©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
gp180, a Host Cell Glycoprotein That Binds Duck Hepatitis B Virus Particles, Is Encoded by a Member of the Carboxypeptidase Gene Family (*)

Kazuyuki Kuroki (1)(§), Frank Eng (2), Takashi Ishikawa (2), Christoph Turck (3)(¶), Fumio Harada (1), Don Ganem (2) (3)(¶)

From the (1)Department of Biophysics, Cancer Research Institute, Kanazawa University, 13-1 Takaramachi, Kanazawa, Ishikawa 920 Japan and Howard Hughes Medical Institute, Departments of (2)Microbiology and (3)Medicine, University of California, San Francisco, California 94143-0502

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Duck hepatitis B virus particles bearing the L and S envelope proteins bind a cellular glycoprotein of 180 kDa (gp180) with high affinity and specificity. Binding is mediated by the pre-S region of the L protein and is blocked by neutralizing but not by non-neutralizing monoclonal antibodies to the virus. These and other properties have suggested that gp180 may be a component of the viral entry machinery. Here we report the purification of gp180 from duck liver and the isolation and characterization of cDNA encoding it. DNA sequence analysis of this cDNA indicates that gp180 is a novel member of the basic carboxypeptidase gene family.


INTRODUCTION

Hepadnaviruses are small enveloped DNA viruses that produce persistent infections of liver cells and cause acute and chronic hepatitis(1) . The prototype member of this virus family is human hepatitis B virus, but related viruses are also found in a variety of other vertebrate species(2) . Although much is known about hepadnaviral genomic replication and gene expression, little is known about the mechanisms by which these viruses enter their host cells. In particular, the cell surface receptor(s) for all of these viruses remain unidentified. We have been attempting to identify components of the hepadnaviral entry pathway, using the duck hepatitis B virus (DHBV)()as a model system. The avian virus was chosen because primary hepatocytes explanted directly from duck liver efficiently support infection with DHBV virions(3, 4) ; for the mammalian viruses, similar primary hepatocytes are poorly available and inefficiently infectable(5, 6) . These primary duck hepatocytes provide both a convenient source of starting material for receptor identification as well as a basis for infectivity assays essential to test the in vivo role of candidate receptor proteins.

We have previously reported that a 180-kDa duck cell glycoprotein (gp180) binds DHBV particles with high affinity and specificity(7) . The species distribution of this protein mirrors the known host range of the virus, and gp180 binding is blocked by neutralizing but not by non-neutralizing monoclonal antibodies to the viral envelope(7) . Of the two viral envelope proteins (L and S)(8, 9) , binding occurs only to the L protein and can be localized to a 65-amino acid region within the so-called pre-S domain of L(10) , a domain previously shown to harbor important determinants for host cell binding(11) . All of these properties are consistent with a potential role for the protein in DHBV infection; however, definitive assessment of the role of gp180 in entry will require further characterization of the protein and its gene. Here we report the purification of gp180 and the isolation of cDNA clones encoding this protein. Nucleotide sequence analysis of this cDNA indicates that the gp180 coding region encodes a novel member of the basic carboxypeptidase gene family.


MATERIALS AND METHODS

Purification of gp180 from Duck Liver

Duck liver cut into small pieces was added to homogenization buffer (50 mM Tris, pH 7.4, 150 mM NaCl) (5 ml/g of liver; typically multiple aliquots of 8 g of tissue/aliquot were extracted) and then homogenized in a Polytron homogenizer (three 30-s bursts on setting 4). The homogenate was clarified by centrifugation for 10 min at 600 g (in an HG-4L rotor at 1500 rpm.) The supernatant was collected and then centrifuged for 10 min at 12,000 g (in an SS34 rotor at 10,000 rpm). The supernatant from this step was then centrifuged for 90 min in a 60 Ti rotor at 37,500 rpm (100,000 g) to generate a microsomal pellet. This pellet was resuspended in homogenization buffer (with the aid of the Polytron homogenizer), and the solution was adjusted to a protein concentration of 1 mg/ml and a final concentration of 1.0% Triton X-100 and incubated on ice for 60 min. Insoluble material was removed by centrifugation for 90 min at 100,000 g; supernatants were then frozen at -80 °C until used.

The supernatant generated from 500 g of liver was then passed over a pre-S-glutathione S-transferase affinity column. DHBV pre-S-glutathione S-transferase fusion protein BE1 (see Ref. 10) was expressed in E. coli, and fusion protein from 4 liters of culture was batch adsorbed onto 5 ml of glutathione-Sepharose as described previously(12) . Liver extract was passed over the column, and the column was then washed with 4 bed volumes of homogenization buffer plus 1% Triton X-100; bound proteins were eluted in 20 mM glutathione (in 60 mM Tris, pH 8.0, 150 mM NaCl, 1% Triton X-100). Fractions were examined by SDS-polyacrylamide gel electrophoresis and Coomassie Brilliant Blue staining, and fractions positive for gp180 were pooled and concentrated by Centricon 100 filtration. This material was adjusted to 1 mM CaCl and 1 mM MnCl and then applied to a 5-ml lentil lectin-Sepharose 4B column (Sigma) equilibrated with 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1 mM CaCl, and 1 mM MnCl; bound material was eluted with homogenization buffer containing 0.1% Triton X-100 and 200 mM -methyl -D-mannopyranoside. This material was then subjected to preparative electrophoresis through 6% SDS-polyacrylamide gels, and the 180-kDa band was identified by staining with Coomassie Brilliant Blue. This preparation method yielded 0.4-0.8 µg of gp180/g of extracted liver.

Proteolysis and Amino Acid Sequence Determination

The 180-kDa protein was recovered from the preparative SDS-polyacrylamide gels by electroelution, precipitated with acetone, and redissolved in 70% formic acid for cleavage with CNBr (12 h). Subsequently the protein fragments were further digested with trypsin at 37 °C for 12 h. Tryptic cleavage fragments were separated on a C18 reversed phase column (Vydac, Hesperia, CA), and selected peaks were subjected to protein sequence analysis with an Applied Biosystems Sequencer, Model 475A (Applied Biosystems, Foster City, CA).

Preparation and Screening of cDNA Libraries

A plasmid-based cDNA library from duck liver poly(A) RNA was prepared by the method of Okayama and Berg(13) , using a plasmid, pSVH, bearing an SV40 origin and early promoter. The plasmid was derived from pSV45H (14) by deletion of a 1.4-kb internal Bam fragment. This plasmid was cleaved with PstI, and poly(dT) tails were added to the resulting 3` termini by incubation with terminal transferase and dTTP. The upstream poly(dT) tail was removed by cleavage at a unique HindIII site 5` to the original PstI site. To this dT-tailed plasmid was annealed poly(A) RNA from uninfected duck liver, and cDNA synthesis was effected by addition of all four dNTPs and recombinant Moloney murine leukemia virus reverse transcriptase (cDNA Synthesis System Plus, Life Technologies, Inc.). Following repair of the termini by incubation with T4 DNA polymerase, products with inserts of about 4 kb or greater in length were isolated by agarose gel electrophoresis, self-ligated, and cloned into E. coli strain HB101.

Colonies were screened by hybridization with degenerate synthetic oligonucleotides labeled at their 5` ends with P by incubation with T4 polynucleotide kinase and [-32P]ATP. The oligonucleotides were derived from the sequences of the peptides isolated for purified gp180; those that were successfully employed were 46 MR (5`-TT(A/G)TC(T/C)TG(A/G/T/C)AG(A/G)AA(A/G/T/C)CC(A/G)TG(A/G/T/C)AC-3`) com-plementary to peptide 3 and 61MR (5`-TA(A/G)TT(A/G/T/C)(G/C)(A/T)(A/G/T/C)GT(A/G)AA(A/G)TC(A/G/T/C)GT(A/G)TC-3`) complementary to peptide 5. This screen yielded one clone, A23 (from about 10,000 colonies screened); this clone contained an approximately 4.3-kb insert that annealed to both oligonucleotides. This clone comprised 2.5 kb of 3`-noncoding sequences and 1.8 kb of coding sequence from the COOH-terminal region of gp180 (Fig. 1). The DNA sequence of A23 confirmed that it indeed encoded both peptides from which the screening oligos were derived. An oligonucleotide (5`-GTTCTCTATGATGGCTTTGGTCTCA-3`) complementary to sequence from the 5` region of A23 was used to prime cDNA synthesis from duck liver poly(A) RNA, and double-stranded cDNA products were cloned into gt11. This library was then screened with A23 probes to identify the overlapping clones J2 and J13 (see Fig. 1). To isolate more 5`-coding sequences, another cDNA library was prepared by priming cDNA synthesis with oligo-primer 2 (5`-GGCAGCAGGTACAGGTCGGTGGTGT-3`) complementary to the 5` region of clone J2. Screening of this library with J2 sequences yielded additional clones J39 and J31. Sequencing of these cDNAs revealed that the coding region was still open at the 5` extremity of the cDNA, indicating missing 5` sequences. All attempts to clone the residual 5` sequences (estimated by comparison with the mRNA size on Northern blots to be about 200 nucleotides) by cDNA cloning and by the 5` rapid amplification of cDNA ends method (15) failed. It was anticipated that the missing sequences would contain both the initiator methionine and the signal peptide, essential elements for correct expression of the cDNA. To allow reconstruction of a functional cDNA, the missing sequences were supplied from genomic clones. A Sau3AI partial digest of DNA from duck embryo fibroblasts was cloned into EMBL3 and was screened with sequences from J31 (see Fig. 1) to yield clones DG1, DG2, and DG7, the last of which clearly spanned the 5` end of the cDNA. To reconstitute the 5` end of the cDNA (see Fig. 2), the following fragments (listed in order from the 5` end of the gene) were ligated together: NcoI-BglI from DG7; BglI-HindIII from J31; HindIII-BglII from J2 (this was the product of a BglII partial digest); and BglII-NcoI from A23. To construct the full-length gp180 expression vectors, the NcoI fragment bearing the entire gp180 coding region was cloned into (i) the unique EcoRI site of pSVH (after blunting all termini with T4 DNA polymerase), to generate pSV180, and (ii) the SmaI site of pBKRSV (Stratagene), to generate pKRSV180. In the case of pKRSV180 (Fig. 2) the lac promoter sequences just downstream of the Rous sarcoma virus promoter (located between NheI and XbaI) were removed by digestion and religation.


Figure 1: Cloning of gp180. Bottom, black bars denote extent of cDNA clones encoding the indicated portions of the gp180 cDNA. pA23 and pD6 are plasmid-based clones; the remaining clones were isolated in phage . Primers 1 and 2 are described in the text and were used to prime cDNA synthesis for the indicated subjacent cDNA clones. probe denotes extent of sequences from clone J31 used to screen genomic clones to reconstruct the 5` end of the gp180 coding region. Center, the structure of the gp180 cDNA. Stippledbox, coding region; openbox, 3` noncoding region. Top, clones of the genomic gp180 locus. Darklines denote overlapping clones bearing genomic DNA fragments annealing to gp180 cDNA probes. A region of clone DG7 is highlighted; a fragment from this region was used to reconstruct the 5` end of the gp180 coding region, which was unclonable as cDNA.




Figure 2: Construction of a full-length gp180 expression vector (pKRSV180). Center, the structure of the gp180 expression vector. Blacklines, vector sequences including the Rous sarcoma virus promoter (RSV promoter) and SV40 polyadenylation sequences as indicated. Stippledbox, gp180 coding region, with indicated restriction sites. Above and below the pKRSV180diagram are depicted the genomic and cDNA clones (respectively) used to reconstruct the vector. Dottedlines denote the contributing fragments from each clone. Note that the entire coding region can be mobilized on a single NcoI fragment. This fact was used to construct the related plasmid pSV180, in which the gp180 coding region is subcloned between the SV40 early promoter and the hepatitis B virus poly(A) signal.



DNA Sequence Analysis

Multiple fragments of gp180 cDNA and genomic DNA were cloned into pGEM3Zf(+), and their DNA sequences were determined on an ABI 370A DNA sequencer. Nucleotide and amino acid sequences were analyzed with the DNASIS software (Hitachi Software Engineering, Yokohama, Japan).


RESULTS

Purification of gp180 from Duck Liver

Our initial protocol for the purification of gp180 from duck liver was based on our earlier analytical procedure for identification of the protein in cultured hepatocytes(7) . The liver was first homogenized in nonionic detergent; after removal of cell and nuclear debris by low speed centrifugation, the cell extract was passed over a DHBV pre-S affinity column created by the binding of a pre-S-protein A fusion protein to IgG-Sepharose. Following elution of the bound material in 2 M MgCl, the eluate was then applied to a ConA-Sepharose column, and bound glycoproteins eluted with -methyl mannose. The eluted gp180 was then further purified by SDS-polyacrylamide gel electrophoresis, and the 180-kDa band electroeluted. However, this procedure resulted in an extremely poor yield of purified gp180 (about 80 µg/kg of liver), which we suspected was due to poor recovery of gp180 from the initial homogenate. Because of this, subsequent work employed a different extraction procedure: homogenization in aqueous buffer and preparation of a crude membrane fraction by differential centrifugation prior to detergent extraction (see ``Materials and Methods''). This membrane fraction was then extracted with Nonidet P-40, and the extract was further purified by pre-S affinity chromatography, lentil-lectin affinity chromatography, and SDS-polyacrylamide gel electrophoresis. (In this scheme, a pre-S-glutathione S-transferase fusion protein immobilized on glutathione-Sepharose was used in the first chromatographic step).

Gel-purified gp180 prepared by each method was used for NH-terminal Edman degradation; no products were identified, suggesting a blocked amino terminus. Accordingly, the same materials were first digested with CNBr, trypsin, or the combination of CNBr plus trypsin, and peptides were purified by high pressure liquid chromatography and sequenced. The sequences of the isolated peptides are shown in . With the exception of peptide 2, all of these sequences were identified within the coding region predicted by the isolated cDNA clones; the origin of peptide 2 remains unclear but is most likely a contaminant. Once these sequences were known, screening of the protein data base immediately suggested that gp180 might be homologous to members of the carboxypeptidase gene family: homologs of peptide 4 were found in mouse carboxypeptidase A; homologs of peptides 3, 5, 6, and 7 were found in rat and human carboxypeptidase H.

Cloning of gp180 cDNA

The sequences of the isolated peptides were used to design synthetic oligonucleotides complementary to the predicted coding sequences; these oligonucleotides displayed considerable degeneracy. Two oligonucleotides (46MR and 61MR; see ``Materials and Methods'') were used to screen a size-selected, oligo(dT)-primed, plasmid-based cDNA library prepared from duck liver poly(A) RNA. A single clone, A23, was isolated that annealed to both probes. This clone, which derived from the 3` region of the cDNA, was used to isolate additional overlapping cDNA clones as described in detail under ``Materials and Methods.'' Ultimately, nearly 7 kb of cDNA was isolated, but sequence analysis revealed that the coding region at the 5` extremity of this cDNA was still translationally open, implying that additional sequences were missing from the 5` end of the clone. Multiple attempts to clone this 5` region (estimated at about 200 nucleotides) from several different cDNA libraries, including some prepared with primers from the most 5` regions of the existing clones, failed. Similarly, all attempts at recovering the 5` end of the cDNA by the PCR-based 5` rapid amplification of cDNA ends method (15) also failed. We do not know the reason for this; perhaps the 5` region contains an RNA structure that retards elongation by reverse transcriptase. In any case, our repeated failures to clone the 5` end as cDNA prompted us to reconstruct this region from genomic sequences (see ``Materials and Methods'' for details). Examination of the sequence of the reconstructed gene (see Fig. 4and Fig. 5) indicates that the homology to known carboxypeptidases extends throughout the 5` region and leaves little doubt that this procedure has accurately reconstructed the authentic 5` end of the gene.


Figure 4: The DNA sequence of the gp180 coding region and predicted amino acid sequence of its product. Potential sites for N-linked glycosylation are boxed, and peptide fragments that were purified and sequenced from native gp180 (see Table I) are underlined. Dashedline indicates putative signal peptide; doublyunderlined segment indicates putative transmembrane domain.




Figure 5: Alignment of gp180 coding domains A, B, and C with the coding region for human carboxypeptidase H (CBPH). Identical amino acid sequences are boxed. Stars, carboxypeptidase residues involved in zinc binding; opencircles, residues involved in substrate binding; closedcircles, residues involved in catalysis, as described in Ref. 19.



The overlapping recombinant cDNA fragments derived from the cloning procedures were assembled into full-length gp-180 expression vectors as described under ``Materials and Methods'' (see also Fig. 2). To verify that this cDNA encodes a functional product we examined cells transfected with gp180 expression vectors for the production of functional pre-S binding activity. One of the gp180 expression plasmids (pKRSV180) bearing a selectable marker encoding G418 resistance was transfected into LMH chicken hepatoma cells; stable G418-resistant colonies were isolated and analyzed for duck gp180 sequences with P-labeled cloned gp180 cDNA. As shown in Fig. 3A, chicken genomic DNA contains sequences that anneal to duck gp180 coding sequences (laneLMH); in six independent clones, an additional band corresponding to the transfected duck gp180 sequences was present. To test for expression, the cells were labeled with [S]methionine for 3 h. Cytoplasmic extracts prepared from these cells were then incubated with an immobilized pre-S-glutathione S-transferase fusion protein, and bound proteins were displayed by SDS-polyacrylamide gel electrophoresis. As shown in Fig. 3B, a 180-kDa protein that comigrates with authentic gp180 (not shown) was precipitated from several of these clones (shown are clones 15, 16, and 35) but not from parental LMH cells, confirming that the assembled clone is active in pre-S binding. Interestingly, Western blotting of untransfected LMH extracts with an anti-gp180 monoclonal antibody raised against the recombinant duck protein()revealed that chicken cells express a 180-kDa protein that is presumably the chicken homolog of duck gp180 (Fig. 3C); however, this protein is inactive in DHBV pre-S binding (Fig. 3B).


Figure 3: A, Southern blotting of G418-resistant LMH clones transfected with pKRSV180. Total genomic DNA from parental LMH cells and from G418-resistant clones 11, 12, 13, 15, 16, and 35 was digested with HindIII, electrophoresed through a 0.8% agarose gel, transferred to nylon membranes, and annealed to P-labeled gp180 probe corresponding to the 2.5-kb BamHI fragment of pKRSV180. B, expression of gp180 cDNA in LMH-gp180 transformants. Parental LMH cells and clone 15, 16, or 35 were labeled with S-methionine, and cytoplasmic extracts were prepared as described (7). Extracts were incubated with the pre-S-glutathione S-transferase fusion protein BE1 (10) linked to glutathione-Sepharose; the bound material was eluted by boiling in Laemmli sample buffer, electrophoresed through an SDS-polyacrylamide gel, and autoradiographed as described (10). The experiment with clones 15 and 16 was conducted separately from that involving clone 35. C, chicken cells contain a gp180 homolog. Total cytoplasmic extracts of LMH-gp180 clone 35 or parental LMH cells were electrophoresed through SDS-polyacrylamide gels, transferred to solid supports, and incubated with anti-duck gp180 monoclonal antibodies 1D11; bound antibody was reacted with rabbit anti-mouse IgG conjugated to horseradish peroxidase, and complexes were detected by enhanced chemiluminescence.



DNA Sequence of gp180

The DNA sequence of gp180 is presented in Fig. 4. The sequence predicts a protein of 1389 amino acids, with a corresponding molecular weight of 153,498, in good agreement with our earlier experimental estimate of 150 kDa for the unglycosylated chains made in vivo in the presence of tunicamycin(7) . Thirteen candidate sites (Asn-X-Ser/Thr) for N-linked glycosylation were identified in the sequence.

Comparison of the amino acid sequence of gp180 with known protein sequences revealed significant homology with members of the basic carboxypeptidase gene family. Fig. 5displays the sequence alignment of gp180 with human carboxypeptidase H (CBPH). As can be seen, gp180 is about 3 times the size of carboxypeptidase H (and all other carboxypeptidases in the GenBank data base), but homologies to the basic carboxypeptidases can be found throughout the protein, in a fashion that indicates that gp180 is actually a head-to-tail array of carboxypeptidase homology domains. This suggests that the gene for gp180 may have evolved by tandem duplication of an ancestral carboxypeptidase coding sequence in the distant past. We designate each of the carboxypeptidase homology units in gp180 as domains A, B and C, with A the NH-terminal unit and C the COOH-terminal unit. Carboxypeptidase H displays amino acid sequence identities of 39, 43, and 29% with gp180 domains A, B, and C, respectively. We note that domain B has conserved the residues known to reside at the carboxypeptidase catalytic center (19) as well as the residues involved in zinc and substrate binding(19) ; this suggests that gp180 has the potential to encode an enzymatically active protease.

Our previous work has shown that gp180 is a membrane protein, found on both internal and plasma membranes of the cell(7) . Fig. 6shows the hydrophobicity analysis of the sequence, plotted according to the method of Kyte and Doolittle(20) . The hydrophobic region at the NH terminus is highly homologous to the known signal sequence of carboxypeptidase H and almost certainly mediates the corresponding ER-translocation function in gp180. At the COOH terminus is a second, still more hydrophobic region (residues 1309-1329) that could serve as a transmembrane domain.


Figure 6: Hydrophobicity plot of gp180 by the method of Kyte and Doolittle (20). Window, 20 amino acids. Hydrophobic regions project above the horizontalline.




DISCUSSION

The results presented here indicate that gp180, a membrane-associated protein initially identified in duck hepatocytes on the basis of its ability to bind DHBV envelope proteins, is actually a member of the basic carboxypeptidase gene family. Basic carboxypeptidases are enzymes that remove basic amino acids (lysine or arginine) from the COOH terminus of polypeptide chains. Several membrane-bound enzymes of this family are known (see Ref. 16 for review), and these play important and diverse roles in biology. CPB-H, the enzyme with the greatest homology to gp180, is found on the membranes of secretory granules in many endocrine and neuroendocrine cells and is involved in the post-translational maturation of insulin and enkephalin from their precursor polypeptides. Carboxypeptidase N, another family member, is a secreted enzyme produced by hepatocytes that cleaves and inactivates the C3a anaphylotoxin in the circulation. Carboxypeptidase M is a plasma membrane enzyme that cleaves the vasoactive protein bradykinin (as well as other substrates) to generate des-Arg bradykinin, a molecule with different receptor recognition properties. As noted above, the sequence of gp180 indicates that the molecule has preserved the residues involved in catalytic activity; recent preliminary investigations()suggest that gp180, which is widely distributed on many tissues of the duck, is indeed an enzymatically active protease. We do not know, however, the normal biological function of gp180 in the uninfected host.

The interaction of gp180 with DHBV envelope proteins displays many of the properties expected for a component of the host machinery involved in viral entry (see Introduction). Because of its affinity and specificity for the pre-S region of the viral L protein, it is tempting to assign to it a role in initial virion binding at the cell surface. However, since gp180 is also found on internal membranes it could be involved in post-internalization events (e.g. within endosomes). In addition, there is reason to believe that gp180 is not the sole host molecule required for viral entry. For example, gp180 is found in several duck cells and tissues not known to be permissive for viral infection (e.g. duck embryo fibroblasts). In addition, we have recently noted that expression of recombinant gp180 in chicken LMH hepatoma cells does not confer susceptibility to DHBV infection.()This suggests that multiple host components may be required to fully reconstitute viral entry, a theme that is emerging in several viral systems (e.g. adenovirus (17) or human immunodeficiency virus(18) ). These other host molecules could be involved in additional virus-cell binding interactions or in the envelope fusion reaction (or both). Clearly, further research will be required to fully define the hepadnaviral entry mechanism and to identify all of its components.

  
Table: gp180 peptide sequences



FOOTNOTES

*
This work was supported in part by Grant AI 31973 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) U25126.

§
Supported by a grant-in-aid for scientific research from the Ministry of Education and Culture of Japan and by the Hokkoku Foundation for Cancer Research. To whom correspondence should be addressed. Tel.: 81-762-62-8151; Fax: 81-762-22-5831.

Investigator of the Howard Hughes Medical Institute.

The abbreviations used are: DHBV, duck hepatitis B virus; kb, kilobase(s).

K. Kuroki, unpublished observation.

F. Eng, unpublished observations.

K. Kuroki, T. Ishikawa, and D. Ganem, unpublished observations.


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