 ◽  
Vol 105 (6) ◽  
pp. 2631-2639 ◽  
Author(s):  
E S Sztul ◽  
J P Hendrick ◽  
J P Kraus ◽  
D Wall ◽  
F Kalousek ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cleavage Site ◽  
Liver Mitochondria ◽  
Essential Elements ◽  
Ornithine Transcarbamylase ◽  
Proteolytic Processing ◽  
Leader Peptide ◽  
Rat Liver Mitochondria

The mitochondrial matrix enzyme ornithine transcarbamylase (OTC) is synthesized on cytoplasmic polyribosomes as a precursor (pOTC) with an NH2-terminal extension of 32 amino acids. We report here that rat pOTC synthesized in vitro is internalized and cleaved by isolated rat liver mitochondria in two, temporally separate steps. In the first step, which is dependent upon an intact mitochondrial membrane potential, pOTC is translocated into mitochondria and cleaved by a matrix protease to a product designated iOTC, intermediate in size between pOTC and mature OTC. This product is in a trypsin-protected mitochondrial location. The same intermediate-sized OTC is produced in vivo in frog oocytes injected with in vitro-synthesized pOTC. The proteolytic processing of pOTC to iOTC involves the removal of 24 amino acids from the NH2 terminus of the precursor and utilizes a cleavage site two residues away from a critical arginine residue at position 23. In a second cleavage step, also catalyzed by a matrix protease, iOTC is converted to mature OTC by removal of the remaining eight residues of leader sequence. To define the critical regions in the OTC leader peptide required for these events, we have synthesized OTC precursors with alterations in the leader. Substitution of either an acidic (aspartate) or a "helix-breaking" (glycine) amino acid residue for arginine 23 of the leader inhibits formation of both iOTC and OTC, without affecting translocation. These mutant precursors are cleaved at an otherwise cryptic cleavage site between residues 16 and 17 of the leader. Interestingly, this cleavage occurs at a site two residues away from an arginine at position 15. The data indicate that conversion of pOTC to mature OTC proceeds via the formation of a third discrete species: an intermediate-sized OTC. The data suggest further that, in the rat pOTC leader, the essential elements required for translocation differ from those necessary for correct cleavage to either iOTC or mature OTC.

scholarly journals Primary structure requirements for correct sorting of the yeast mitochondrial protein ADH III to the yeast mitochondrial matrix space.

1987 ◽  
Vol 7 (1) ◽  
pp. 294-304 ◽  
Author(s):  
D Pilgrim ◽  
E T Young
Keyword(s):  
Amino Acids ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Proteolytic Processing ◽  
Mitochondrial Matrix ◽  
Wild Type ◽  
Mature Form ◽  
Amino Terminal ◽  
The Matrix

Alcohol dehydrogenase isoenzyme III (ADH III) in Saccharomyces cerevisiae, the product of the ADH3 gene, is located in the mitochondrial matrix. The ADH III protein was synthesized as a larger precursor in vitro when the gene was transcribed with the SP6 promoter and translated with a reticulocyte lysate. A precursor of the same size was detected when radioactively pulse-labeled proteins were immunoprecipitated with anti-ADH antibody. This precursor was rapidly processed to the mature form in vivo with a half-time of less than 3 min. The processing was blocked if the mitochondria were uncoupled with carbonyl cyanide m-chlorophenylhydrazone. Mutant enzymes in which only the amino-terminal 14 or 16 amino acids of the presequence were retained were correctly targeted and imported into the matrix. A mutant enzyme that was missing the amino-terminal 17 amino acids of the presequence produced an active enzyme, but the majority of the enzyme activity remained in the cytoplasmic compartment on cellular fractionation. Random amino acid changes were produced in the wild-type presequence by bisulfite mutagenesis of the ADH3 gene. The resulting ADH III protein was targeted to the mitochondria and imported into the matrix in all of the mutants tested, as judged by enzyme activity. Mutants containing amino acid changes in the carboxyl-proximal half of the ADH3 presequence were imported and processed to the mature form at a slower rate than the wild type, as judged by pulse-chase studies in vivo. The unprocessed precursor appeared to be unstable in vivo. It was concluded that only a small portion of the presequence contains the necessary information for correct targeting and import. Furthermore, the information for correct proteolytic processing of the presequence appears to be distinct from the targeting information and may involve secondary structure information in the presequence.

scholarly journals The Herpes Simplex Virus Type 1 UL20 Protein and the Amino Terminus of Glycoprotein K (gK) Physically Interact with gB

2010 ◽  
Vol 84 (17) ◽  
pp. 8596-8606 ◽  
Author(s):  
Vladimir N. Chouljenko ◽  
Arun V. Iyer ◽  
Sona Chowdhury ◽  
Joohyun Kim ◽  
Konstantin G. Kousoulas
Keyword(s):  
Amino Acids ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Herpes Simplex Virus Type ◽  
Protein Complex ◽  
Amino Terminus ◽  
Amino Terminal ◽  
Glycoprotein K ◽  
Simplex Virus

ABSTRACT Herpes simplex virus type 1 (HSV-1) glycoprotein K (gK) and the UL20 protein (UL20p) are strictly required for virus-induced cell fusion, and mutations within either the gK or UL20 gene cause extensive cell fusion (syncytium formation). We have shown that gK forms a functional protein complex with UL20p, which is required for all gK and UL20p-associated functions in the HSV-1 life cycle. Recently, we showed that the amino-terminal 82 amino acids (aa) of gK (gKa) were required for the expression of the syncytial phenotype of the mutant virus gBΔ28 lacking the carboxyl-terminal 28 amino acids of gB (V. N. Chouljenko, A. V. Iyer, S. Chowdhury, D. V. Chouljenko, and K. G. Kousoulas, J. Virol. 83:12301-12313, 2009). This work suggested that the amino terminus of gK may directly or indirectly interact with gB and/or other viral glycoproteins. Two-way coimmunoprecipitation experiments revealed that UL20p interacted with gB in infected cells. Furthermore, the gKa peptide was coimmunoprecipitated with gB but not gD. Three recombinant baculoviruses were constructed, expressing the amino-terminal 82 aa of gKa together with either the extracellular portion of gB (30 to 748 aa), gD (1 to 340 aa), or gH (1 to 792 aa), respectively. Coimmunoprecipitation experiments revealed that gKa physically interacted with the extracellular portions of gB and gH but not gD. Three additional recombinant baculoviruses expressing gKa and truncated gBs encompassing aa 30 to 154, 30 to 364, and 30 to 500 were constructed. Coimmunoprecipitation experiments showed that gKa physically interacted with all three truncated gBs. Computer-assisted prediction of possible gKa binding sites on gB suggested that gKa may interact predominantly with gB domain I (E. E. Heldwein, H. Lou, F. C. Bender, G. H. Cohen, R. J. Eisenberg, and S. C. Harrison, Science 313:217-220, 2006). These results imply that the gK/UL20p protein complex modulates the fusogenic properties of gB and gH via direct physical interactions.

Mitotic arrest caused by the amino terminus of Xenopus cyclin B2

1993 ◽  
Vol 13 (3) ◽  
pp. 1480-1488
Author(s):  
H M van der Velden ◽  
M J Lohka
Keyword(s):  
Sea Urchin ◽  
Kinase Activity ◽  
Histone H1 ◽  
Amino Terminus ◽  
Protein Kinase Activity ◽  
Truncated Protein ◽  
Amino Terminal ◽  
Egg Extracts ◽  
The Stability

Progression through mitosis requires the inactivation of the protein kinase activity of the p34cdc2-cyclin complex by a mechanism involving the degradation of cyclin. We have examined the stability in Xenopus egg extracts of radiolabeled Xenopus or sea urchin B-type cyclins synthesized in reticulocyte lysates. Xenopus cyclin B2 and sea urchin cyclin B were stable in metaphase extracts from unfertilized eggs but were specifically degraded following addition of Ca2+ to the extracts. The degradation of either cyclin was inhibited by the addition of an excess of unlabeled Xenopus cyclin B2 but not by the addition of a number of control proteins. A truncated protein containing only the amino terminus of Xenopus cyclin B2, including sequences known to be essential for cyclin degradation in other species, also inhibited cyclin degradation, even though the truncated protein was stable in extracts following Ca2+ addition. The addition of the truncated protein did not stimulate histone H1 kinase activity in extracts but prevented the loss of H1 kinase activity that normally follows Ca2+ addition to metaphase extracts. When the amino-terminal fragment was added to extracts capable of several cell cycles in vitro, progression through the first mitosis was inhibited and elevated histone H1 kinase activity was maintained. These results indicate that although the amino terminus of cyclin does not contain all of the information necessary for cyclin destruction, it is capable of interacting with components of the cyclin destruction pathway and thereby preventing the degradation of full-length cyclins.

DIRECTED MUTAGENESIS IN THE STUDY OF THE REQUIREMENTS FOR FACTOR VIII ACTIVITY IN VITRO AND IN VIVO

1987 ◽  
Author(s):  
Randal J Kaufman ◽  
Debra D Pittman ◽  
Louise C Wasley ◽  
W Barry Foster ◽  
Godfrey W Amphlett ◽  
...  
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Cleavage Site ◽  
Factor Xa ◽  
Cleavage Sites ◽  
Thrombin Cleavage ◽  
Terminal Fragment ◽  
Cofactor Activity

Factor VIII is a high molecular weight plasma glycoprotein that functions in the blood clotting cascade as the cofactor for factor DCa proteolytic activation of factor X. Factor VIII does not function proteolytically in this reaction hut itself can be proteolytically activated by other coagulation enzymes such as factor Xa and thrombin. In the plasma, factor VIII exists as a 200 kDa amino-terminal fragment in a metal ion stabilized complex with a 76 kDa carboxy-terminal fragment. The isolation of the cENA for human factor VIII provided the deduced primary amino acid sequence of factor VIIT and revealed three distinct structural domains: 1) a triplicated A domain of 330 amino acids which has homology to ceruloplasmin, a plasma copper binding protein, 2) a duplicated C domain of 150 amino acids, and 3) a unique B domain of 980 amino acids. These domains are arranged as shown below. We have previously reported the B domain is dispensible far cofactor activity in vitro (Toole et al. 1986 Proc. Natl. Acad 5939). The in vivo efficacy of factor VIII molecules harboring the B domain deletion was tested by purification of the wildtype and modified forms and infusion into factor VIII deficient, hemophilic, dogs. The wildtype and the deleted forms of recombinant derived factor VIII exhibited very similar survival curves (Tl/2 = 13 hrs) and the cuticle bleeding times suggested that both preparations appeared functionally equivalent. Sepharose 4B chromatography indicated that both factor VIII molecules were capable of binding canine plasma vWF.Further studies have addressed what cleavages are necessary for activation of factor VIII. The position of the thrombin, factor Xa, and activated protein C (AFC) cleavage sites within factor VIII are presented below, site-directed ENA medicated mutagenesis has been performed to modify the arginine at the amino side of each cleavagesite to an soleucine. In all cases this modification resulted in molecules that were resistant to cleavage by thrombin at the modified site. Modification of the thrombin cleavage sites at 336 and 740 and modification of the factor Xa cleavage site at 1721 resulted in no loss of cofactor activity. Modification of the thrombin cleavage site at either 372 or 1689 destroyed oofactor activity. Modification of the thrombin cleavage site at 336 resulted in a factor VIII having an increased activity, possibly due to resistance to inactivation. These results suggest the requirement of cleavage at residues 372 and 1689 for cofactor activity.

scholarly journals Negative Regulation of DNA Replication by the Retinoblastoma Protein Is Mediated by Its Association with MCM7

1998 ◽  
Vol 18 (5) ◽  
pp. 2748-2757 ◽  
Author(s):  
Jacqueline M. Sterner ◽  
Susan Dew-Knight ◽  
Christine Musahl ◽  
Sally Kornbluth ◽  
Jonathan M. Horowitz
Keyword(s):  
Amino Acids ◽  
Dna Replication ◽  
Protein Complexes ◽  
Replication Assay ◽  
Amino Terminal ◽  
Licensing Factor ◽  
Human Proteins ◽  
Carboxy Terminal ◽  
Related Proteins

ABSTRACT A yeast two-hybrid screen was employed to identify human proteins that specifically bind the amino-terminal 400 amino acids of the retinoblastoma (Rb) protein. Two independent cDNAs resulting from this screen were found to encode the carboxy-terminal 137 amino acids of MCM7, a member of a family of proteins that comprise replication licensing factor. Full-length Rb and MCM7 form protein complexes in vitro, and the amino termini of two Rb-related proteins, p107 and p130, also bind MCM7. Protein complexes between Rb and MCM7 were also detected in anti-Rb immunoprecipitates prepared from human cells. The amino-termini of Rb and p130 strongly inhibited DNA replication in an MCM7-dependent fashion in a Xenopus in vitro DNA replication assay system. These data provide the first evidence that Rb and Rb-related proteins can directly regulate DNA replication and that components of licensing factor are targets of the products of tumor suppressor genes.

Amino-terminal TACE prodomain attenuates TNFR2 cleavage independently of the cysteine switch

2005 ◽  
Vol 288 (6) ◽  
pp. L1132-L1138 ◽  
Author(s):  
Caitriona A. Buckley ◽  
Farshid N. Rouhani ◽  
Maryann Kaler ◽  
Barbara Adamik ◽  
Feras I. Hawari ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Inhibitory Activity ◽  
Catalytic Domain ◽  
Amino Terminus ◽  
Mucin Production ◽  
Inactive State ◽  
Amino Terminal ◽  
Switch Mechanism ◽  
Cysteine Switch

TNF-α-converting enzyme (TACE, ADAM17) cleaves membrane-associated cytokines and receptors and thereby regulates inflammatory and immune events, as well as lung development and mucin production. For example, the TACE-mediated cleavage of the type II 75-kDa TNF receptor (TNFR2) generates a soluble TNF-binding protein that modulates TNF bioactivity. TACE is synthesized as a latent proenzyme that is retained in an inactive state via an interaction between its prodomain and catalytic domain. Although the formation of an intramolecular bond between a cysteine in the prodomain and a zinc atom in the catalytic site had been thought to mediate this inhibitory activity, it was recently reported that the cysteine-switch motif is not required. Here, we hypothesized that the amino terminus of the TACE prodomain might contribute to the ability of the prodomain to maintain TACE in an inactive state independently of a cysteine-switch mechanism. We synthesized a 37-amino acid peptide corresponding to TACE amino acids 18–54 (N-TACE18–54) and assessed whether it possessed TACE inhibitory activity. In an in vitro model assay system, N-TACE18–54 attenuated TACE-catalyzed cleavage of a TNFR2:Fc substrate. Furthermore, N-TACE18–54 inhibited constitutive TNFR2 shedding from a human monocytic cell line by 42%. A 19-amino acid, leucine-rich domain, corresponding to TACE amino acids 30–48, demonstrated partial inhibitory activity. In summary, we have identified a subdomain within the amino terminus of the TACE prodomain that attenuates TACE catalytic activity independently of a cysteine-switch mechanism, which provides new insight into the regulation of TACE enzymatic activity.

scholarly journals A Cyclin-Binding Motif within the Amino-Terminal Homology Domain of EBNA3C Binds Cyclin A and Modulates Cyclin A-Dependent Kinase Activity in Epstein-Barr Virus-Infected Cells

2004 ◽  
Vol 78 (23) ◽  
pp. 12857-12867 ◽  
Author(s):  
Jason S. Knight ◽  
Nikhil Sharma ◽  
Danielle E. Kalman ◽  
Erle S. Robertson
Keyword(s):  
Amino Acids ◽  
Epstein Barr Virus ◽  
Kinase Activity ◽  
Cyclin A ◽  
Carboxy Terminus ◽  
Barr Virus ◽  
Amino Terminal ◽  
Infected Cells ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is a virus-encoded latent antigen essential for primary B-cell transformation. In this report we demonstrate that although the carboxy terminus of EBNA3C predominantly regulates cyclin A-dependent kinase activity, the region of greatest affinity for cyclin A lies within the EBNA3 amino-terminal homology domain of EBNA3C. Detailed mapping studies employing both in vitro binding assays and coimmunoprecipitation experiments implicated a small region of EBNA3C, amino acids 130 to 159 within the EBNA3 homology domain, as having the greatest affinity for cyclin A. The EBNA3 homology domain has the highest degree of amino acid similarity (approximately 30%) between the EBNA3 proteins, and, indeed, EBNA3B, but not EBNA3A, showed binding activity with cyclin A. We also show that EBNA3C binds to the α1 helix of the highly conserved mammalian cyclin box, with cyclin A amino acids 206 to 226 required for strong binding to EBNA3C amino acids 130 to 159. Interestingly, EBNA3C also bound human cyclins D1 and E in vitro, although the affinity was approximately 30% of that seen for cyclin A. Previously it was demonstrated that full-length EBNA3C rescues p27-mediated suppression of cyclin A-dependent kinase activity (J. S. Knight and E. S. Robertson, J. Virol. 78:1981-1991, 2004). It was also demonstrated that the carboxy terminus of EBNA3C recapitulates this phenotype. Surprisingly, the amino terminus of EBNA3C with the highest affinity for cyclin A was unable to rescue p27 suppression of kinase activity and actually downregulates cyclin A activity when introduced into EBV-infected cells. The data presented here suggests that the amino terminus of EBNA3C may play an important role in recruiting cyclin A complexes, while the carboxy terminus of EBNA3C is necessary for the functional modulation of cyclin A complex kinase activity.

scholarly journals The influenza hemagglutinin insertion signal is not cleaved and does not halt translocation when presented to the endoplasmic reticulum membrane as part of a translocating polypeptide.

1987 ◽  
Vol 104 (6) ◽  
pp. 1705-1714 ◽  
Author(s):  
J Finidori ◽  
L Rizzolo ◽  
A Gonzalez ◽  
G Kreibich ◽  
M Adesnik ◽  
...  
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Signal Sequence ◽  
In Vitro Translation ◽  
Signal Peptidase ◽  
Endoplasmic Reticulum Membrane ◽  
Hydrophobic Region ◽  
Amino Terminal ◽  
Influenza Hemagglutinin

The co-translational insertion of polypeptides into endoplasmic reticulum membranes may be initiated by cleavable amino-terminal insertion signals, as well as by permanent insertion signals located at the amino-terminus or in the interior of a polypeptide. To determine whether the location of an insertion signal within a polypeptide affects its function, possibly by affecting its capacity to achieve a loop disposition during its insertion into the membrane, we have investigated the functional properties of relocated insertion signals within chimeric polypeptides. An artificial gene encoding a polypeptide (THA-HA), consisting of the luminal domain of the influenza hemagglutinin preceded by its amino-terminal signal sequence and linked at its carboxy-terminus to an intact prehemagglutinin polypeptide, was constructed and expressed in in vitro translation systems containing microsomal membranes. As expected, the amino-terminal signal initiated co-translational insertion of the hybrid polypeptide into the membranes. The second, identical, interiorized signal, however, was not recognized by the signal peptidase and was translocated across the membrane. The failure of the interiorized signal to be cleaved may be attributed to the fact that it enters the membrane as part of a translocating polypeptide and therefore cannot achieve the loop configuration that is thought to be adopted by signals that initiate insertion. The finding that the interiorized signal did not halt translocation of downstream sequences, even though it contains a hydrophobic region and must enter the membrane in the same configuration as natural stop-transfer signals, indicates that the HA insertion signal lacks essential elements of halt transfer signals that makes the latter effective membrane-anchoring domains. When the amino-terminal insertion signal of the THA-HA chimera was deleted, the interior signal was incapable of mediating insertion, probably because of steric hindrance by the folded preceding portions of the chimera. Several chimeras were constructed in which the interiorized signal was preceded by polypeptide segments of various lengths. A signal preceded by a segment of 111 amino acids was also incapable of initiating insertion, but insertion took place normally when the segment preceding the signal was only 11-amino acids long.(ABSTRACT TRUNCATED AT 400 WORDS)

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