Comparative Nucleotide and Amino Acid Sequences Among Seven Hepatitis C Virus cDNA Isolates in Japan and U.S.A.
Wei‑Yun ZHENG, Shintaro KURIHARA, Kouichi MORITA, Mariko TANAKA and Akira IGARASHI
Abstract: The nucleotide and deduced amino acid sequence homology of hepatitis C virus (HCV) among seven HCV CDNA isolates in Japan and U.S.A. have been compared and discussed. It was found that Okamoto's nucleotide sequence (J1) has high homology (>94.1%) with American (CHIRON) in both nucleotide and amino acid sequence. Kato's nucleotide se‑
quence (JGC) was close to Takamizawa's nucleotide sequence (JBK), with similarity of
>87.0% for both nucleotide and amino acid. CHIRON was closer to JGC and JBK than to Okamoto's nucleotide sequence (J6). J6 was different from each of the other six isolates.
The high degree of sequence conservation in the 5' non‑coding region among six isolates (>92.8%) except Takeuchi's nucleotide sequence (JYK) has been observed. The low level of nucleotide and amino acid sequence variation in the putative core protein and the variability of putative envelope glycoprotein among seven isolates have been observed.
Key words: Hepatitis C virus, Nucleotide and amino acid sequence homology
Department of Virology, Institute of 7>φleal Medicine, Nagasaki University, 1‑12‑4 Sakamoto, Nagasaki 852, Japan
INTRODUCTION
Hepatitis C virus (HCV) is supposed to be an enveloped virus with about 10 Kb
positive―sense, single―stranded RNA genome. Its genome organization is predicted to resem―
ble that of the flavivirus and pestivirus, therefore classified as a genus in the family Flaviviridae (Wengler, 1991). A 5'non‑coding segment of 341 nucleotides precedes a con‑
tmuous open reading frame (ORF) of about 9030 nucleotides which is followed by a 54 nucleotides long 3'non‑coding region. Genomic RNA is probably translated into a single polyprotein of about 3010 amino acids which is processed into functional proteins (Plagemann, 1991).
Recently, isolations of several HCV CDNA clones from human patient's sera and HCV infected chimpanzee have been reported in the U.S.A. and Japan as follows: CHIRON group (Choo et at., 1991) entire sequence of CHIRON 9,379 Kb; Kato's group (1990) entire sequence of JGC 9,413 Kb; Takeuchi's group (1990) partial sequence of JYK 1,323 Kb; Okamoto's group partial sequence of Jl and J4 1,863 Kb (Okamoto et at., 1990a) and entire sequence of J6
Received for Publication, August 27, 1992.
Contribution No.2715 from the Institute of Tropcial Medicine, Nagasaki University.
9,589 Kb (Okamoto et at., 1991); and Takamizawa's group (1991) entire sequence of JBK 9,416 Kb,
respectively. It is valuable and feasible to compare the homology of HCV cDl岨sequences
among these seven isolates, although some of them have been compared partly before (Kato et at., 1990; Okamoto et al, 1991; Takamizawa et al.t 1991).
In this paper, significance of the genome variabilities among seven isolates of HCV will
=
be discussed in order to identify and analyze the functions of HCV genome, and to unders‑
tand immunology and epidemilogy of HCV.
MATERIALS AND METHODS
Information of HCV Nu亡Ieotide Sequence: Nucleotide sequence data of seven HCV
CDNA isolates were obtained from following publications: CHIRON (Choo et al., 1991), JGC (Kato et al., 1990), JBK (Takamizawa et al., 1991), J6 (Okamoto et at., 1991), Jl and J4 (okamoto et at., 1990a), and JYK (Takeuchi et al., 1990)
comparison of Nucleotide and Amino Acid Sequences Among Seven HCV CDNA Isolates: According to the method reported by Okamoto et al (1991), the HCV
nucleotide sequence was divided into following genes: 5'non―coding region, core (C) protein,
envelope (E) protein, NSl―NS5 protein regions and ‑3'non―coding region.
The homology comparison of HCV nucleotide and deduced amino acid sequence were performed by using DNASIS Version 7.00 computer programing software (HITACHI, 1991).
RESULTS
Figure la shows the 5'non‑coding region nucleotide sequence homology of six HCV
●
CDNA isolates. The homology among all compared isolates was very high (>92.8%) except TYK. The homology search indicated that JYK (1,323 Kb) nuclotide sequence start from putative core protein region. The putative C protein showed high conservation among all com‑
pared HCV CDNA isolates (nucleotide >80.9%, amino acid >90.6%: Fig. Ib). From putative E to NSl‑NS5 protein region, J6 showed low degree homology in nucleotide and amino acid sequences compared with other isolates (nucleotide < 69.5%, amino acid < 73.6%) except NS3 region, which showed slightly higher homology (nucleotide 70.0%, amino acid 80.3%: Figs. 1, 2 and 3).
The J6 showed very low percentage of nucleotide sequence homology especially in 3 non‑
coding region (<42.9%), while the conservation between the JGC and JBK was very high (92.3%: Fig. 3C).
The nucleotide and amino acid sequence conservation of E protein was lower than C protein region among all isolates especially in J6 which was the lowest. In E protein region, the J4 is closely related to JYK, JGC and JBK (nucleotide >90.9%, amino acid >91.4%), whereas only 74.5% (nucleotide average count) and 77.3% (amino acid average count) homologies were conserved to Jl and CHIRON. The Jl is closely related to CHIRON
(nucleotide 94.1%, amino acid 97.0%, whereas only 73.捕(nucleotide average count) and
76.8% (amino acid average count) homologies were conserved to JYK, JGC and JBK. The J6
5 ‑Non‑coding region (a)
C protein region (b)
E protein region (c)
Fig. 1. Nucleotide sequence homology of 5'non‑coding region(a), and nucleotide and amino acid sequence homology of C protein (b) and E protein (c) regions.
Fi即res in the left lower half show nucleotide sequence homology and those in the right upper half show amino acid sequence homology, respectively, represented in homology percent.
―― ――――
J1 1C!
―― ― 〓 〓―
Lj監 CHIROト
Jffii
〓 ―― 〓― ――――
JGC
―〓 ― ― ― ―――
JBK
Jl ― J4 ― J6 CHIRO叫JYK JGC JBK
99.1
― ―― ――――
94.9 98.8
97.6
―■■―■■―――■―――,〓―――‑
99.1
二:こ二二こ二=―=
94.6
―‑――l――――,
99.
―――― ― ―
98.5
‖m
95.2
92.8
93.5
―― ―= ―
97.3
‥ ― ―― ‥― ―
99.1 qR.,i
Jl
―∴―〓― 〓
J4
―― ―――― ―
J6 :HIRON
OT3 JGC IRK"
Jl l―国―
89.5 80.9 97.9 89.9 89.9
90.9
96.9
iZ日
81.8 89.7 96.2
―〓 ― ―――
95.8 96.^
91.1 91.6
iiZI
81.8 83.0
81―8
83.1
――̲―二こ―二=
97.9 96.9 90.6
iZ日
90.2 90.4 90.2
97.4
=―――
98.4
――――――――=
92.7
97.4
iiZ主
――,―,,――――■―■―――――――――――――――――――■――――――――■
93.9 92.8
97.4 97.9 91.1 97.4 98.4 ヽ､ヽ 95.6
97.4
――――――⊥―二―二二二▲=
97.9
―――― 〓〓― O〓――――
92.1
二二二二二=｢――――――■
97.4
― ― ― ―――
98.4
――― ―― ― ― ――
97.9
i己主―
Jl m J6
｣立二二―==
二HIRON
JYK
―― 〓〓 〓
JGC JBK
Jl 】 J4 ― J6 CHIRON― JYK JGC ― JBK
73.6 62.5 94.1 73.5 73.2
73.5
76.8
iZ日
62.5 75.4 93.2
90.9 9 .6
60.4 53.3
iiZ日
62.9 62.6
60.8 60.8
97.0
〓 〓∴―=二 〓――〓 ―
77.8
〓 ― 〓― 〓 ―=―一―:――〓 〓―
60.4
iii岳
74.3 75.6 75.0
77.8 92.4 53.8
――――――――― ― ― ―
77.8
＼
85.1 87.8
76.8 93.4 53.3
―――――― ― ― ― ―
79.3 92.9
iiZ主
― ― ― ― ―――
90.6
75.8 91.4
―― ― ― 〓―――
53.3
― ― ― ― ―
78.3 90.9
〓 ― …―― ―
93.4
NSl protein region (a)
NS2 protein region (b)
NS3 protein region (c)
Fig. 2. Nucleotide and amino acid sequence homology of NSl protein (a), NS2 protein (b) and NS3 protein (c) regions.
Figures in the left lower half show nucleotide sequence homology and those in the right upper half show amino acid sequence homology, respectively, represented in homology percent.
J6 CHIRON
―― ‥ ― ―― ― ―
JGC JBK
J6
,,‑‑,,ー
66.5
69.5 69.4
CHIRON
,〓―――■――‑
69.4
ii⊆≡日
――――――――――――――――――――――――――――――――――――――――,■‑―
73.5 72.0
JGC 70.3 77.9
､,,‑,､
87.0
JBK
― ― ― ― ― ― ― ― ―
72.6
― ― ― ―――‥
80.0
==― 〓
88.5
:≡l―
LJ見
― ――― ―〓
CHIRON
――〓―――――――――――――‑――,――――――,
JGC
― ―――‖ ―――
JBK
J6
― ― ― ― ――――
､‑｢｢､
56.7
― 二:二――――――――――――――――――――――――――――――――̲
59.6
――― ―――〓― ―
59.4
CHIRON
― ― ―― 〓 巾― ――――
57.8
―=― ―一
㌔‑‑,,
―――― ―― ――― ―― ―
71.0
―― ― 〓―――〓 ― ― ―
71.1
JGC
―――― ―― ―――― ―
60.6 77.3
〓 ― ― 〓 ― ―
i⊇≡日
〓 ―〓〓 ―――― 〓 〓
91.4
JBK
〓 ―――〓―〓〓 ――
59.6
―― ― ― ― ――
76.2
――――――――― ⊥一
92.4
――■=―=二――――― ―
i=lさl―
――― ― ―― ― ― ― ―
J6
― ― ― ―― 〓 〓
CHIRON
―― 〓 ― ―
JGC
―― ― ―■― ―
̲IRK
J6
―――― ― ―――― ――
ii⊆:≡主
―― ――― ――一
69.9
――― ―――――一二
70.1
― ―― ― ― ――O
了0.0
〓― ―― ― ――― ―――
CHIRON
̲ ―― ―〓〓―― ――――― ― ―――
80.3
―――――:― ― 二――
､ ‑,､
―― 〓‖ 〓 ――
79.8
――― 〓 ――
78.6
JGC
― ―― 〓 〓 ―――
80.3
―■――――■,―■●〓――――――――――■l――――――――――l――――――――――――――――――――,
91.5 1=ii張
―――― ― ― ――
90.6
J BI(こ
――一――一一
80.3〓
―― ――――― 〓―〓 ― ――
91.8
―――― ― ――
95.
―― ― 〓 ――
､ ‑,｢､
―― ― 〓―‖〓
NS4 protein region (a)
NS5 protein region (b)
3 ‑Non‑coding region (c)
Fig. 3. Nucleotide and amino acid sequence homology of NS4 protein (a) and NS5 protein (b) regions, and nucleotide sequence homology of 3'non‑coding region (c).
Figures in the left lower half show nucleotide sequence homology and those in the right upper half show amino acid sequence homology, respectively, represent by the homology percent.
J6 CHIRON
JGC JBK
J6
､,＼
67.8 67.6 67.3
CHIRON 72.8
＼‑,‑,,､
79.1 79.6
JUし
―― ― ― ― ― ― ―――― ――――――
73.6 86.7
,ー‑ ＼,
92.1
JBK
―― ― ― ―O―二O ――
73.3 86.9
二二二二=
97.5
J6 CHIRON
JGC IRK
J6
ii張
58.1 59.3 58.6
C―11RON
E]皇*
＼‑,＼｢
wm 79.4
JGC 59.7 84.1
i張
92.7
JBK
―――――,―〓―――――――――――,――,――――――――――――――――,――,
71.5 84.5
――――――――――――――――――〓‑
95.2
J6 CHIRON
JGC JBK
J6
i張
29.6 38.1 42.9
CHIRON
＼ ,＼
73.7
77.8
JUし
―〓―〓 ―
､‑‑‑､
92.3
JBK
has a highest degree of variation among seven isolates (nucleotide <60.8%, amino acid
<53.3%: Fig. 1C). Homologies from NSl to 3J non‑coding region also showed moderate variabile and hypervariable regions among each four isolates (Figs. 2 and 3).
DISCUSSION
According to the homology comparison, Jl showed that its nucleotide and ammo acid sequence has high homology (more than 94.1%) with CHIRON. Therefore, Jl should belong to the CHIRON subgroup HCV. (Kato et at., 1990). J6 was different from each of the other three isolates (CHIRON, JGC and JBK) in entire sequence (Okamoto et aL, 1991). JGC was
close to JBK, similar in >87.0% for both nucleotide and amino acid sequence in entire se―
quence. J4 and JYK were also close to JBK and JGC (nucleotide >85.1%, amino acid
>90.9%: Figs. 1, 2 and 3). Our homology comparison resembles with those by Okamoto et al.
(1991) and Takamizawa et al. (1991). On the basis of sequence similarity therefore, there would be at least three subtypes of HCV genome. Recently, Okamoto et al. (1992) indicated that the HCV genome sequences can be classified into four genotypes: CHIRON and Jl as genotype 1; JGC, JBK, JYK and J4 as genotype 2; J6 as genotype 3; J8 as genotype 4 (this sequence was not included in our sequence comparison). Our homology comparison gave the result compatible with this classification in general.
Due to the high degree of nucleotide and amino acid sequence conservation in the 5 non‑coding region among compared six isolates ( > 92.8%) except JYK, primers deduced from this region could be useful for the specific detection of HCV RNA by reverse transcription
polymerase chain reaction (RT‑PC町HCV antibody detection system using core protein an‑
tigen have already been developed and available as a commercial assay kit (Abbot, U.S.A.;
Ortho Diagnostic Systems, Tokyo, Japan). According to the low level of nucleotide and amino acid sequence variation in the core protein region, the products have been used to
.
detect HCV antibody by the ELISA (Okamoto et aL, 1990b, Chiba et al., 1991) and by western blotting method for early diagnosis of HCV infection. Using core protein antigen for HCV antibody detection is more sensitive than C‑100 antigen which was derived from NS3 and NS4 protein region (Harada et al, 1991). The significance of the apparent nucleotide and amino acid sequence variabilities in the E protein region is unclear. It could be a conse‑
●
quence of immune selection and needs to be considered in the future development of HCV vaccines (Plagemann, 1991).
Because its genome organization and predicted virion structure closely resemble those of the flaviviruses and pestiviruses, HCV has been proposed to be classified in the Family Flaviviridae as a new third genus (Plagemann, 1991; Wengler, 1991). The nucleotide and
amino acid sequence variation of the E protein region indicated the possibility of multiple sub―
types assay. Recently, researcher in France and Thailand also published partial sequences of HCV (Kremsdorf et al., 1991; Mori et al., 1992). Each HCV subtype would be expected to have distinct epidemiological distribution, and this phenomenon would influence the choice of materials best suited for diagnosis and immunoprophylaxis in the geographical. area at issue
(Okamoto et al., 1991). The authors are now determining the sequence of HCV CDNA which has been derived from the HCV infected patient in Nagasaki Prefecture in order to clarify the local strain of HCV in Japan.
AcKNOWLE D GMENTS
This study was supported by a Grant｢in Aid for Non A‑non B Hepatitis Study from Ministry of Health and Welfare of Japan. The first author was supported by Monbusho Scholarship of Japan.
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