Download Effect of frameshift mutation in the pre

Journal of General Virology (1994), 75, 917-923. Printed in Great Brita#l
917
Effect of frameshift mutation in the pre-C region of hepatitis B virus on
the X and C genes
Seong Kee Kim, 1 Sung Key Jang 2 and H y u n e M o Rho ~*
1Department o f Molecular Biology and Research Center for Cell Differentiation, Seoul National University,
Seoul 151-742 and 2Department o f Life Science, Pohang htstitute o f Science and Technology, Pohang 790-784,
Korea
We have previously cloned a mutant hepatitis B virus
(HBV) genome which had one thymidine addition in the
pre-C region resulting in a frameshift mutation in the
pre-C region and fusion of the X and C genes. We
constructed plasmids containing serially deleted and/or
back-mutated (authentic) pre-C regions to study the
effect of the frameshift mutation. COS cells transfected
with plasmids containing the frameshifted pre-C region
produced a 21K C protein (P21c) but not a 22K partially
processed pre-C protein (P22). On the other hand, COS
cells transfected with plasmids containing the back-
mutated pre-C region produced P22. This result was
also observed in HepG2-K8 cells producing the mutant
HBV particles, Therefore, the pre-C region of HBV is
likely to be non-essential for virus replication, COS cells
transfected with the plasmid containing a fused X-C
open reading frame (ORF) produced a 40K X-C fusion
protein. This X-C fusion protein exerted transcriptional
trans-activation. These results suggest that the mutant
HBV has a C gene with a defective pre-C region and a
fused X-C ORF, and hence cannot synthesize 16K
HBeAg (P16e).
Genomic DNA of hepatitis B virus (HBV) has four open
reading frames (ORFs) which are designated pre-C/C, P,
pre-S/S and X (Ganem & Varmus, 1987; Tiollais et al.,
1985), In HBV the coding region for the C protein (P21c)
is preceded by an in-phase ORF termed the pre-C region
(Ou et al., 1986). Translation initiation from the pre-C
initiation codon produces a pre-C protein (P25) that
contains the entire C protein sequence and a 29-residue
amino-terminal extension (pre-C region). It has been
demonstrated that the first 19 amino acids of the pre-C
region form a signal sequence to direct the pre-C protein
to the endoplasmic reticulum, where it is cleaved, forming
the partially processed pre-C protein (P22) (Bruss &
Gerlich, 1988; Garcia et al,, 1988, Junker et al., 1987; Ou
et al., 1986, 1988; Roossinck et al., 1986; Weimer et al.,
1987). P22 is further processed by a protease(s) at its
arginine-rich carboxy-terminal domain and secreted as
HBV e antigen (HBeAg) (Pl6e) (Ou et al., 1986, 1988;
Salfeld et al., 1989). In the course of HBV infection, the
presence of HBeAg is generally correlated with active
virus replication and often liver disease. On the other
hand, the clearance of HBeAg and subsequent rise of the
homologous anti-HBe antibody indicate termination of
HBV replication and remission of liver disease (Hoofnagle et al., 1981 ; Realdi et al., 1980).
The smallest ORF, X, is conserved among all
mammalian hepadnaviruses and has been shown to be
expressed during virus infection (Kay et al., 1985~
Meyers et al., 1986; Moriarty et al., 1985). It has been
demonstrated that the X protein is a transcriptional
activator of various enhancer promoter combinations,
including the HBV enhancer and C gene promoter, the
simian virus 40 (SV40) enhancer and early promoter, and
the /q-interferon gene (Spandau & Lee, 1988; Twu &
Schloemer, 1987; Zahm et al., 1988).
A novel HBV protein of 35K to 40K was discovered in
HBV-transfected HepG2 cells (Bchini et al., 1990) and in
core particles isolated from HBV-infected livers (Farza
et al., 1988; Feitelson, 1986). It was characterized by
Western blotting and proposed to be an X-C fusion
protein. However, HBV does not have a fused X-C
ORF. The biological function and the mechanism of
production of such an X-C fusion protein are as yet
unknown. The possibility of ribosomal frameshifting
causing X C fusion protein production has been excluded (Lo et al., 1990).
Previously, we have cloned a mutant HBV (adr)
genome from a Korean hepatitis B patient (Choi et al.,
1984) and determined the complete sequence of 3213
nucleotides (nt) (Rho et al., 1989). The DNA sequence
analysis revealed the addition of one thymidine at nt
1821 of the pre-C region, which resulted in a frameshift
mutation in the pre-C region and fusion of the X and C
genes. In this paper, we characterized the proteins
expressed from the pre-C/C and fused X-C ORFs of the
mutant HBV. As the fused X-C ORF contained a region
0001-1967 © 1994SGM
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918
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Fig. 1. The nucleotide sequence of a pre-C region and mapping of the 5" deletion of the pre-C region in plasmlds. (a) Nucleotlde
sequences of the pre-C region. Deleted 5' ends are shown by arrowheads. Three initiation codons are underlined and numbered: 1, ATG
codon of possible elongated pre-C protein; 2, ATG codon of frameshifted (authentic) pre-C protein: 3, ATG codon of C protein; 4,
two termination codons of frameshifted pre-C protein. The site of one thymidine addition is indicated as a vertical arrowhead. The
encoded amino acid sequence is shown above the nucleotide sequence. Termination of the frameshifted pre-C protein is marked by an
asterisk. (b) Schematic diagram of recombinant plasmids containing the serially deleted and/or back-mutated pre-C region with the
C gene. Vector pUC19 sequences are shown as a thin line. Three ATG codons and endpoints of 5' deletion mutants are the same as
in (a). Horizontal dashed lines indicate the region deleted with Bal 31 nuclease. The filled circles indicate the T deletion mutation.
Abbreviations: B, BamHI: H, HindlII ; Hc, HinclI ; S, SalI.
encoding part o f the X protein, we also tested whether
the fused X - C O R F products possess trans-activating
activity.
In the m u t a n t HBV, the putative initiation c o d o n o f
the elongated pre-C region at nt 1758 was located 53 nt
upstream o f the initiation c o d o n o f the authentic pre-C
region at nt 1811 (Fig. 1 a). Therefore we tested whether
the m u t a n t H B V contained an elongated pre-C region
instead o f the authentic pre-C region which m a y be
involved in generating HBeAg. To investigate the
utilization o f the A U G c o d o n s o f the pre-C region for
the H B V core antigen (HBcAg) and H B e A g synthesis,
we constructed r e c o m b i n a n t plasmids containing serially
deleted a n d / o r b a c k - m u t a t e d (authentic) pre-C regions
(Fig. 1). To construct 5' deletion m u t a n t plasmids,
p U S C P containing the C gene and pre-C region was
opened with B a m H I and serially deleted with Bal 31
nuclease. After filling o f the staggered ends with the
K l e n o w fragment, B a m H I linkers were attached. The
resulting series o f 5' deletion m u t a n t s was selected by
D N A sequencing (data not shown). To recover the
p r o m o t e r region, the SV40 late p r o m o t e r region o f
p U S C P was cloned between B a m H I and H i n d I I I sites o f
the deletion m u t a n t plasmids. M u t a n t s with endpoints at
- 180 (pUSCP1), - 129 ( p U S C P 3 ) and - 3 2 (pUSC)
were selected (numbering system starts with the A o f the
C gene initiation codon). F o r the construction o f
plasmids p U S C P 1 0 and p U S C P 3 0 containing the backm u t a t e d pre-C region, site-directed mutagenesis was
performed using P C R ( K a m m a n n et al., 1989; K i m
et al., 1992b; Saiki et al., 1988) with a mutagenic primer.
Plasmids p U S C P 1 0 and p U S C P 3 0 were selected by
D N A sequencing (data not shown).
Each of the vectors was introduced into C O S cells
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HBV cannot synthesize pre-C region-derived HBeAg.
This observation was further confirmed by Western
blotting experiments.
Characterization of core antigens by Western blotting
(Sambrook et al., 1989) was performed to estimate the
relative size of the processed antigens. We expressed the
C gene in Escherichia coli (Choi et al., 1991). The
recombinant core particles produced by E. coIi cells were
purified (Kim & Rho, 1992) and used as an Mr marker.
A rabbit polyclonal anti-HBc antiserum against recombinant HBcAg was prepared using techniques reported
previously (Sambrook et al., 1989). As shown in Fig.
2(a), COS cells transfected with pUSCP1, pUSCP3 and
pUSC produced only a 21K protein (P21c), whereas
COS cells transfected with pUSCP10 produced a minor
22K protein (P22) as well as a 21K protein (P21c). After
deletion of the ATG codon region of the elongated preC (pUSCP30), the 22K protein became the major species.
The 22K protein (P22) then becomes cleaved by a
protease(s) and secreted into the medium (Ou et at.,
1986, 1988; Salfeld et al., 1989). The production of P22
from pUSCP10 was much lower compared with that
from pUSCP30 (Table 1 and Fig. 2). This result suggests
that the A U G codon of the elongated pre-C may
interfere with translational initiation at the A U G codon
of the pre-C, but not with the A U G codon of the
C protein. We have also established a mutant-HBVproducing cell line, HepG2-KS, by transfection of
mutant HBV DNA into HepG2 cells (Kim et al., 1992a).
In HepG2-K8 cells, only the 21K protein was detected
(Fig. 2a). These results confirm that our mutant HBV
has a defective pre-C region, and hence cannot synthesize
Table 1. Relative levels of expression of HBcAg and
HBeAg in recombinant plasmid-transfected COS cells
Radioactivity (c.p.m.)t
Plasmid*
Cell extract
Culture medium
pUC19
pUSCPI
pUSCP10
pUSCP3
pUSCP30
pUSC
pSVXC
607
17262
19184
24 624
25423
46012
11786
532
1163
3234
1089
14622
1426
1125
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* Plasmlds are described in Fig. 1 (b) and 4(a).
"~ Cell extracts and culture media were one-fifth of the cell lysate,
and one-fifth of the supernatant, respectively, from a 100 mm dish. The
values are the mean of three determinations.
using the calcium phosphate method (Chen & Okayama,
1987). At 72 h post-transfection, cells and culture media
were collected separately. Antigens from COS cells
transfected with the recombinant plasmids were assayed
with the Abbott HBe radioimmunoassay kit (Abbott
Laboratories) (Table 1). Note that this assay cannot
differentiate between HBcAg and HBeAg. In the case of
plasmids pUSCP10 and pUSCP30 (Fig. 1) which
contained the C gene with the back-mutated (wild-type)
pre-C region, HBcAg/HBeAg was detected in both cell
extracts and culture media. These results agreed with the
reports of previous studies (Bruss & Gerlich, 1988 ; Ou
et al., 1986; Roossinck et al., 1986). In the case of
plasmids pUSCP 1, pUSCP3 and pUSC, HBcAg/HBeAg
was detected only in cell extracts but not in the culture
media (Table 1). These results suggest that our mutant
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Fig. 2. Western blotting analysis of HBV antigens. Extracts of COS cells transfected with recombinant plasmids and HepG2 cells
producing the mutant HBV particles were separated by S D S - P A G E and transferred to a nitrocellulose filter. Core proteins (P21c and
P22) (a) and X - C fusion protein (b) were detected by an anti-HBcAg antiserum and 125I-labelled Protein A. The COS control refers
to pUC19-transfected COS cells. M r values are shown on the right. M and D indicate the positions of the monomer (21K) and dimer
(42K) forms of P21c purified from
E. coli.
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Short communication
HBV (adw2)
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Fig. 3. Genetic organizationof X and C ORFs in various hepadnaviruses. Numbers indicatemap positions. Sequenceswere taken from
the followingreferences:HBV(adw2) (Valenzuelaet al., 1980);mutant
HBV (adO (Rho et al., 1989); HBV-71 (Tong et al., 1990); DHBV
(Mandart et aL, 1984).
the 16K HBeAg, in agreement with the radioimmunoassay results (Table 1). This implies that the
mutant HBV did not utilize the AUG codon of the
elongated pre-C region for HBcAg and HBeAg synthesis.
Kozak (1986) proposed that the optimal context for
initiation is CCPuCCAUGG (Pu, purine). However, if
the sequence surrounding the first AUG triplet is
suboptimal, some 40S subunits bypass that site and
initiate translation further downstream. Translation
initiation from the AUG codon of the elongated pre-C
(in the context GGUUAAUGA) may therefore be
suboptimal.
Several laboratories have found HBV variants
mutated in the pre-C region (Brunetto et aI., 1989, 1990;
Carman et al., 1989; Fiordalisi et al., 1990; Li et al.,
1990; Okamoto et al., 1990; Riamondo et al., 1990;
Tong et al., 1990). Most of these HBV variants contained
a TAG stop codon formed by a point mutation in the
distal pre-C region. We have previously established a cell
line that produces such mutants (Kim et al., 1992a) and
therefore the pre-C region of the HBV is likely to be nonessential for virus replication. Introduction of a frameshift and nonsense mutations in the duck hepatitis B
virus (DHBV) pre-C region demonstrated that expression of the region is not essential for viral genome
replication (Chang et al., 1987; Schlicht et al., 1987). The
variant HBV-~I with a nonsense mutation in the distal
pre-C region had an in vitro replication capacity (Tong
et al., 1991). These results suggest that HBV may not
require HBeAg for its replication. The HBV variant
contained a TAG stop codon in the distal pre-C region
due to a G to A transition. However, our HBV with one
thymidine addition at nt 1821 has not only a C gene with
a defective pre-C region but also a fused X-C ORF. Our
mutant HBV is different from HBV-~I (Tong et al.,
1990) and DHBV (Mandart et al., 1984) in its genetic
organization (Fig. 3). The HBV-e 1 has a C gene with a
defective pre-C region and wild-type X gene. The DHBV
has only a pre-C/C ORF. Furthermore, the 5' end of the
DHBV C gene shows no homology with the X gene of
mammalian hepadnaviruses (Feitelson & Miller, 1988).
Therefore, the DHBV C gene cannot be considered as a
fusion between the C and X genes.
To determine whether an X-C fusion protein is
synthesized, we constructed the plasmid pSVXC which
contained a fused X-C ORF under the control of the
SV40 promoter. Extracts of COS cells transfected with
pSVXC contained HBcAg/HBeAg activity (Table 1). In
RNA analysis experiments, a 1.9 kb transcript which had
the fused X-C ORF coding sequences was detected (data
not shown). However, a transcript of approx. 1 kb which
encodes the C protein, transcribed from the initiation site
of C mRNA, was not detected. Approximately 70 % of
the transcripts from a region near the initiation site of the
X mRNA did not stop at the HBV transcription
termination signal (data not shown). This value was
higher than that in a previous report (Guo et al., 1991)
which showed that 50 % of X gene transcripts could read
through the transcription termination signal. As shown
in Fig. 2 (b), COS cells transfected by pSVXC produced
a 40K X-C fusion protein. Bands above the 40K band
appear to be due to proteins interacting non-specifically
with the anti-HBcAg antiserum. The fused X-C ORF
could encode an X-C fusion protein of 359 amino acids,
consisting of 151 amino acids encoded by the wild-type
X ORF and 208 amino acids encoded by the pre-C/C
ORF region.
Previous results have shown that the carboxyl-terminal
amino acids of X protein are dispensable for its function
as a transcriptional trans-activator (Levrero et al., 1990;
Ritter et al., 1991); we therefore examined the transactivating activity of the X-C fusion protein. The
reporter plasmid pSV2CAT containing the SV40 enhancer and early promoter driving the chloramphenicol
acetyltransferase (CAT) gene was cotransfected with test
plasmids into HepG2 cells, and a CAT assay was then
performed (Gorman et al., 1982). Plasmid pHX contained the wild-type X gene (Won et al., 1989) under the
control of the HBV enhancer/X promoter and the
poly(A) signal of the herpes simplex virus thymidine
kinase gene (HSV TK). Plasmid pHXC, on the other
hand, was similar to pHX but contained the fused X-C
ORF instead of the X gene (Fig. 4). Plasmid pHXCM is
similar to pHXC except that the poly(A) signal sequence
UAUAAA was mutated to UACAAG. In the case of
plasmid pHXC, the poly(A) signal is located inside the C
gene. To exclude the effect of the TATAAA sequence
when it encounters the transcription termination signal,
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(a)
HBV
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Fig. 4 Transcriptional trans-actlvation of SV40 enhancer/promoter by
the fused X-C ORF product. (a) Schematic maps of test plasmids pHX,
pHXC and pHXCM, the correct constructions of which were confirmed
by DNA sequence analysis. Numbers in parentheses indicate map
positions on the HBV genome. E/P, HBV enhancer/X promoter; open
boxes, ORFs; open circles, HBV poly(A) signal: sohd circles, herpes
simplex virus TK poly(A) signal; star, mutated HBV poly(A) signal. (b)
CAT assay for the trans-activation function of the X C fusion protein.
HepG2 cells were cotransfected with 5 lag of pSV2CAT and 15 lag of
each of the plasmids pUC19, pHX, pHXCM or pHXC. Activation
(fold) represents the ratio of the percentage of chloramphenlcol
acetylation in cotransfection with the X and X-C gene expression
vectors to that with control plasmid pUC19. All values are given as the
mean+s.D, obtained from experiments carried out in triplicate.
this sequence was mutated to TACAAG. These mutations do not change any amino acids of the X-C fusion
protein. In a SV40 mutant, deletion of the entire
AAUAAA sequence shifted polyadenylation to just
downstream from the next most proximal AAUAAA
(Fitzgerald & Shenk, 1981). A single mutation, a U to G
transversion in the AAUAAA hexanucleotide of the
E1A RNAs of adenovirus, decreases the efficiency of
RNA cleavage of the primary transcript by over 95 %
(Montell et al., 1983). Therefore, in the case of plasmid
pHXCM, over 70 % of the transcripts transcribed from
a region near the initiation site of the X mRNA may stop
at the HSV TK transcription termination signal.
As shown in Fig. 4, the CAT activities derived from
cells cotransfected with pHX, pHXC or pHXCM were
about sevenfold higher than those from cells cotrans-
921
fected with the control vector pUC19. CAT activity in
pHXCM-transfected cells was slightly higher than in
pHXC-transfected cells. Therefore, it is evident that the
X-C fusion protein expressed from the fused X C ORF
using the X promoter is active in trans-activation. These
results suggest that the mutant HBV contains a fused
X-C ORF. As the fused X-C ORF contains the entire X
and pre-C/C genes, the X-C fusion protein might have
the functions of both the X and pre-C proteins. In
HepG2-K8 cells, six HBV-specific transcripts of 4.0, 3-5,
2.2, 2.1, 1.2 and 0"9 kb were detected (Kim et at., 1992a)
and the 4-0 kb species may encode the X-C fusion
protein during the life cycle of the mutant HBV, The
cDNA cloning and sequence analysis of X mRNA
showed that an elongated X ORF encoding 193 amino
acids (21K) was created by a frameshift mutation at a 3'
region of the wild-type X ORF and the formation of an
in-frame termination codon (TAA) resulted from polyadenylation (Kim et at., 1992). This elongated X gene
product exerted transcriptional trans-activation (Kim
et al., 1992a). These results suggest that an X-C fusion
protein and/or an elongated X protein can be expressed
by the mutant HBV and may be involved in the
regulation of virus and/or host gene expression. However, it remains unknown whether the X-C fusion
protein is expressed during the life cycle and has a
biological function. Experiments are in progress to
investigate whether HBV with a defective pre-C region is
infectious and expresses the X-C fusion protein during
its replication.
This work was supported in part by research grants from the Korean
Ministry of Education and from KOSEF through the Research Center
for Cell Differentiation (93-4-1).
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Short communication
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(Received 19 July 1993; Accepted 26 October 1993)
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