JPH0692996A - Protein constituting hepatitis c virus particle and its manifestation - Google Patents

Abstract

PURPOSE:To identify an envelope protein to become a target of a neutralizing antibody, existing on the surface of hepatitis C virus(HCV) particle and to utilize the protein for trial preparation of vaccine usable for diagnosis of infection with virus and prevention of crisis. CONSTITUTION:An envelope protein of two HCVs is processed from N end 980 amino acid residue to be encoded in a transcription decoding frame of HCV genome and has an amino acid sequence from N end 192th to 383rd of the transcription decoding frame of HCV genome and about 35,000 molecular weight and an amino acid sequence of about 350 starting from 384th and about 79,000 molecular weight. A core protein of HCV has an amino acid sequence from N end 1st to 191st and about 22,000 molecular weight.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention is a major causative virus of post-transfusion non-A non-B hepatitis, and it has become clear that hepatitis C virus (HCV; (Hereinafter referred to as HCV) Proteins constituting particles and their expression and identification. More specifically, the present invention identifies an envelope protein present on the surface of virus particles and targeted by neutralizing antibodies by expressing a viral protein in vitro using a cloned HCV genomic cDNA, and thus It is intended to be used for trial production of vaccines used for diagnosis and prevention of viral infections.

[0002]

BACKGROUND OF THE INVENTION Hepatitis C is one of viral hepatitis that constitutes post-transfusion hepatitis. HCV, which is a causative virus of hepatitis C, has become a problem in many infected people. HCV infection was found to be common in hepatitis, liver cirrhosis and primary biliary cirrhosis, which were previously thought to be alcoholic and autoimmune, and, like hepatitis B (HBV), transition from chronic hepatitis to cirrhosis. Moreover, it has become clear that the risk of developing hepatocellular carcinoma is high. About two-thirds or more of liver cancer patients have HCV antibody or HCV genomic RN
A is detected. At present, it is known that interferon (IFN) is effective for the treatment of such hepatitis C itself, and there is virus elimination data, but it is also difficult as a so-called specific drug, and its diagnosis, therapeutic drug, blood transfusion The development of purifying agents, vaccines, etc. is desired.

[0003] Hepatitis C has only recently been found to have a viral gene that causes it, and it is now open to diagnosis and prevention. A portion of the HCV gene was isolated by Chiron, USA in 1989 [Choo et al., Scien.
ce, 244, 359 (1984)]. Based on this information, research on HCV was accelerated, and in Japan, cDNA was isolated from the serum of Japanese patients and cloned to clarify the HCV gene sequence unique to Japanese. The present inventors have found that HCV (HC
VJ) gene has been analyzed and the entire structure has been clarified [Kato et al. Proc. Acad. Natl. Sci. USA 87 , 9
524 (1990)]. According to it, HCV has an RN of about 10 Kb.
A virus, the single-stranded RNA consists of about 9500 bases, and is a region read by amino acids (transcriptional frame:
The ORF is 9030 bases and the number of amino acids is 3010. However, as described above, although the entire gene of HCV has been clarified, no virus particle has been recognized, and the identification and analysis of the protein constituting the virus particle has not been achieved.

Therefore, the object of the present invention is to analyze the action of HCV on infected cells at the level of virus-produced proteins, and
In particular, the focus is on the envelope protein, which is present on the surface of the virus and is the target of neutralizing antibodies, for its identification and analysis.

[0005]

[Means for Solving the Problems] The inventors of the present invention have conducted various studies in order to achieve the above-mentioned object and found that a viral protein is expressed in vitro using a cloned HCV genomic cDNA. By HC
Among the HCV proteins encoded by the V gene,
We have found that the proteins that make up the virus particles, especially the envelope proteins that are present on the surface of the virus and that are the target of neutralizing antibodies, are processed and produced accurately.
That is, it was discovered that the action of signalase existing in the microsomal membrane fraction is essential for this processing, and two types of proteins are expressed as membrane proteins of virus particles. Furthermore, the viral core protein is also expressed by this processing action. It was revealed that they were produced, and the regions on the HCV gene encoding these viral particle constituent proteins were identified.

[0006] The present invention is an HCV particle processed from the N-terminal 980 amino acid residues encoded by the transcription frame of the HCV genome by expressing a viral protein in vitro using the cloned HCV genomic cDNA. HCV having a molecular weight of about 22000, which has an amino acid sequence from the N-terminal 1st to 191st of the HCV genome open reading frame
Core protein (p22), which has an amino acid sequence from the N-terminal 192nd to 383rd of the open reading frame of the HCV genome, has a molecular weight of about 35,000, and starts from the 384th and has an amino acid sequence of about 350. About 7
Two HCV envelope proteins of 0000 (gp3
5, gp70) was identified (see FIG. 8 below).

In the present invention, processing of putative structural proteins of HCV was investigated using an in vitro expression system. That is, an RNA transcript used for cell-free translation was prepared from a cDNA containing a region encoding the N-terminal 980 residue of a viral polyprotein precursor, and subsequently translated in the presence of a microsomal membrane (in vitro transcription / translation). ). in
Processing of in vitro translation products proceeded co-translationally (with translation) in the presence of microsomal membranes, yielding four major products (p22, gp35, gp70, p19).
Note that p19 is the mR used in the in vitro translation system.
It is the product resulting from the artificial cleavage of the 3'end of NA. The product was similarly translated from a cDNA construct shorter than the N-terminal 980 residues (deletion form) and compared with the analysis of the processed protein from the N-terminus of the polyprotein precursor to NH ₂ -p22. -It was suggested that they are arranged in the order of gp35-gp70-p19-COOH. For protein analysis after processing, polyacrylamide gel electrophoresis (SDS
-PAGE) followed by fluorography. The N-terminal amino acid sequence is determined by the Edman method.

In order to predict and analyze each translation product as a virus envelope protein or a virus core protein, gene constitution on the genome of other similar flaviviruses, comparison of hydrophilicity profiles of amino acid sequences, glycosylation signal This is possible based on the presence or absence of (N−X−T / S).

The portion corresponding to the signal sequence is gp35
And found in the N-terminal upstream of gp70 [J. Mol. Bio
l. 184, 99-105 (1985)]. In addition, due to the presence of a transmembrane region in the region where the hydrophobic amino acid sequence continues,
And a transmembrane portion was deduced in the C-terminal upstream region of gp70. Through this, the transmembrane portion is gp35 and gp
It is considered to play a role of sending 70 into the endoplasmic reticulum. Furthermore, the site having a possibility of glycosylation was judged because there is a site where N-glycosylation is likely to occur with respect to the NXT / S sequence.

[0010]

INDUSTRIAL APPLICABILITY The present invention is intended to identify an envelope protein that is present on the surface of HCV particles and is a target of neutralizing antibodies, and to utilize it for trial production of a vaccine used for diagnosis and prevention of viral infection. .

[0011]

【Example】

Example 1 (1) Construction of expression plasmid The 81-bp 5'non-coding region of the HCV cDNA sequence and the 2940-bp coding region are contained, that is, HCV.
The expression vector pC980 encoding the N-terminal 980 residues of the open reading frame (ORF) of E. coli, and a series of deletion mutants thereof were constructed as follows. First, HCV-J clone 2 [Ka
to, N. et al. Proc. Natl. Acad. Sci. USA 87, 9524-95
28 (1990)] was added to pTZ containing the T7 phage promoter immediately upstream of the multiple cloning region.
An expression vector pC163 was constructed by subcloning the 18U vector [Toyobo Co., Ltd.] into the EcoRI site. Next, the NarI-HindIII fragment of pC163 was replaced with NarI-HindIII of HCV-J clone 3 (same as above) to obtain pC511.
Was produced. The MluI-HindIII fragment of pC511 was HCV-
Replaced with the MluI-HindIII fragment of clone 33 (Id.) Containing part of the cDNA clone for J (Id.), PC74
I got 0. Then, the BssHII-HindIII fragment of pC740 was set to H
It was replaced with the BssHII-HindIII fragment of CV-J clone 6 (Id.) To construct pC980. pC980 to NruI-Cla
I fragment and SmaI-BamHI fragment were deleted,
The Klenow fragment of polymerase I was used to fill in the restriction enzyme-cutting protruding ends, and then the fragments were ligated using Sph-I linker (Takara Shuzo) to construct pN124 and pN340. pN124 was further digested with SphI to delete the 3'end of 187 base pairs. The construction diagram of pC980 is shown in FIGS. 1 and 2.

(2) In Vitro Transcription All purified plasmids were cleaved at the unique HindIII site of the multicloning region downstream of the inserted cDNA or at appropriate restriction enzyme sites in the insert. , Straightened. These were used as templates for in vitro transcription. According to previous reports, T7 RNA polymerase (Uni
ed States Biochemical) to synthesize RNA transcripts in vitro [Hijikata et al., J. Virol. 64 , 4632].
-4639 (1990)]. PC5 shortened with BamHI or HincII
11 and pN340 shortened with BssHII or NotI as a template and transcribed in vitro to give C281, C38
1, N340B and N340N were obtained. A map of each deletion construct obtained by the above-mentioned treatment and used in the in vitro processing analysis and a list of the expression plasmid construction strategies are shown in FIG.

(3) In vitro processing assay and protein analysis [ ³⁵ S] methionine (Amersham) for labeling was added in the presence or absence of canine pancreatic microsomal membrane (Promega),
Using rabbit reticulocyte lysate (Amersham), pC9
80 transcripts were translated. Then, the translation mixture sample was subjected to 30 ° C. at 0 ° C. in the presence or absence of a surfactant (1% sodium deoxycholate / 1% Triton X-100).
Treatment with proteinase K (200 μg / ml) for minutes.
After centrifugation of the mixture at 10,000 xg for 20 minutes, samples of the precipitate and supernatant were analyzed by fluorography after SDS / PAGE.

The results of the in vitro processing assay using pC980 are shown in FIG. The translation product of pC980 (C980) is processed and a (up to 70 k) only when the microsomal membrane is present during translation in vitro.
Da), b (35 kDa), c (22 kDa), d (19 kDa) 4
Two major degradation products were produced (Fig. 4, lane 5). The unprocessed form synthesized in the absence of microsomes was not detected except as a form that could remain at the migration start of the gel (Fig. 4, lane 3). These four major products a, b, c, d were all found in the precipitated microsomal membrane fraction. Products a and b were resistant to proteinase K treatment, while products c and d were degraded by the enzyme (Fig. 4, lane 7). Resistance to proteinase treatment was lost by detergent treatment (Fig. 4, lane 9), suggesting that products a and b migrate into microsomes.

(4) Gene Mapping of Processed Products on the HCV Genome To determine the sequence order of the four processed products encoded by the HCV ORF, various deletions of C980 obtained in (2) above were determined. Using the demutated mutant, the precursor protein was processed in vitro in the same manner as in (3).
That is, each transcript of various deletion mutants of C980 was translated in vitro in the presence or absence of microsome membrane, and the sensitivity of the reaction product to proteinase K was examined (FIG. 5). In vitro translation products C740, C
511, C381, C281, C163 have a series of C-terminal deletions of C980, 740, 511, 38, respectively.
It contained an N-terminal sequence of 1,281,163 residues. p
N124 and N340, which are translation products of N124 and pN340, have the N-terminal sequences of residues 123 and 339 of C980 substituted with the amino acid sequence Met-Pro.
pN124-ORF lacks the base sequence for the 59C-terminal residue of pC980. Also, N340B and N34
0N lacked the C-terminal sequence of residues 252 and 438 of N340, respectively.

The proteinase resistance product a (70 kDa) is
Since this protein was detected only in the processed form of C740, N124, N340, N340B (FIG. 5, lanes 3, 18, 21, 24), this protein was identified in the HCV ORF N
The terminal residues are located at approximately 340 to 740, and the region contains a sequence which can be a signal sequence of the protein. In addition, another proteinase resistance product b (35 kDa) is C2
81, C163, N340, N340B, N340N were not detected in the processed form (FIG. 5, lane
12, 15, 21, 24, 27), a smaller proteinase-resistant product (30 kDa) was detected in the processed form of C281, indicating that this protein is HCV.
It is shown that the N-terminal residues of the ORF are located approximately at 163 to 381. The band for product c (22 kDa) is N-
Terminal deletion mutants N124, N340, N340
This protein is derived from the N-terminus of the HCV ORF, as it was lost in the processed form of B, N340N (FIG. 5, lanes 17, 20, 23, 26). Product d (19 kDa)
Was detected only in C980 and in the processed form of N340, which has the same C-terminal sequence as C980 (FIG. 5, lane 20).
It is suggested that it is encoded in the C-terminal region of the ORF. From these results, the sequence order of the processed proteins in the HCV ORF is NH ₂ -c (22 kDa) -b.
It is concluded that it is (35 kDa) -a (70 kDa) -d (19 kDa) -COOH.

(4) Endoglycosidase H Treatment of Proteinase-Resistant Product A proteinase-resistant translation product of a deletion mutant of C980 was prepared according to the report of Ruiz-Linares et al. [Ruiz-Linares et a
L., J. Virol., 63 , 4199-4209 (1990)], endoglycosidase H (Boehringer Mannheim) treatment. FIG. 6 shows the results of SDS / PAGE analysis after removing N-linked high-mannose glycans that can be bound by the treatment. As shown in FIG. 6, the size of the proteinase resistant products a and b decreased after treatment with endoglycosidase H, suggesting that both products are glycoproteins. The molecular weights of the deglycosylated forms of a and b were estimated to be about 21 kDa and 38 kDa, respectively (FIG. 6, lanes 2, 4, 6, 8,
12). As expected from the deletion analysis, the C281 and N340N products were reduced by deglycosylation to approximately 10 kDa and 22 kDa, respectively (FIG. 6, lanes 10, 14). Product a,
b was designated as gp35 and gp70, respectively.

(5) Determination of N-terminal amino acid sequences of gp35 and gp70 In order to determine the exact cleavage point of the HCV polyprotein precursor, proteinase resistance of C281 and N340N in vitro.
The ro translation product was used to examine the N-terminal amino acid sequences of gp35 and gp70, respectively. ^The proteinase-resistant translation product labeled with ³ H-labeled amino acid was deglycosylated with endoglycosidase H and electrophoresed on SDS / PAGE.
Electroblotted onto lon membrane (Millipore). The radiolabeled product on the membrane was removed and subjected to manual Edman degradation. The radioactive amino acid release at each cycle of degradation was plotted (Figure 7). The amino acid sequence determined from the ³ H-labeled amino acid peak in the Edman degradation cycle was compared to the amino acid sequence decoded from the C980-ORF. Sequencing of the in vitro processed products from C281 and N340N identified the N-terminal residues of gp35 and gp70 as Tyr-192 and His-384 of the sequence decoded from the C980-ORF, respectively. .

The amino acid sequence decoded from HCV 980-ORF and the region corresponding to the processed protein in HCV ORF are shown in FIG. gp35
And the HCV ORF just upstream of the N-terminus of gp70
Residues 175-191 and 370 in the coding sequence of
A sequence corresponding to the signal sequence was found at position -383 (Fig. 8, double line). In addition, C of gp35 and gp70
Two parts were observed in the terminal region that act as transmembrane parts (Fig. 8, underlined), suggesting that both products gp35 and gp70 are type I membrane-bound proteins.

[Brief description of drawings]

FIG. 1 shows a construction diagram of pC980.

FIG. 2 shows a construction diagram (continuation) of pC980.

FIG. 3: List of construction strategies for expression plasmids and in v
A map of the deletion construct used for in vitro processing analysis is shown.

FIG. 4 S of the in vitro transcribed translation of pC980
The processing assay result by DS / PAGE is shown. Lane 1; product translated in the absence of microsome membrane without RNA (control) Lane 2; product translated in the presence of microsome membrane without RNA (control) Lane 3; in vitro transcript of pC980 Product translated in the presence lane 4; supernatant fraction of product translated from in vitro transcript of pC980 in the presence of microsome membrane lane 5; precipitation fraction of product translated from in vitro transcript of pC980 in the presence of microsome membrane lane 6 After translation of an in vitro transcript of pC980 in the presence of a microsomal membrane, and then supernatant fraction of a proteinase K-treated product in the absence of a surfactant, lane 7; after translation of an in vitro transcript of pC980 in the presence of a microsomal membrane. , A product treated with proteinase K in the absence of detergent Precipitated fraction Lane 8: pC980 in vitro transcript was translated in the presence of a microsomal membrane, and was then treated with proteinase K in the presence of a surfactant. Supernatant fraction of Lane 9; pC980 in vitro transcript was present in a microsomal membrane. After the translation, the precipitate fraction of the proteinase K-treated product in the presence of a surfactant

FIG. 5: SDS / PAG of various deletion mutants of C980
The in-vitro processing assay result by E is shown. Lanes 1, 4, 7, 10, 13, 16, 19, 22, 25: Translation in the absence of microsome membrane Lanes 2, 5, 8, 11, 14, 17, 20, 23, 26: Translation in the presence of microsome membrane 3, 6, 9, 12, 15, 18, 21, 24, 27: Proteinase K treatment after translation in the presence of microsome membrane

FIG. 6: Proteinase-resistant translation product products of deletion mutants of C980 (-, lanes 1, 3, 5, 7, 9, 11, 13), after treatment with endoglycosidase H (eH) (+, lane 2,
4, 6, 8, 10, 12, 14) shows the analysis results by SDS / PAGE.

FIG. 7 shows a plot of radioactive amino acid release during each cycle of Edman degradation of in vitro processed products from C281 and N340N.

FIG. 8 shows the amino acid sequence decoded from the C980-ORF and the processed protein in the HCV ORF.

Claims (3)

[Claims] 1. An envelope protein of hepatitis C virus having a molecular weight of about 35,000, which has an amino acid sequence from the N-terminal 192nd position to the 383rd position of the open reading frame of hepatitis C virus genome. 2. A hepatitis C virus envelope protein having a molecular weight of about 70,000, which starts from the N-terminal 384 of the open reading frame of the hepatitis C virus genome and has an about 350 amino acid sequence. 3. A hepatitis C virus core protein having a molecular weight of about 22000, which has an amino acid sequence from the N-terminal 1st to 191st of the open reading frame of hepatitis C virus genome.