Journal of General Virology (1994), 75, 3279 3284.
Printed in Great Britain
Full-length sequence of the genome of hepatitis C virus type 3a:
comparative study with different genotypes
N o r i k o Y a m a d a , ~ Kiyomi Tanihara, 1 M a s a s h i M i z o k a m i , 2 Kenichi Ohba, 2 Akira T a k a d a , a
M i k i h i r o Tsutsumi 3 and T a k a y a s u D a t e 1.
1 Department o f Biochemistry, Kanazawa Medical University, Uchinada, Ishikawa 920-02, 2 Second Department o f
Internal Medicine, Nagoya City University, Kawasumi Mizuho, Nagoya 467 and 3 Department o f Gastroenterology,
Department o f Internal Medicine, Kanazawa Medical University, Uchinada, Ishikawa 920-02, Japan
Hepatitis C virus (HCV) type K3a (type 3a), which
represents a minor genotype in Europe, the U.S.A. and
Asia, appears to be significantly distributed throughout
Australia and Brazil. We amplified the HCV-K3a/650
genome by reverse transcription polymerase chain
reaction in ten overlapping fragments and determined
the nucleotide sequences. The total sequence was 9454
bases in length and contained an open reading frame of
3021 amino acids, which is 10 or 11 amino acids longer
than in HCV type 1 and 12 amino acids shorter than the
Hepatitis C virus (HCV) is the major causative agent of
non-A, non-B hepatitis (Choo et al., 1989). A number of
HCV strains from different areas of the world have been
isolated and subjected to sequence analysis. The viral
genome is a positive strand RNA molecule, approxi-
mately 9400 nucleotides in length, containing an open
reading frame (ORF) which encodes 3010 to 3033 amino
acid residues (Kato et al., 1990; Choo et aL, 1991;
Takamizawa et al., 1991; Okamoto et al., 1991, 1992b).
By analogy with flaviviruses, this polypeptide can be
divided into a 5' structural region, consisting of the
putative core and envelope proteins, and a 3' region
corresponding to non-structural (NS1 to NS5) proteins.
HCV shows substantial nucleotide sequence variation
throughout the viral genome and has been classified into
several genotypes to date. Since we discovered that HCV
isolates could be assigned to two genotypes and four
subtypes based on analysis of a portion of the NS5
domain (Enomoto et al., 1990), many variants have been
isolated and recent phylogenetic analysis of the NS5
region has led us to classify HCV into six major groups
and 11 subtypes (Simmonds et al., 1993a). HCV has also
been grouped into several distinct genotypes on the basis
of sequence variation at the 5' non-coding region (5'
The sequence data from this article have been deposited with the
GSDB,DDBJ, EMBL,NCBIDNA databasesunderaccessionnumber
D28917.
0001-2580 © 1994 SGM
3279
sequence of type 2. These differences were due to the
different lengths of both the putative envelope protein
E2 and the NS5A regions, whose nucleotide lengths
differ between types 1 and 2 also. Phylogenetic analysis
of the putative core region and a portion of NS5B
encoding the Gly-Asp-Asp motif indicated that HCV-
K3a closely matched the corresponding type 3a group.
The deletion and addition of amino acids in both E2 and
NS5A may be associated with their pathobiological
features.
NCR; Bukh et al., 1992; Chan et al., 1992; Simmonds et
al., 1993b), core gene (Simmonds et al., 1993b), E1 gene
(Bukh et al., 1993) and other regions (Okamoto et al.,
1991, 1992; Chan et al., 1992; Simmonds et al., 1992).
Previously, we described a scheme of HCV types- PT
(prototype), K1, K2a, K2b and K3a (Enomoto et al.,
1990; Nakao et al., 1991; Takada et al., 1993b) which
correspond to types la, lb, 2a, 2b and 3a, respectively
(Chan et al., 1992). Other groups use different schemes.
In this paper we use the terminology as described by
Chan et at. (1992) to distinguish between different genetic
variants of HCV.
The major HCV types distributed widely around the
world are types la and lb. Type 2 has also been found in
almost all countries, but it preferentially distributes in
the Far East (Takada et al., 1992a, 1993a; Bukh et al.,
1993). Types 4, 5 and 6 are found mainly in Africa (Cha
et al., 1992; Bukh et al., 1992, 1993; Simmonds et al.,
1993a). Type 3a, which was first found in isolates from
Thailand (Mori et al., 1992), and was then reported from
Europe (Chan et al., 1992) and the U.S.A. (Lee et al.,
1992), appears to be a common genotype worldwide with
the exception of Japan (Takada et al., 1993b).
Here we report the full sequence of type 3a (HCV-
K3a/650) and compare it with other isolates.
We previously reported the world distribution profile
of HCV types obtained from a limited number of
collections and demonstrated that their distribution
3280 Short communication
Table 1. Distribution o f H C V type 3a in different
countries*
No. of
Country Occurrence samples
Australia 13 57
Brazil 5 58
Spain 2 26
U.S.A. 2 63
Japan 3 376
China 0 59
Thailand 0 14
* In other countries such as G e r m a n y (13 samples), France (31) and
Egypt (25), no type 3a was detected in our samples.
appears to differ from country to country. However,
some isolates could not be classified (Takada et al.,
1992a, b). Cloning and sequence analysis in the region of
NS5B of three unclassified samples revealed that they
were all part of a third group named K3a (Takada et al.,
1993 b) which corresponds to type 3a (Chart et al., 1992).
A specific probe was synthesized from the cloned
fragment and used for slot-blot analysis of PCR
products. Table 1 shows the world distribution of HCV
type 3a. Type 3a seems to be a minor genotype in most
countries but it is significantly distributed in Australia
and Brazil. In contrast to the finding by Mori et al.
(1992), no type 3a was detected in our collection from
Thailand. Because our samples were not representative,
the distribution of type 3a appears to be localized in
specific areas even within one country. Recently, we
found three Japanese patients with HCV type 3a.
In order to clone the type 3a HCV genome, cDNAs
were obtained by reverse transcription polymerase chain
reaction (RT PCR) using samples (HCV-K3a/650)
(107 HCV/ml) from the U.S.A. Amplified fragments
were obtained from both the NS5B (Enomoto et al.,
1990) and 5' NCR (Okamoto et al., 1990) regions, since
their sequences had been determined previously. The
primer used for cDNA synthesis was a random hexamer,
except for synthesis of the 3'-end fragment, for which
oligo-(dA)l4 was used. A major difficulty with PCR
cloning is the possibility that mismatches between the
primers and the variant sequence will prevent ampli-
fication. To overcome this problem, the sequences of
PCR primers for the side whose sequence had not been
defined were selected from among relatively conserved
regions of different genotypes. Most of the PCR primers
were also designed to contain a restriction enzyme site
near their 5' end to assist in subsequent cloning. We
amplified ten overlapping fragments: they were between
nucleotides 1 to 362, 41 to 1597, 1449 to 2584, 2456 to
3664, 3607 to 4795, 4685 to 5443, 5304 to 6418, 6414 to
8286, 7635 to 8725 and 8447 to 9454. The 5' terminal
sequence of the 5' NCR was obtained by using a 5'-
Amplifinder race kit (Clontech) followed by PCR
according to the manufacturer's protocol with minor
modifications. The nucleotide sequences in these fra-
% gments were determined using an automated DNA
sequencer (ABI 373A-70).
22,8
8.6
The resulting HCV-K3a genome was 9454 bases in
7-7 length and contained an ORF encoding a polypeptide of
3.2 3021 amino acids (aa). The genome was organized from
0.8
0
5' to 3' as genes C (191 aa), E1 (192aa), E2-NS1
0 (433 aa), NS2 (170 aa), NS3 (677 aa), NS4A (54aa),
NS4B (261 aa), NS5A (452aa) and NS5B (59t aa),
based on cleavage sites for the processing of the precursor
polyprotein obtained from types la and lb (Hijikata et
al., 1991a, 1993; Grakoui et al., 1993a, b). The ORF of
HCV-K3a is 10 and 11 amino acids longer than that of
types la and lb, respectively (Kato et al., 1990; Choo et
al., 1991), and 12 amino acids shorter than that of types
2a and 2b (Okamoto et al., 1991, 1992). These differences
are due mainly to deletion or addition to the E2-NS1
and NS5A regions.
Comparative analysis of nucleotide sequences was
carried out to clarify the relationships between our
isolate and other isolates. Phylogenetic trees for the core
protein region and a portion of the NS5B region in types
1 to 3 were constructed using evolutionary analysis
software (Ina; ODEN ver 1.1, National Institute of
Genetics Mishima, Japan, 1990). In both cases variants
associated into three major groups-types 1, 2 and 3 -
and each group split into two major clusters (Fig. 1).
Based on analysis of the NS5 region, we had previously
classified HCV isolates corresponding to the phylo-
genetic groups PT/K1 and K2, the first being subdivided
into PT and K1 and K2 comprising K2a and K2b
(Enomoto et al., 1990). Phylogenetic analysis supports
this classification and has also been confirmed by other
research groups. These analyses indicated that HCV-
K3a/650 is most closely related to E-groups such as E-
bl, E-b2, E-b3 and E-b7 and next is closely related to
HCV-TR, which includes type 3a and type 3b, re-
spectively (Simmonds et al., 1993a, b). The NS5B
sequence of HCV-K3a/650 analysed in this study was
more than 93 % identical to all other reported sequences
in group 3a and about 80 % identical to that in group 2b.
The phylogenetic trees constructed from the E 1 regions
were similar and essentially the same. Classification
using partial sequencs of the genome is controversial, but
the determination of full genomic sequences of HCV is
impractical for most laboratories. The full genomic
sequence of our subtype 3a may be useful when new
sequences are determined and analysed.
The nucleotide and deduced amino acid sequences of
the genes in our isolate were compared within ten
different regions with those of other HCV genotypes (la,
lb, 2a and 2b). As shown in Table 2, nucleotide sequences
 Short communication 3281

(a) (b)
~ H HCV1
1a I L ~ HCVH
HC-J1 t HC-J1
HCT18 r - - HCV-BK
CVTh
la HCT23
HCVH lb ~_~MjH~IjK 1
HCT27 HCV-JT
T-1
lb T-7
I L ~ H H CHCV-J
V'BK
HCVJK1
C-J4 I-1 "L
HCVJ I %==,,=o
HCVT

"'t
3b I [ T.0
"!"-10
CV.JT
3a HCV-TR
K3a1650
3b HCV-TR
2a
2a I HC-J5 I
[ 2b HC-J6
HC-J7
I
2b ~ _ .
K2b
HC-J7
I HC-J8 I~ K2b-1
HC-J8
o.1oo o o.:Ioo
Fig. 1. Phylogenetic tree for the HCV core (a) and NS5B (b) regions of HCV-K3a/650 and previously reported isolates. The
DDBJ/GenBank/EMBL accession numbers of the clones are as follows: HCV-1, M62321 ; HCV-J1, D00831 ; HCVH, M67463; HCVJ,
D00574; HCV-J, D90208; HCV-BK, M58335; HCVT, M84754; HCV-JT, Dl1168; HCVJK1, X61596; HC-J4, D00832; HC-J5,
D 10075 ; HC-J6, D00944; HC-J7, D 10077; HC-J8, D 10988; E-b 1, D 10123 ; HCV-TR, D 11443. The lower scale refers to the number
of the nucleotide substitutions per site.

T a b l e 21 Comparison o f the nucleotide and deduced amino acid sequences o f ten different regions o f the H C V - K 3 a
genome with the corresponding regions o f seven other H C V isolates*

Percentage nucleotide (deduced amino acid) identity against:

Region HCV- 1 HCV-J HCV-JT HACV-BK HC-CHINA HC-J6 HC-J8

(aa)t (la) (1 b) (lb) (lb) (1 b) (2a) (2b)
5' NCR 92 92 92 91 92 89 88
C (191) 82 (89) 80 (89) 82 (89) 82 (89) 81 (89) 80 (85) 78 (84)
E1 (192) 64 (66) 65 (67) 64 (66) 65 (67) 64 (65) 59 (54) 56 (54)
E2-NS1 (433) 66 (72) 65 (71) 66 (72) 66 (70) 65 (70) 63 (66) 63 (68)
NS2 (170)~ 54 (52) 59 (59) 57 (61) 57 (57) 56 (56) 54 (50) 53 (48)
NS3 (677) 69 (80) 71 (82) 70 (82) 69 (82) 70 (81) 68 (81) 68 (80)
NS4A (54) 69 (76) 67 (74) 65 (74) 66 (72) 68 (76) 65 (65) 61 (61)
NS4B (261) 69 (79) 69 (77) 69 (76) 67 (76) 69 (76) 65 (71) 62 (70)
NS5A (452) 63 (59) 64 (72) 64 (71) 64 (71) 64 (72) 59 (62) 61 (62)
NS5B (591) 71 (75) 71 (76) 71 (76) 71 (76) 70 (75) 69 (75) 69 (75)
* HCV-1 (Choo et al., 1991), HCV-J (Kato et al., 1990), HCV-JT (DDBJ/GenBank/EMBL no. D11168), HCV-BK (Takamizawa et al., 1991),
HC-CHINA (DDBJ/GenBank/EMBL no. L02836), HC-J6 (Okamoto et al., 1991) and HC-J8 (Okamoto et al., 1992) were compared for nucleotide
sequence identities throughout the entire genome except for the 3' NCR. The relevant genotype designation is indicated in parentheses below each
isolate.
"~ The number of (deduced) amino acids encoded in HCV-K3a/650.
:~ The NS2 region is processed into at least two proteins (Grakoui et al., 1993a).

in the 5' N C R o f H C V - K 3 a / 6 5 0 s h o w 91 to 9 2 % i d e n t i t y to the n u c l e o t i d e s e q u e n c e s o f t y p e 1 a n d t y p e 2,

i d e n t i t y to t h o s e o f t y p e s l a a n d l b a n d 8 9 % a n d 8 8 %
i d e n t i t y to t h o s e o f t y p e 2a a n d t y p e 2b, respectively.
M o s t o f the s e q u e n c e h e t e r o g e n e i t y in the 5' N C R is
f o u n d in the vicinity o f the 5' e n d b e t w e e n p o s i t i o n s 1
a n d 18. O t h e r r e g i o n s in the 5' N C R s h o w 93 % a n d 9 1 %
respectively.
W h e n H C V - K 3 a / 6 5 0 w a s c o m p a r e d at the a m i n o a c i d
level w i t h o t h e r isolates, the gene e n c o d i n g the p u t a t i v e
c o r e p r o t e i n w a s 89 % a n d 85 % i d e n t i c a l to t y p e 1 a n d
t y p e 2, respectively. H o w e v e r , p u t a t i v e e n v e l o p e p r o t e i n s
3282 Short communication

(a)
80 90 i00 ii0 120 140 150
K3a/650: TFFKQGWGPLTDA NITGPSDDKPYCWHYAPRPCGIVPALNVCGPVYCFTPSPVVVGTTDAKGAPTYTWGA
HCVI(Ia): -D-D ..... ISY- -GS-- -QR ...... P-K ....... KS .................. RS ...... S--E
HCV-J(Ib): DE-A ..... I-HD EGS-- -QR ................ SQ .................. RF ...... S--E
HCV-J6(2a): EA-RV---A-Q-ED-V-N-E-MR ...... P--Q--V-S-SS .................. RL ......... E
HCV-J8(2b): DD-RI---T-EYET-V-NDG-MR ...... p ......... RT .................. KQ-V .... ---E

150 160 170 180 190 200 Total aa

K3a/650: NKTDVFLLESLRPPSGRWFGCTWMNSTGFVKTCGAPPCNIYGDGRDAQNESDLVCPTD 433
HCV(Ia): -D .... V-NNT---L-N ............ T-V ...... V-G- AGNNTLH .... 427
HCV-J(ib): -E---L--SNT---Q-N ............ T .... G ..... G- VGNNT ...... 427
HCV-J6(2a): -E ...... N-T---Q-S ........... YT ...... i-R-RA -FNASM--L .... 431
HCV-J8(2b): -E ...... N-T---R-A ........ G---T ........ R-RK -YNSTI--L .... 431

(b)

260 270 380 390 400 410

K3a/650: DAELVDANLLWRQEMGSNITR ALAALAEKSFPSSKPQEENSSSSGVDTQSSTASKVLPSPG
HCVI (la) : .... IE .......... G .... ---E--TR--G-- ST~GITG-NTTTSSEPAPSGCP
HCV-J (ib) : --D-ID .......... G .... ---E--T-T-G-- G--AVDSG-ATGPPDQASDDGD
HCV-J6 (2a) : Y-VDM .... F --GDV-- --QQ--I--FGQPP-SGDSGL-T-A-AAD- G-RTP-DEL
HCV-J8 (2b) : Y-CDM .... F --GDV-- V-REM-D-VLSPLQDMNDSGH-T-A--GGD IVQQPSDET

420 430 440 450 Total aa

K3a/650: EESDSESCSSMPPLEGEPGDPDL SCDSWSTVS DSEEQSVVCC 452
HCV(Ia): PD--A--Y . . . . . . . . . . . . . . . . DG ...... SEANAED--- 448
HCV-J(ib): KG--V--Y . . . . . . . . . . . . . . . . DG ...... GEAGED .... 447
HCV-J6(2a): ALSETG-I ......... ". . . . . . EPEQVELQPPPQGGVVTPGSGSG .... C- EEDD ..... 466
HCV-J8(2b): AASEAG-L ............... EPEQVELQPPSEGECEVIDSDSK ....... Q-D ..... 466

Fig. 2. Comparison of the deduced amino acid seuence of a portion of the putative E2 NS1 gene product (a) and a portion of the NS5A
gene product (b). Sequences of HCV-K3a/650 were compared with those of HCV-1 (Choo et al., 1991), HCV-J (Kato et al., 1990), HC-
J6 (Okamoto et al., 1991) and HC-J8 (Okamoto et al., 1992). Amino acid sequences are aligned from the cleavage site and numbered
according to processing results by Grakoui et al. (1993a, b). Sequences between 271 and 370 are abbreviated. Amino acid residues
identical to those in HCV-K3a/650 are denoted by a dash ( ) .

E 1 and E2-NS 1, which contain a hypervariable region at The deduced amino acid sequences of the E2- and
the N terminus (Hijikata et al., 1991b), show fewer NS5A-encoded proteins are of interest since these regions
similarities within the two groups. The NS3 gene show type-specific deletions or additions in several
products, which have protease and probably RNA positions. As shown in Fig. 2 (a), the optimal alignment
helicase activities, are more conserved than other regions indicated that several gaps were present in the middle of
such as the core protein. All residues required for E2 NS1. The total number of amino acid residues in E2
protease activity reported thus far are conserved in both for types 1, 2 and 3a was estimated to be 427, 431 and
the NS2 and NS3 regions. The NS4A and NS4B gene 433, respectively. The other type la groups, HC-J1 and
products, p27 and p70, respectively (Grakoui et al., HCVH, showed the same gaps as found in HCV1, while
1993a, b), show about 61 to 77% identity in types 1 and other type lb groups, HCV-BK, HCV-JT and HCV-
2. The 50 amino acid residues at the N-terminus of NS4B CHINA, showed the same additions or deletions as
varied considerably, showing 50% identity to the found in HCV-J1. Since the cDNA of HCV-K3a/865,
corresponding region in other genotypes. The NS5A another isolate of type 3a from Australia, had an almost
gene product of HCV-K3a/650 shows less similarity to identical sequence in the E2 region, these deletions and
those encoded by other genotypes because of varied additions could be type-specific.
sequences between the middle region and the C terminus Vaccinia virus transient-expression assays revealed
(Fig. 2b). The nucleotide sequence of NS5B (a putative that the E2 gene encodes 70K (gp70) and 88K (gp88)
RNA polymerase gene) of HCV-K3a/650 is the same proteins containing N-linked glycans (Grakoui et al.,
length as in other genotypes and shows about 75% 1993 b). The latter glycoprotein sequence extends into the
identity to types 1 and 2. NS2 region. The cleavage site of the N-terminus of gp70

is between amino acids 383 and 384, while that of
E2 NS2 is predicted to be near the middle of the E2 gene
where type-specific additions or deletions in the amino
acid sequence are found. Alternative proteolytic pro-
cessing or other post-translational modifications are also
thought to occur in the E2-NS2 region, leading to the
production of multiple forms of E2, possibly with distinct
biological functions in HCV replication (Grakoui et al.,
1993 b). Thus, it is possible that these addition or deletion
sequences in the E2 region may affect the properties of
the multiforms of E2 which in turn changes the
immunoresponse, proliferation and stability of the virus
particle in blood.
The deduced amino acid sequences in the NS5A
regions also show type-specific additions and deletions
(Fig. 2b). Type 2 has the largest NS5A gene of the
reported genotypes (a 20 amino acid insertion at its C
terminus in NS5A) whereas type lb has the smallest one.
The regions between aa 260 and 263 and aa 384 and 388
are missing in types 2 and 1, respectively, whereas type 2
has 20 amino acids inserted between aa 433 and 434.
Furthermore, the sequence between aa 375 and 412
shows hypervariation among different genotypes,
although only a few amino acids are substituted within
the same genotype. For example, in the HCV-K3/865
genome, Ala at aa 373 is substituted by Arg.
Two proteins are derived from the NS5 region through
processing by the NS3-encoded protease; these are
NS5A (58K) and the C-terminal NS5B (66K; Grakoui et
al., 1993b; Manabe et al., 1994). NS5B is predicted to
contain the RNA-dependent RNA polymerase activity
on the basis of the presence of the characteristic Gly-
Asp-Asp sequence (aas 2747 to 2749) and surrounding
conserved motifs (Poch et al., 1989). The function of the
NS5A product has not yet been elucidated. Recently, we
demonstrated that in samples from patients with chronic
hepatitis only a single 84K protein specifically reacted
with antibodies to the NS5B protein (Tsutsumi et al.,
1994). This 84K protein is 18K larger than the NS5B
product obtained by vaccinia virus transient-expression
assays. Therefore, it is possible that the NS5B product is
post-translationally modified or that alternative pro-
cessing occurs in human liver cells. In flaviviruses the
NS5 region is not processed further but remains as a
single polypeptide of about 100K (Chambers et al.,
1990); it would thus be interesting to determine whether
the C terminus of the NS5A region, which shows type-
specific heterogeneity in length, is the component of
native RNA polymerase isolated from human hepato-
cyts.
There are several lines of evidence to indicate that
infection with different HCVs leads to different clinical
pictures. HCV type 2 groups seem to be less able to
replicate in human liver compared to type lb, because of
Short communication 3283
a significantly lower concentration of RNA in the blood
(Yoshioka et al., 1992). It has been reported that type 2a
and 2b groups are more likely to respond to interferon
than type lb (Takada et at., 1992b; Yoshioka et al.,
1992; Kanai et al., 1992). Although there are no data to
link regions in the viral genome with each clinical
picture, we hope that the deletion and addition of amino
acid sequences in both E2 and NS5A may be associated
with different pathobiological features.
We thank Dr Lawrence Lumeng, Indiana University, U.S.A. and
Dr Geoff Farrell, University of Sydney, Australia for gifts of serum
samples from patients with HCV. This work was supported by grants
from the Hokkoku Cancer Research Foundation, Ishikawa, Japan and
the Foundation for Promotion of Cancer Research backed by the
Japan shipbuilding Industry Foundation.
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(Received 18 April 1994; Accepted 4 July 1994)