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Journal of Virology, November 1998, p. 8789-8796, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
The 3'-Untranslated Region of Hepatitis C Virus
RNA Enhances Translation from an Internal Ribosomal Entry
Site
Takayoshi
Ito,1
Stanley M.
Tahara,2 and
Michael M. C.
Lai1,2,*
Howard Hughes Medical
Institute1 and
Department of Molecular
Microbiology and Immunology,2 University of
Southern California School of Medicine, Los Angeles, California
90033-1054
Received 26 May 1998/Accepted 12 August 1998
 |
ABSTRACT |
Translation of most eukaryotic mRNAs and many viral RNAs is
enhanced by their poly(A) tails. Hepatitis C virus (HCV) contains a
positive-stranded RNA genome which does not have a poly(A) tail but has
a stretch of 98 nucleotides (X region) at the 3'-untranslated region
(UTR), which assumes a highly conserved stem-loop structure. This X
region binds a polypyrimidine tract-binding protein (PTB), which also
binds to the internal ribosome entry site (IRES) in HCV 5'-UTR. These
RNA-protein interactions may regulate its translation. We generated a
set of HCV RNAs differing only in their 3'-UTRs and compared their
translation efficiencies. HCV RNA containing the X region was
translated three- to fivefold more than the corresponding RNAs without
this region. Mutations that abolished PTB binding in the X region
reduced, but did not completely abolish, enhancement in translation.
The X region also enhanced translation from another unrelated IRES
(from encephalomyocarditis virus RNA), but did not affect the
5'-end-dependent translation of globin mRNA in either monocistronic or
bicistronic RNAs. It did not appear to affect RNA stability. The free X
region added in trans, however, did not enhance
translation, indicating that the translational enhancement by the X
region occurs only in cis. These results demonstrate that
the highly conserved 3' end of HCV RNA provides a novel mechanism for
enhancement of HCV translation and may offer a target for antiviral
agents.
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INTRODUCTION |
All eukaryotic cellular mRNAs and
many viral RNAs have a cap structure at the 5' end and a poly(A) tail
at the 3' end, both of which, individually or in concert, play
essential roles in the regulation of translation. Each of these
elements works by associating with a specific RNA-binding protein, and
together they function synergistically to stimulate translation (see
reference 29 for a review). In the yeast system, a
translation initiation factor, eIF4G, which is a subunit of eIF4F,
associates with the poly(A)-binding protein (Pab1p) (34).
This interaction mediates the ability of the poly(A) tail to stimulate
translation in vitro (35). These data support the model that
the 5' and 3' ends of mRNA interact via eIF-4G and Pab1p. However, some
viral RNAs lack either the cap structure, poly(A) tail, or both; the
mechanism of translational regulation in these RNAs is still unclear.
Hepatitis C virus (HCV) is now recognized as the principal agent of
parenterally transmitted non-A, non-B hepatitis. The viral genome has
been cloned and sequenced (9) and shown to be a 9.5-kb
single-stranded, positive-sense RNA encoding a large polyprotein with a
size of about 3,008 to 3,037 amino acids (see reference 10 for a review). Although the overall sequence of
HCV RNA shows significant diversity within the coding region among
various isolates (see reference 8 for a review), the
5'-untranslated region (5'-UTR) and the 3'-end 98 nucleotides (nt)
(termed X region) are highly conserved (7, 22, 33).
Conceivably, both the 5'- and 3'-end sequences are important for viral
RNA replication and/or translation. The 5'-UTR of HCV RNA has been
shown to form extensive secondary structures (6, 37). In
this region, the majority of HCV genotypes possess five AUG codons,
which are not used for initiation of translation. Tsukiyama-Kohara et
al. demonstrated that the HCV 5'-UTR could regulate translation
initiation in an internal ribosome entry site (IRES)-dependent manner
(37) as in the picornaviruses (19). Furthermore,
the HCV 3'-UTR does not have a poly(A) tail but has a variable
poly(U-C) stretch plus a conserved X region at the 3' end (22,
33), which forms a three-stem-loop structure and binds a
polypyrimidine tract-binding protein (PTB) (4, 17, 36). PTB
binding requires strict primary sequence in the loops and stem
structures (17). Interestingly, PTB has also been reported
to bind to the IRES regions of several RNA viruses, including HCV, and
regulate their translation (1, 2).
The experiments in this study are designed to examine whether the X
region of HCV 3'-UTR plays a role in the IRES-dependent translation of
HCV RNA. By using an in vitro translation system in rabbit reticulocyte
lysate and transfection in mammalian cells, we showed that the HCV X
region specifically enhanced the IRES-dependent translation from the 5'
end of HCV RNA. This effect does not depend on the primary sequence of
the 5' end of HCV RNA, since translation from another IRES of an
unrelated virus (encephalomyocarditis virus [EMCV]) was also
enhanced, although at a lower efficiency. Furthermore, we showed that
this enhancement effect was not due to stabilization of RNA by the X
region. This effect was seen only in cis, and PTB binding to
the X region may be partially responsible, suggesting an interaction
between the IRES sequence and the X region, possibly mediated by PTB
and other proteins. In contrast, the X region did not stimulate the
5'-end-dependent translation from other RNAs.
Our results indicate that the X region of HCV RNA, probably together
with its RNA-binding proteins, can stimulate the IRES-dependent translation from HCV RNA, thus providing a new mechanism for regulating HCV translation. The 5'- and 3'-end interaction in HCV RNA translation may provide a potential target for antiviral therapy.
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MATERIALS AND METHODS |
Plasmid constructions.
Plasmid pHCV-5CL, which contains a T7
promoter, the 5'-UTR, and the entire core protein-encoding region of
HCV-1b strain (1 to 914 nt) and the luciferase gene fused in frame, was
made from pT-gHCV (17) and pGL-basic vector (Promega). The
cDNA fragment containing the T7 promoter and the 5'-UTR and core region
of HCV were made by PCR with pT-gHCV as a template and appropriate
primers containing the restriction enzyme sites. After digestion with the appropriate restriction enzymes, this PCR product was cloned into
the pGL-basic vector. The cDNA representing the 3'-end 98 nt (X region)
of the HCV RNA and an XbaI site at the 5' end and a
BamHI site at the 3' end was made by PCR from a plasmid,
HCV-X(+) (17), and subcloned into the pCR 2.1 vector
(Invitrogen) by the TA cloning method (41). The resulting
plasmid, pCR-X(+)-XBI, was confirmed by sequencing (30). To
generate pHCV-5CL-X, pCR-X(+)-XBI was digested by XbaI and
subcloned to pHCV-5CL. The site-directed mutants pHCV-5CL-Xa and
pHCV-5CL-Xg were generated by subcloning the PCR fragments containing
the corresponding mutations in the plasmid HCV-X(+), as previously
described (17).
Plasmid pGL-EMCV, which contains the IRES of EMCV and the luciferase
gene under the T7 promoter, was made from pTF7.25EMC-1 (13)
and pGL-basic vector by the strategy used for pHCV-5CL. To construct
plasmid pGL-
-globin, which contains the 5'-UTR and the coding region
of -globin gene fused in frame to the luciferase (LUC) gene under
the T7 promoter, the globin gene was amplified from phG
(32) by PCR and subcloned into pGL-basic vector. pGL-EMCV-X and pGL-globin-X were generated by insertion of the X region into
pGL-EMCV and plasmid pGL-globin, respectively.
To construct the plasmid for bicistronic RNA, pCAT-5CL, the
chloramphenicol acetyltransferase (CAT) gene in the pOPI3CAT vector
(Stratagene) was digested by
NotI and subcloned into the
NotI
site, which had been artificially introduced between
the T7 promoter
and the 5'-UTR of HCV in pGL-5CL. To generate
pCAT-5CL-X, the
X gene was inserted at the
XbaI site of the
pCAT-5CL; the resulting
pCAT-5CL-X and pCAT-5CL plasmids transcribe
bicistronic RNAs containing
a CAT gene preceded by the vector sequence
(6 nt) and a LUC gene
preceded by the 5'-UTR and the core-encoding
sequence of HCV genome.
Thermodynamic calculation of RNA secondary structure.
The
optimal and suboptimal secondary structures for the wild-type and
mutant X region of HCV RNA molecules were predicted by the method of
Zuker (42) with the MulFold program in the Genetics Computer
Group sequence analysis software package.
In vitro RNA transcription and translation.
Plasmids were
linearized by digestion with various enzymes, including XbaI
(for pHCV-5CL, pGL-EMCV, pGL-globin-X, and pCAT-5CL), HpaI (for pHCV-5CL, pGL-EMCV, and pCAT-5CL),
BamHI (for pHCV-5CL-X, pCAT-5CL-X, and related mutants), or
EcoRV (for pGL-EMCV-X and pGL-globin-X). RNA was
transcribed by T7 RNA polymerase according to the protocol supplied by
the manufacturer (Promega). After transcription, 1 U of RQ DNase I
(Promega) was added to the reaction mixture to digest DNA templates,
extracted with phenol-chloroform, and precipitated with ethanol-7.5 M
ammonium acetate. The concentration of RNA was determined by
spectrophotometry. To generate capped RNAs, the mMESSAGE kit (Ambion)
was used for in vitro transcription reactions.
In vitro translation was carried out in micrococcal nuclease-treated
rabbit reticulocyte lysates (Flexi; Promega). Translation
reactions (25 µl) were programmed with 2 µg of RNA, 10 µl of lysates,
0.5 U of
RNase inhibitor (RNasin; Promega), 2 mM dithiothreitol,
20 µM amino
acid mixture minus methionine, and various concentrations
of KCl in the
presence of 1 µl of [
35S]methionine (10 mCi/ml; NEN)
and carried out at 30°C for 90 min.
For RNAs containing an IRES
sequence (e.g., HCV-5CL and [EMCV])
and for
-globin RNAs,
translation was carried out in the presence
of 120 and 70 mM KCl,
respectively. At the end of the reactions,
stop buffer (50 µg of
RNase A per ml, 10 mM EDTA [pH 7.5]) was
added to the reaction
mixture. Aliquots of the translation products
were separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) on 7.5 to
~10% polyacrylamide gels.
Primer extension.
Two micrograms of each of the various RNAs
was incubated with rabbit reticulocyte lysates in the presence of 120 mM KCl under the conditions for in vitro translation. RNA was extracted
from the lysates 30 and 90 min after the reaction by using TRIZOL
(GIBCO BRL), and one-half of the total RNA was analyzed by primer
extension with a 32P-end-labeled primer
(5'-AACACTACTCGGCTAGCAGT-3') complementary to the 5'-UTR of
HCV RNA as previously described (17). HCV-5CL-X RNA was used
to determine the linear range in which primer extension was performed.
Cell culture and transfection.
Huh7 cells (25), a
human hepatoma cell line, were seeded onto 35-mm-diameter tissue
culture dishes 48 h prior to transfection. Cells (80% confluent)
were infected with recombinant vaccinia virus vTF7-3 (expressing T7 RNA
polymerase) (14) in 100 µl of Dulbecco's modified
Eagle's medium (DMEM) at a multiplicity of infection (MOI) of 10. After incubation at 37°C for 1 h, the virus inoculum was removed
and replaced with a mixture containing 2.5 µg of linearized plasmid
DNA and 15 µl of DOTAP (Boehringer Mannheim) in 75 µl of 20 mM
HEPES buffer (pH 8.0), followed by the addition of 2 ml of DMEM
supplemented with 10% fetal bovine serum. After a 24-h incubation at
37°C, the cells were washed with phosphate-buffered saline twice and
harvested with 200 µl of the cell lysis buffer (Promega) and prepared
for CAT (31) and LUC (12) assays.
LUC and CAT assays.
Cellular lysates of transfected Huh7
cells (from approximately 3 × 105 cells) were
centrifuged at 10,000 × g for 5 min. Ten microliters of the supernatant was mixed with 100 µl of luciferase assay reagent (Promega), and the LUC activity was measured after 20 s by
Luminometer (Berthold). For the LUC assay of the in vitro translation
product, 5 µl of reaction mixture was used.
For the CAT assay, cellular lysates were incubated at 60°C for 10 min, and 100 µl of cell extract was mixed with 15 µl of
0.25 M
Tris-HCl (pH 7.4), 3 µl of [
14C]chloramphenicol (0.025 mCi/ml; Dupont NEN), and 5 µl of
n-butyryl
coenzyme A (5 µg/µl; Sigma). After incubation for 8 h at 37°C,
the mixture
was extracted with 300 µl of xylenes (Mallinckrodt
Chemical Works).
The phase of xylenes was back-extracted twice
with 100 µl of 0.25 M
Tris-HCl (pH 8.0), and 200 µl of upper xylenes
was measured for the
14C count in a scintillation counter (Beckman Instruments
model
LS6000IC).
Statistical analysis.
Data from the repeated experiments
were averaged and expressed as means ± standard deviations. The
effects of the X region on translation were analyzed by Student's
t test for paired samples. P < 0.05 was
taken as the level of statistical significance.
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RESULTS |
The X region of HCV RNA enhances its translation in monocistronic
RNAs.
In order to investigate the biological function of the
3'-end 98-nt sequence (X region) on the translation of HCV RNA, we compared the translational efficiency of several synthetic RNAs which
contain the authentic 5'-end sequence of HCV RNA, but different sequences at their 3' ends. Because the 5' end of the core
protein-encoding sequence is part of the IRES structure of HCV RNA
(27, 28) and because the 3' end of the core protein-encoding
region has a strong PTB-binding site (18), we included both
the 5'-UTR and the entire core protein-encoding region (1 to 914 nt
from the 5' end of the viral RNA) as part of the 5'-end sequence in all
of these constructs. These constructs represent the authentic 5'-end
structure of HCV RNA at the translation initiation site. The core
protein sequence was fused in frame with the LUC gene. Thus, the
core-LUC fusion protein is the primary translation product of these
constructs. HCV-5CL-X mimics the native HCV RNA in structure, consisting of the entire 5'-UTR at the 5' end, the X region at the 3'
end, and the coding sequence for the core-LUC fusion protein in the
middle (Fig. 1A). HCV-5CL-Vec has 160 nt
of vector sequence at the 3' end, whereas HCV-5CL-Xa and -Xg contain
mutations in the X region which affect PTB binding (17). The
secondary structure of the HCV-5CL-Xa RNA is predicted to be the same
as that of the wild type, but the stem-loop 2 structure of the X region
is predicted to be altered in the HCV-5CL-Xg RNA (Fig. 1B). Neither of
these two mutant RNAs binds PTB (17). In vitro translation
of these RNAs was carried out in rabbit reticulocyte lysates in the
presence of 120 mM potassium chloride, which is the physiological salt concentration and allows HCV RNA translation in an IRES-dependent manner (3, 5). The translation products were examined by SDS-PAGE autoradiographs of the core-LUC fusion protein (Fig. 1C) and
the LUC activity of the fusion protein (Fig. 1D). The results showed
that the translation level of HCV-5CL-X was three- to fivefold higher
than that of the control RNAs (HCV-5CL and HCV-5CL-Vec), while the
mutations in the X region that affected PTB binding reduced the level
of, but did not completely abolish, the translational enhancement.
These results indicate that the 3' end 98 nt of HCV RNA serves as a
translational enhancer for its own RNA, and this enhancement may
involve, at least partially, the sequence or structure of the
PTB-binding site in this region.
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FIG. 1.
Functional analysis of the HCV X region by in vitro
translation. (A) Schematic diagrams of pHCV-5CL and its related
plasmids used in this study. pHCV-5CL contains T7 promoter (large open
arrow), the 5'-UTR (single line), and core-encoding region (open box)
of the HCV 1b strain fused to LUC genes (closed box) in the pGL vector
(Promega). pHCV-5CL-X, -Xa, and -Xg contain, in addition, the X region
and its mutants, respectively, at the 3' end. The plasmids were
linearized with the appropriate restriction enzymes and transcribed
with T7 RNA polymerase to generate transcripts. (B) Computer-predicted
secondary structures of the X region and its mutants in HCV-5CL-X, -Xa,
and -Xg RNAs (17). SL2, stem-loop 2. (C) In vitro
translation products of various RNAs separated by SDS-PAGE on 7.5%
polyacrylamide gels. In vitro translation was carried out in rabbit
reticulocyte lysates at 120 mM KCl. An arrow indicates the core-LUC
fusion protein. Computer imaging was generated by Adobe Photoshop,
version 3.0. (D) Relative LUC activity of the translation products of
various RNAs. The LUC activity of HCV-5CL RNA is artificially set at
100%. The columns and bars represent the means and standard deviations
of two sets of triplicate studies. The asterisks indicate that the
translational enhancement of these RNAs compared to the translational
level of HCV-5CL RNA is significant. *, P < 0.05;
**, P < 0.01.
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To investigate whether the translational enhancement by the X region
was due to possible stabilization of mRNAs, we monitored
the stability
of these RNAs in rabbit reticulocyte lysates during
in vitro
translation, by primer extension study with a primer
complementary to
the 5'-UTR sequence. Primer extension was performed
under the condition
in which the primer-extended products reflected
the amounts of RNA
(HCV-5CL-X RNA) in a linear relationship within
the range of RNA
amounts used in this study (Fig.
2A). The
results
showed that the amounts of all three RNAs decreased at
approximately
the same rate (Fig.
2B), indicating that these three RNAs
had
comparable stabilities and that the translation enhancement effect
by the X region was not due to RNA stabilization by this region.
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FIG. 2.
Primer extension study of the HCV RNA constructs. (A)
Calibration of the primer extension reactions. Decreasing amounts of
HCV-5CL-X RNA were used in the primer extension reactions with a 5'-UTR
primer, yielding a 265-nt product (arrow). (B) RNA stability of HCV-5CL
(lanes 1 to 3), 5CL-Vec (lanes 4 to 6), and 5CL-X (lanes 7 to 9) RNA in
rabbit reticulocyte lysates. Two micrograms of each RNA was used in in
vitro translation in rabbit reticulocyte lysates. Reactions were
stopped at 0 min (lanes 1, 4, and 7), 30 min (lanes 2, 5, and 8), and
90 min (lanes 3, 6, and 9). RNAs were extracted, and half of the
amounts from each time points were used in primer extension experiments
as in panel A.
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To examine whether this translation enhancement effect of the X region
was also seen in intact cells, the plasmid DNAs (pHCV-5CL
and
pHCV-5CL-X) which had been linearized by the appropriate restriction
enzymes were transfected into Huh7 cells infected with the recombinant
vaccinia virus expressing T7 RNA polymerase (vTF7-3) (
14).
The
translation efficiency of the transcribed RNA containing the X
region (HCV-5CL-X) was found to be two- to threefold higher than
those
of HCV-5CL and 5CL-Vec (Fig.
3). Thus,
the X region can
enhance translation in both rabbit reticulocyte
lysates and intact
cells.
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FIG. 3.
Effects of the X region on HCV translation in vivo.
Linearized plasmids were transfected into Huh7 cells infected with a
recombinant vaccinia virus expressing T7 RNA polymerase. Relative LUC
activities in the lysates were determined 24 h after transfection.
The columns and bars represent the means and standard deviations of
three independent transfections. *, P < 0.05 compared with HCV-5CL.
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The X region of HCV RNA enhances translation of another
IRES-containing RNA but not non-IRES-dependent translation.
To
analyze the mechanism of translational enhancement by the X region, we
next asked whether the IRES derived from unrelated RNAs such as EMCV
RNA (20) can be stimulated by the HCV X sequence (Fig.
4A). The EMCV-X construct is a chimeric
RNA which consists of the entire 5'-UTR of EMCV RNA, LUC coding
sequence, and the X region of HCV. An in vitro translation study under
the same condition (120 mM KCl) described above showed that the
presence of the X region at the 3' end also enhanced the translation of RNAs by approximately twofold, compared with that of the corresponding RNAs containing vector sequences at the 3' end or no 3'-UTR at all
(Fig. 4B and C). These results indicate that the translational enhancement by the HCV X region is not restricted to the HCV 5'-UTR sequence.
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FIG. 4.
Effects of the X region on translation from an EMCV
IRES. (A) Schematic diagrams of the plasmids used. pGL-EMCV contains
the T7 promoter (large open arrow), the 5'-UTR of EMCV (single line),
and LUC genes (closed box) in the pGL vector. pGL-EMCV-X contains, in
addition, the X region of HCV at the 3' end. The plasmids were
linearized with the appropriate restriction enzymes and transcribed
with T7 RNA polymerase to generate transcripts. (B) In vitro
translation products of the various RNAs were separated by SDS-PAGE on
7.5% polyacrylamide gels. In vitro translation was performed in rabbit
reticulocyte lysates at 120 mM KCl. An arrow indicates the LUC protein.
Computer imaging was generated by Adobe Photoshop, version 3.0. (C)
Relative levels of LUC expression of the various RNAs. The activity of
the EMCV transcripts is set at 100%. The columns and bars represent
the means and standard deviations of two sets of triplicate studies.
**, P < 0.01 compared with EMCV RNA.
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We next asked whether the HCV X region can also enhance the translation
of RNAs without IRES. We constructed RNAs containing
the natural 5'-UTR
of
-globin mRNA, which does not contain IRES
and is translated by a
5'-end-dependent mechanism, and the entire
globin-coding sequence fused
to LUC reporter gene followed by
a different 3'-UTR (Fig.
5A). The
-globin-X RNA construct
contains
the X region of HCV at the 3'-end, whereas the
-globin
construct
does not have a 3'-UTR. Both uncapped and capped RNAs were
translated
at 70 mM KCl in rabbit reticulocyte lysates, which allowed
more
efficient translation of RNAs without an IRES (
3,
5)
(described
below). Figure
5B and C showed that these RNAs, both capped
and
uncapped, were translated to approximately the same extent. The
presence of the HCV X region slightly increased translation efficiency
over that of the RNA without a 3'-UTR, but this difference was
not
statistically significant. These results combined indicate
that the X
region of HCV can enhance only the IRES-dependent translation
but not
the 5'-end-dependent translation, including the cap-dependent
translation.
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FIG. 5.
Effects of the X region on -globin translation from
the 5'-UTR of the -globin gene. (A) Schematic diagrams of the
plasmids used. pGL-globin-X contains T7 promoter (large open arrow),
the 5'-UTR (single line) and coding region (open box) of -globin
gene fused to LUC genes (closed box), and the X region of HCV in the
pGL vector. The plasmids were linearized with the appropriate
restriction enzymes and transcribed with T7 RNA polymerase to generate
uncapped and capped RNAs. (B) In vitro translation products of uncapped
(left) and capped (right) RNAs separated by SDS-PAGE on 7.5%
polyacrylamide gels. Translation was performed in rabbit reticulocyte
lysates at 70 mM KCl. An arrow indicates the -globin-LUC fusion
protein. Computer imaging was generated by Adobe Photoshop, version
3.0. (C) Relative LUC expression of uncapped (left) and capped (right)
RNAs. -Globin RNA is set at 100%. The columns and bars represent
the means and standard deviations of two sets of triplicate studies.
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Specific enhancement of the IRES-dependent translation by the X
region in bicistronic RNAs.
The results presented above showed
that the HCV X region can enhance the IRES-dependent translation, but
not the 5'-end-dependent translation. To rule out the trivial
explanation that these two types of RNA have completely different RNA
structures, we combined these two types of translation in bicistronic
RNA constructs. These bicistronic RNAs contain a minimum vector
sequence (8 nt) preceding the CAT reporter, followed by the HCV 5'-UTR
sequence preceding the HCV core-LUC fusion gene (Fig.
6A). The CAT gene is translated in the
5'-end-dependent manner, while the core-LUC fused gene is translated in
the IRES-dependent manner. Figure 6B and C showed that when translation
was carried out under the physiological salt concentration (120 mM
KCl), the translation of core-LUC from CAT-5CL-X, which was translated
from an IRES, was three- to fivefold higher than that from RNAs without
the X region (CAT-5CL and CAT-5CL-Vec), similar to the translational enhancement observed with the monocistronic RNAs (Fig. 1). In this
experiment, there was a marginally significant enhancement of
translation by the presence of the vector sequence at the 3' end of RNA
(CAT-5CL-Vec). Determination of the significance of this observation
was not further pursued. Under the same conditions, the CAT translation
was barely detectable; nevertheless, it can be seen that the presence
of the 3' X sequence in CAT-5CL-X RNA did not enhance the CAT
translation at all.
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FIG. 6.
Effects of the X region on translation from bicistronic
RNAs. (A) Schematic diagrams of plasmids used. pCAT-5CL contains the T7
promoter (large open arrow), the CAT gene (hatched box), the 5'-UTR
(single line), and the core protein-encoding region (open box) of HCV
fused to a LUC gene (closed box) in the pGL vector. pCAT-5CL-X
contains, in addition, the X region at the 3' end. The plasmids were
linearized with the appropriate restriction enzymes and transcribed
with T7 RNA polymerase to generate transcripts. (B) In vitro
translation products of RNAs with 50 mM KCl (left) or 120 mM KCl
(right) after separation by SDS-PAGE on 10% polyacrylamide gels. The
core-LUC fusion protein (upper arrow) and CAT (lower arrow) are
indicated. (C) Relative LUC expression of RNAs with 50 mM KCl (left) or
120 mM KCl (right). The columns and bars represent the means and
standard deviations of two sets of triplicate studies. *,
P < 0.05; **, P < 0.01 (compared
with CAT-5CL RNA).
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To evaluate more precisely the effects of the X gene in these
bicistronic RNAs, we also performed translation under the low-salt
condition (50 mM KCl). This condition allowed the efficient translation
of the CAT gene, which represents the 5'-end-dependent translation,
from the bicistronic RNA, similar to the monocistronic RNA (Fig.
4).
The results showed that the translation level of the CAT gene
from
CAT-5CL-X RNA was not significantly different from those
from the other
two RNAs (Fig.
6B and C), indicating that the X
region did not enhance
the 5'-end-dependent translation. The core-LUC
was translated very
inefficiently under this condition; nevertheless,
there was still a
marginal but statistically significant enhancement
of translation by
the X region. Thus, the results obtained with
the bicistronic RNAs are
consistent with those from the monocistronic
RNAs and suggest that
IRES-dependent, but not 5'-end-dependent,
translation can be enhanced
by the X region. These results further
suggest that the enhancement of
the IRES-dependent translation
by the X region was not because of the
stabilization of the RNA
by the X region, since the 5'-end-dependent
translation from the
same RNA was not enhanced by the presence of the X
region.
To determine whether the translation-enhancement effects of the X
region were also observed in intact cells, these bicistronic
RNAs were
transfected into Huh7 cells infected with the recombinant
vaccinia
virus expressing T7 RNA polymerase. The results show
that the LUC/CAT
ratio from CAT-5CL-X RNA was significantly higher
than that from the
other RNAs by approximately twofold, confirming
the results of in vitro
translation, although the level of enhancement
was lower in intact
cells (Fig.
7). These results indicate
that
the X region of HCV 3'-UTR can specifically enhance the
IRES-dependent
translation in vitro and in vivo.
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FIG. 7.
Effects of the X region on translation in vivo from
bicistronic RNA constructs. Linearized DNAs were transfected into Huh7
cells infected with a recombinant vaccinia virus expressing T7 RNA
polymerase. Luciferase and CAT activities were determined at 24 h
posttransfection. The relative LUC and CAT activities of the various
RNAs and their LUC/CAT ratios are shown. The columns and bars represent
the means and standard deviations of three independent transfections.
*, P < 0.05 compared with CAT-5CL RNA.
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The X region added in trans does not have effects on
translation.
To investigate the possible mechanism of the
translational regulation by the HCV X region, we asked whether the X
region has any effects on translation when added in trans.
Various amounts of X region RNA transcript were added to in vitro
translation reaction mixtures containing CAT-5CL RNA, which did not
have a 3'-UTR, and the LUC activity was determined. Figure
8A showed that the free X region added in
trans did not significantly enhance the translation of RNA
without the 3'-UTR, suggesting that the HCV X region can enhance the
IRES-dependent translation only as a cis-acting element. We
then studied whether the X region added in trans could
inhibit the translational enhancement effect of the X region in the
CAT-5CL-X. If the enhancement effect of the X region is mediated by a
trans-acting factor, then the free X region added in
trans is expected to deplete this factor and inhibit CAT-5CL-X translation. The results showed that the X region added in
trans did not inhibit the LUC activity of CAT-5CL-X RNA
(Fig. 8B). These results combined indicate that the translation
enhancement effect of the HCV X region works only in cis and
may involve not only translation factors but an RNA element as well.
View larger version (13K):
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|
FIG. 8.
The trans effects of the X region on
translation. A 1- to 10-fold excess of HCV X(+) RNA (17) was
added to rabbit reticulocyte lysate containing CAT-5CL RNA (A) or
CAT-5CL-X RNA (B). In vitro translation was carried out at 120 mM KCl.
Translation without free HCV-X(+) RNA [( )] is set at 100%. The
columns and bars represent the means and standard deviations of three
independent translation reactions.
|
|
| |
DISCUSSION |
In this study, we have demonstrated that the X region in HCV
3'-UTR enhanced HCV RNA translation in rabbit reticulocyte lysates and
in intact mammalian cells (Fig. 1). This enhancement was not due to RNA
stabilization by the X region (Fig. 2). Mutations in the X region which
affected PTB binding reduced, but did not completely abolish, the
translational enhancement, suggesting that the PTB-3'-UTR interaction
may be at least partially involved in the regulation of HCV
translation. Furthermore, this region also enhanced translation from
another IRES derived from EMCV RNA, but did not affect the 5'-end-dependent translation of -globin mRNA (Fig. 4 and 5). Bicistronic RNAs which contain an IRES between two reporter genes confirmed that the X region enhanced only IRES-dependent translation (Fig. 6). Thus, the X region in HCV 3'-UTR very likely regulates HCV
RNA translation in natural infection.
Because of the absence of a poly(A) tail in HCV RNA, the mechanism of
regulation of HCV RNA translation is predicted to be different from
those of most eukaryotic mRNAs and other viral RNAs. The X region in
HCV 3'-UTR binds PTB, which recognizes the primary sequences and stem
structures in this region (17, 36). PTB has also been
reported to interact with the IRESs of many RNA viruses, including HCV
and EMCV (1, 2), and it was thought to be a translation
factor (1, 2, 15, 16, 38-40), although this claim has been
disputed (21, 27). The PTB-3'-UTR complex may play a role
similar to that of the Pab1p-poly(A) tail complex in the translation of
eukaryotic mRNAs. Our data from in vitro translation in rabbit
reticulocyte lysates indeed showed that the X region enhanced
translation nearly fivefold, when compared with control RNAs without
the X region, at the physiological salt concentration (23)
(Fig. 1 and 6C). When translation was carried out at a nonphysiological
salt concentration (50 to 70 mM KCl), in which translation was
predominantly from the 5' end of RNA, the enhancement effect of the X
region on translation from the HCV 5'-UTR was only marginal (Fig. 6B).
Thus, the effects of translational enhancement by the X region are most
significant when translation is strictly IRES dependent.
When RNAs were expressed in mammalian cells, the X region enhanced HCV
translation two- to threefold (Fig. 3), but not the three- to fivefold
stimulation as seen in translation in reticulocyte lysates, suggesting
either that the in vitro and in vivo conditions are different or that
the HCV RNA may undergo 5'-end-dependent translation as well as
IRES-dependent translation in vivo. Regardless, our data clearly showed
that the X region enhanced HCV RNA translation both in vitro and in
vivo. This enhancement may be mediated, at least partially, by PTB
interacting with the X region. However, mutations in the PTB-binding
site did not completely abolish the translational enhancement by the X
region. This may have been due to the binding of a residual amount of
PTB (17). Alternatively, other translation factors or
primary sequence or secondary structure of X region RNA may also be
involved in this enhancement.
In vitro translation studies with another IRES-containing RNA from an
unrelated virus (EMCV) and non-IRES RNAs (-globin) showed that the X
region in HCV 3'-UTR enhances the IRES-dependent translation only (Fig.
4 and 5). These results were confirmed by the experiments with
bicistronic RNAs (Fig. 6), which also indicated that this enhancement
was not because of the stabilization of RNA by the X sequence. Direct
measurement of RNA stability in rabbit reticulocyte lysates also
indicated that the X region does not protect the 5' end of HCV RNA from
degradation (Fig. 2). Thus, the most likely mechanism of the
translational enhancement is that the X region interacts with the
IRES-specific translational machinery. Recently, Pestova et al.
reported that the mechanism of initiation of HCV translation is
different from that of other IRES-containing RNAs, e.g., EMCV RNA, but
is similar to that of prokaryotic RNAs (26). This may
explain why the level of enhancement by the X region on EMCV
translation (two- to threefold) is lower than that on HCV translation
(fivefold). The fact that PTB binds to the IRES of both HCV and EMCV
RNA (1, 16) suggests that PTB may be involved in the
translational enhancement by the X region in both cases. The X region
in HCV 3'-UTR may interact with these IRESs through PTB. Our recent
data showed that the binding of PTB to the X region is at least 100 times stronger than that to the 5'-UTR of HCV (18). Thus,
the binding of PTB to the X region may be the most significant factor
involved in the translational enhancement. However, the role of PTB in
this 3'-UTR-enhanced translation has not yet been directly tested. Also, it is likely that other protein factors and/or RNA structures are
involved in this enhancement as well. It should be noted that most of
the RNAs used in this study included the entire core protein-coding region. Recently, we found that the inclusion of this stretch of
sequences in the RNAs is crucial for demonstrating the translational enhancement effects of the X region (18). Since the core
protein-coding region also binds PTB (18), this observation
further suggests the importance of the PTB-X region interactions in
translational enhancement. Also, this observation suggests that the
translational enhancement observed in this study likely reflects the
regulation of translation in the virus-infected cells, because natural
HCV infection has to utilize full-length viral RNA, including the core
protein-coding region, for translation.
This study suggests that the functions of the X region may be similar
to that of poly(A) in translation. However, poly(A) added in
trans can inhibit translation from capped polyadenylated mRNAs and stimulate translation from capped RNA without a poly(A) tail
(24). In contrast, the X region does not affect the
translation of HCV RNA with or without the X region when it is added in
trans (Fig. 8), suggesting
that the mechanism of translational enhancement by the X region is
different from that of the poly(A) tail. The function of poly(A) is
thought to be mediated through Pab1p (11, 34). In yeast, a
poly(A) tail enhances the mRNA translation by interacting with eIF4G
through Pab1p (34), suggesting that mRNA can be circularized
by the interaction between Pab1p and eIF4G. Recently Craig et al. also
reported that interaction of poly(A)-binding protein with PAIP, which
is an eIF4G homolog, enhances translation in human cells
(11). This PAIP binds eIF4A to enable the 3' end of mRNA to
communicate with the 5' end. Whether the function of the X region is
mediated through PTB by a mechanism similar to that of poly(A)-Pab1p
requires more stringent tests. Nevertheless, the failure of the X
region to act in trans may be due to the possibility that
PTB is in excess in rabbit reticulocyte lysates, so that PTB could not
be completely depleted by the exogenous X RNA, whereas the
poly(A)-binding protein is in limited supply. In any case, the
enhancement of translation of HCV RNA by its 3' end may provide a novel
target for anti-HCV agents.
| |
ACKNOWLEDGMENTS |
We thank Daphne Shimoda for assistance in preparing the
manuscript.
This work was partially supported by research grant AI 40038 from the
National Institutes of Health. T.I. is a Research Associate and
M.M.C.L. is an Investigator of the Howard Hughes Medical Institute.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Microbiology and Immunology, University of Southern
California School of Medicine, 2011 Zonal Ave., HMR-503, Los Angeles,
CA 90033-1054. Phone: (323) 442-1748. Fax: (323) 342-9555. E-mail: michlai{at}hsc.usc.edu.
| |
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Abstract
Introduction
