Immunoreactive core peptides of hepatitis C virus produced in Escherichia coli and in vitro DNA amplification-restricted transcription-translation system

Journal of Virological Methods Journal of Virological Methods 59 (1996) 91-98

Immunoreactive core peptides of hepatitis C virus produced in Escherichia coli and in vitro DNA amplification-restricted transcription-translation system Mariko EsumPT*, Nakanobu Hayashi”, Hidenori Takahashi”, Toshio Shikata”, Mitsuhiko Moriyamab, Yasuyuki Arakawab, Tatsuo Eta”, Tsukasa Nishihara”, Chikateru Nozaki”, Kyosuke MizunoC aFirst Department of Pathology, Nihon University School ofMedicine, Oyaguchikami-machi, Itabashi-ku, Tokyo bThird Department of Internal Medicine, Nihon University School of Medicine, Tokyo, Japan ‘Chemo-sero-therapeutic Research Institute, Kumamoto, Japan

173, Japan

Received 23 January 1996

Abstract Three kinds of hepatitis C virus (HCV) core peptides were produced directly and efficiently in E. co/i: 1- 120 aa of the C region as NCC, l-157 aa as NCCT and l-190 aa as NCCL. These peptides were estimated to be 16, 22 and 24 kDa, respectively, by SDS-polyacrylamide gel electrophoresis. The processing to produce p22 core protein observed in insect cells and mammalian systems did not occur in E. coli. These peptides were similarly reactive with serum antibody from patients with hepatitis C. A mutant clone of NCC recombinant plasmid pKNCC4 was obtained, whose product, NCC4, was more stable in the E. coli lysate and was highly immunoreactive with sera of hepatitis C patients. This stable immunoreactive core peptide produced by pKNCC4 is useful for the detection of anti-HCV core antibody. Immunoreactive core peptides were also produced by DNA amplification-restricted transcription-translation. Five kinds of cDNA from C to El region were amplified and transcribed in vitro, and these five transcripts were then translated in vitro using rabbit reticulocyte lysate: 1- 120 aa as 17 kDa of Cl, 1 - 155 aa as 21 kDa of C2, 1- 174 aa as 22 kDa of C3, 1-192 aa as 24 kDa of C4, and l-213 aa as 26 kDa of C5. Cotranslational processing using microsomal membranes occurred in peptides C4 and C5 to produce p22 the same size as C3. These results indicate that the C-terminus of the mature core protein p22 may be generated at around aa 174 by cleavage with the signal peptidase. Keywords:

Hepatitis

C virus; Core protein;

E. coli;

In vitro transcription;

* Corresponding author. Tel.: + 81 3 3972 811 I; fax: + 81 3 3972 8830; e-mail: [email protected] 0166-0934/96/$15.00

0 1996 Elsevier Science B.V. All rights reserved

PII SO166-0934(96)02025-3

In vitro translation

1. Introduction Hepatitis C virus (HCV) is a major causative agent of post-transfusion non-A, non-B hepatitis (NANBH). The complete genome of HCV was cloned molecularly (Kato et al., 1990; Choo et al., 1991; Takamizawa et al., 1991; Okamoto et al., 1991, 1992; Hayashi et al., 1993) and diagnostic assays for anti-viral antibody were developed. In particular, the C region of HCV was found to have useful antigenicity for the detection of antiviral antibody. The core region of the HCV genome was expressed and its product was processed to a protein of 22 kDa in monkey COS cells (Harada et al., 1991) insect cells (Chiba et al., 1991) and an in vitro translation system using a reticulocyte lysate and microsomal membranes (Hijikata et al., 1991). The mature core protein p22 is useful for antibody detection in the early phase of infection, and therefore gives a high positive rate in NANBH patients. Recently, the core protein has been well characterized; it has two distinct linear antigenic determinants within aa l-20 and aa 30-47, and a conformational determinant within aa l-69 (Goeser et al., 1994). In addition to ~22, a smaller core protein p16 was generated in a codon 9-dependent manner by cleavage of its C-terminal hydrophobic domain (Lo et al., 1994). The smaller core protein was located in the nuclei whereas p22 was located in the cytoplasm (Suzuki et al., 1995). We expressed various lengths of C region of the cloned HCV-N genome (HCV type 1b) (Hayashi et al., 1993) in Escherichiu coli and in vitro translation system using reticulocyte lysate. We predicted the C-terminus of the mature core protein p22 and isolated a mutant clone encoding 120 aa of the immunoreactive core peptide which was stable in E. coli.

2. Materials

and methods

2.1. Plusmid

construction

To construct three kinds of recombinant mid, HCV core regions encoding amino

plasacid

residues 1~ 120, 1~ 157 and 1~ 190, shown in Fig. la, were amplified from pN4.1 (accession No. D13406 in DDBJ/EMBL/GenBank) of HCV-N (Hayashi et al., 1993) with primers (sense: 5’ATGGAATTCGTCGACCCCATGGTCATGAGCACGAATCCTAAACCCCAA-3’, antisense: 5’-TCAGGATCCAAGCTTTCAACCCAAATTACGCGACGTACGCCGGGG-3’, antisense: 5’-CAGAATTCGGATCCAAGCTTTCAAACCantiCGGACACCATGTGCCAAGGCCC-3’, sense: 5’-TTTGTCGACGGATCCCAAGCTTCAGGAAGCTGGTACGGTCAGACAGGA3’, respectively) by 35 cycles of 30 s at 94°C 1 min at 55°C and 2 min at 72°C. The amplified DNA fragments were digested with NcoI and HindIII, and inserted into NcoI/HindIII site of pKK233-2 (Pharmacia, Uppsala, Sweden) to construct pKNCC, pKNCCT and pKNCCL, respectively. These plasmids transformed E. coli. JM109 competent cells.

2.2. DNA amplcjicution-restricted transcription -trunslution (DA RTT) DARTT was carried out as described previously (Mackow et al., 1990). Briefly, five kinds of cDNA, as shown in Fig. la, were amplified from pN4.1 by PCR using a T3 promoter sequence-anchored primer, 5’-TATTAACCCTCACTAAAGGGATGAGCACGAATCCTAAA-3’ and reverse primers, Cl20 (5’-ACCCAAATTACCl55 (5’-GACACCATGTGCGCGACC-3’) CAAGG-3’), C 174 (5’-AAAAGAGCAACCGGGCA-3’), Cl92 (5’-ATGAGCGGAAGCTGGTA-3’), C2 13 (5’-CACAATGCTTGAGTTGG3’), respectively. Samples of 1 pg each of the amplified DNAs were in vitro transcribed with 50 units of T3 RNA polymerase (Stratagene, CA) at 37°C for 60 min. Then 5 pug of the synthesized run-off transcript RNAs were in vitro translated using 20 ~1 of rabbit reticulocyte lysate (Promega, WI) at 30°C for 60 min. Translated samples were diluted in sample buffer and analyzed by SDSpolyacrylamide gel electrophoresis (PAGE) and immunoblotting. Translations were also undertaken using 3 ~1 of [35S]methionine (Amersham,

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El I

I ’

I

W 160

170

180

190

rE’

GVRVLEDGVlW~T~NMP~~F~IFLLFdLSCLTVP&S@iEV -

c2

t

tt

f c3 c4

Fig. 1. Expression of truncated HCV core peptides (a) and predicted cleavage sites of C-terminus kinds of plasmid, pKNCC, pKNCCT and pKNCCL were constructed for expression in E. co/i, (Cl -C5) containing T3 promoter sequence at the top were used for expression by DARTT. cleavage sites with signal peptidase and underlined residues at - 3 and - 1 position indicate rule (von Heijne, 1983). The cleavage site of N-terminus of El protein is also shown (Hijikata bottom represent C-terminal regions of DARTT products C2, C3 and C4.

UK) in the presence or absence of 2 ~1 of canine pancreatic microsomal membranes (Promega). Analysis of 35S-labeled proteins was undertaken by SDS-PAGE followed by fluorography.

of HCV core protein (b). (a) Three and five kinds of truncated cDNAs (b) Arrows indicate three possible the accordance with the ( - 3, - I) et al., 1991). The three lines at the

pel, West Chester, PA) at 37°C for 1 h. The filters were then visualized by 3,3’-diaminobenzidine.

3. Results and discussion 2.3. Immunoblot

analysis

To analyze products in E. coli, E. coli lysates were prepared by agitation with glass beads. After centrifuging the lysates at 6000 x g for 15 min, the supernatants and pellets were subjected to SDSPAGE. Transfer of a gel to a nitrocellulose filter was followed by incubation of the filter with serum from a hepatitis C patient at 37°C for 1 h, and then incubation with peroxidase-conjugated F(ab’)2 fragment of goat anti-human IgG (Cap-

3.1. Expressions E. coli

of three kinds of core peptide

in

We constructed three plasmids, pKNCC, pKNCCT and pKNCCL to express the HCV core regions: 1~ 120 aa (NCC), 1- 157 aa (NCCT) and 1- 190 aa (NCCL), respectively, directly in E. coli (Fig. la). As shown in Fig. 2a, these three core peptides were produced efficiently in the inclusion body as proteins of 16, 22 and 24 kDa, respec-

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59 (1996) 91-98

(W

(4 123456

kDa -

46 -

-

30 -

2

4

6

‘1’,, _>

Fig. 2. Protein analysis of transformed E. coli JM109 cells by SDS-PAGE with Coomassie brilliant blue (CBB) staining (a) and its immunoblot analysis (b). (a) pKNCC-, pKNCCTand pKNCCL-transformed E. co/i lysates were prepared, and the supernatant (lanes 1, 3, 5) and the pellet (lanes 2, 4, 6) of the lysates were subjected to electrophoresis on SDS,‘15% polyacrylamide gel. Lanes 1, 2: NCC; lanes 3, 4: NCCT; lanes 5, 6: NCCL. (b) Lanes 2, 4, 6 in (a) were blotted and immunostained with serum of a hepatitis C patient, peroxidase-conjugated F(ab’)2 fragment of goat anti-human IgG and 3,3’-diaminobenzidine. Molecular weight markers are shown in the middle.

tively. Immunoblot analysis showed that these peptides were similarly immunoreactive with serum from a hepatitis C patient (Fig. 2b), and these core peptides had the same positive rate of reactivity with sera from NANBH patients. Thus, the shortest peptide, NCC, consisting of the first 120 aa, has sufficient antigenicity to detect antiHCV core antibody in the sera of the patients. Recent studies on epitope mapping of HCV core antigen also demonstrate that antigenic determinants were located in the N-terminal 69 aa. The shortest peptide NCC may be a useful and sufficient antigen for detection of anti-HCV core antibody in the sera of the patients. These results also indicate that the processing to produce p22 HCV core protein observed in eukaryotic expression systems (Harada et al., 1991; Chiba et al., 1991; Hijikata et al., 1991) did not occur in E. coli, and suggest that p22 may consist of more or less than 157 aa, but less than 190 aa. As the molecular weight of the first 19 1 aa of the core region is calculated to be 21 kDa and the N-terminus of the next E protein expressed in

the in vitro translation system is aa 192 (Hijikata et al., 1991) cleavage between aa positions 191 and 192 may be responsible for the production of ~22. However, the molecular weights of core peptides were estimated by SDS-PAGE to be more than the calculated values in this work, i.e. 16 kDa vs. 13.6 kDa for NCC (120 aa), 22 kDa vs. 17.3 kDa for NCCT (157 aa) and 24 kDa vs. 20.7 kDa for NCCL (190 aa). Therefore, p22 core protein may be produced by cleavage at about aa position 157 in eukaryotic systems. 3.2. Expression of jive kinds qf’ core peptide by DARTT To identify the other cleavage in the C-terminal processing of the core protein ~22, DARTT (Mackow et al., 1990) was applied to the putative core protein-encoding cDNA (Fig. la). Five kinds of truncated transcript were synthesized in vitro (Fig. 3a) and translated in vitro using rabbit reticulocyte lysate. Fig. 3b shows that immunoreactive core peptides were synthesized as 17 kDa

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w

ClC2C3C4C5

Cl - C2C3 C4C5

9.5 kb a:$ 2.4

1:;

kDa

1.4 0.24

(c 1

Control

M-

M-M-M-

M-

_g25kDa

-

69'

- 46 - 30 - 21.5

Fig. 3. Analysis of DARTT products. (a) Formaldehyde/l% agarose Immunoblot analysis of in vitro translated products. (c) Fluorography without (-) microsomal membranes.

for Cl, 21 kDa for C2, 22 kDa for C3,24 kDa for C4 and 26 kDa for C5, as observed in the E. coli expression system. In the absence of microsomal 3sS-labeled products also showed membranes, products of similar molecular size (Fig. 3~). However, co-translational processing of C4 and C5 occurred in the presence of microsomal membranes; 24 kDa of C4 and 26 kDa of C5 were processed to 22 kDa (Fig. 3~). The other products Cl, C2 and C3 were not changed in the presence or absence of microsomal membranes. A minor population of processed core peptides was also observed in lanes C4 and C5 of immunoblot analysis even without microsomal membranes (Fig. 3b), probably due to the minor component of microsomal membranes included in the rabbit reticulocyte lysate. These results indicate that another cleavage at about aa position 174 with the signal peptidase generates the mature core protein ~22. While this manuscript was in peparation,

gel electrophoresis of truncated of ‘%-labeled in vitro translated

in vitro transcripts. (b) products with (M) and

(Santolini et al., 1994) reported similar results and predicted the cleavage site to be between aa 173 and 174. The pattern of amino acids near signal sequence cleavage sites has been analyzed and this has revealed the ( - 3, - 1) rule for defining the cleavage site and the important hydrophobic region at residues from - 6 to - 13 (von Heijne, 1983, 1985) According to this hypothesis, there are three possible cleavage sites at about aa position 174 in the HCV core sequence: - ATG/ - , - GCS/ - (pointed out by Santolini et al., 1994) and - SFS/ (Fig. lb). However, no typical hydrophobic region is found upstream of these sites. This cleavage may be special as for targets of signal peptidase whereas the typical recognition pattern is clearly found in the cleavage of N-terminus of E2 (Fig. lb). Another smaller core protein p16 was not generated in this work, for codon 9 of the HCV-N (type lb) core protein sequence used here was arginine but not lysine (Lo et al., 1994).

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3 4

5 6 7 8 9101112

KDa

1 5

9

Fig. 4. Protein analysis of pKNCC-transformed JMl09 cells after incubation at 37°C (a) and its immunoblot analysis (b). (a) Mutant clones pKNCC4 (lanes l-4) pKNCC5 (lanes 5-8) and pKNCC6 (lanes 9912) transformed JM109 cells, and the lysates were nxxbdted at 37°C for 0 h (lanes I, 5, 9) I h (lanes 2, 6, IO), 2 h (lanes 3, 7, I I) and 4 h (lanes 4, 8, 12). Then the lysates were subjected to SDS/l5% PAGE and stained with CBB. (b) \ I Lanes I, 5, 9 were transferred to a nitrocellulose filter and treated as described in Fig. 2b.

DARTT was first applied to identify the recognition sites of rotavirus neutralizing monoclonal antibodies (Mackow et al., 1990). We carried out in vitro translation of DARTT in the presence of microsomal membranes, so this sophisticated technique is also useful for analysis of post-translational processing and glycosylation sites. 3.3. A mutant clone of pKNCC production of core peptide

with more stable

Of the pKNCC clones, 90% produced core peptide of 16 kDa, while the rest produced NCCs of various other molecular sizes. It was found that one of these mutant clones, named pKNCC4, produced a much more stable immunoreactive core peptide with a larger molecular size in E. coli. As shown in Fig. 4a, NCC4 was detected at a high level for 4 h after incubation of an E. coli lysate at 37°C whereas two other mutants, NCC5 and NCC6, were degraded within 1 h. As judged by SDS-PAGE, the molecular weight of NCC4 was higher, and that of NCC6 was lower than that of NCCS (16 kDa), but all three were immunoreactive with serum from a hepatitis C patient (Fig. 4b). Nucleotide sequence analyses indicate that pKNCC4 had a base substitution in codon 94 (GCA-ACA) and a base deletion in

codon 109. This deletion caused a frame shift mutation from 110 aa to 120 aa and reading through of a designed stop codon so that the TGA codon 44 base downstream of the vector sequence was used as a stop codon. A mutant clone pKNCC6 had a base substitution in codon 23 (AAG-ACG) and a base insertion in codon 113 generating a stop codon in codon 115. The clone pKNCC5 with a designed stop codon in codon 121 had a base substitution in codon 15 (ACC-GCC). N-terminal analysis of these mutant core peptides showed that the first Met designed in a PCR primer was similarly deleted and all the N-termini were the same, Val, which was the second aa derived from a PCR primer. Thus NCC4 may be stable in an E. coli lysate because of modification of the C-terminal region. Stability such as that of NCC4 is essential for practical use of the product as an antigen in establishing a method for assay of anti-HCV core antibody. Table 1 shows that NCC4 was a useful diagnostic probe for HCV infection by examining patients’ sera. We established previously an ELISA for detection of anti-core antibody using a fusion protein of the NCC with p-galactosidase produced in E. coli (Hiraga et al., 1994). NCC4 generated similar results in detecting anti-core antibody to those earlier, and showed no addi-

M. Esumi et al. / Journal of Virological Table 1 Detection

of anti-NCC4

Diagnosis

antibody No.

in patients Positive

no. (“/0)

NCC4” Acute hepatitis Post-transfusion Sporadic Chronic liver disease Chronic hepatitis Liver cirrhosis Hepatocellular carcinoma

with NANBH

HCV-IIb

18 28

1I (39%)

14 (78%) 11 (39%)

86 108 50

86 (100%) 99 (92%) 45 (90%)

86 (100%) 101 (94%) 45 (90%)

13 (72%)

“NCC4 was solubilized by deoxycholate from the pellet of E. coli, then purified by ion-exchange chromatography and used for an ELISA. Samples of IOO-fold diluted serum were added to each well coated with NCC4, and then the bound antibody was detected by a second incubation with peroxidase-conjugdted anti-human IgG. The enzyme activity was quantitated by measuring the absorbance at 450nm with tetramethylbenzidine as a substrate. The cut-off value was determined as the mean value +6 S.D. of normal sera. bSecond generation anti-HCV antibody was also measured by Ortho ELISA kit.

tional reactivity against patients’ sera caused by C-terminal modification. NCC or NCC4 seemed to include almost all the epitopes against antiHCV core antibody of patients (Goeser et al., 1994). Therefore, NCC or NCC4 is a useful probe for the detection of HCV infection.

Acknowledgements This work was supported in part by Health Science Research Grants (Non-A, Non-B Hepatitis Research Grants).

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