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Journal of Virology, November 2000, p. 10827-10833, Vol. 74, No. 22
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Comparative Analysis of Translation Efficiencies of Hepatitis C
Virus 5' Untranslated Regions among Intraindividual Quasispecies
Present in Chronic Infection: Opposite Behaviors Depending on
Cell Type
Julien
Laporte,1
Isabelle
Malet,1
Thibault
Andrieu,1
Vincent
Thibault,1
Jean-Jacques
Toulme,2
Czeslaw
Wychowski,3
Jean-Michel
Pawlotsky,4
Jean-Marie
Huraux,1
Henri
Agut,1 and
Annie
Cahour1,*
Laboratoire de virologie, C.E.R.V.I., UPRES
EA 2387, Hôpital Pitié-Salpêtrière, 75651 Paris
Cedex 13,1 INSERM U386, IFR Pathologies
Infectieuses, Université Victor Segalen,
Bordeaux,2 CNRS-UMR 8526,
IBL/Institut Pasteur de Lille, 59021 Lille
Cedex,3 and Service de
Bactériologie-Virologie, Hôpital Henri Mondor,
Université Paris XII, Créteil,4
France
Received 17 May 2000/Accepted 22 August 2000
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ABSTRACT |
Hepatitis C virus (HCV) RNA translation initiation is dependent on
the presence of an internal ribosome entry site (IRES) that is found
mostly in its 5' untranslated region (5' UTR). While exhibiting the
most highly conserved sequence within the genome, the 5' UTR
accumulates small differences, which may be of biological and clinical
importance. In this study, using a bicistronic dual luciferase
expression system, we have examined the sequence of 5' UTRs from
quasispecies characterized in the serum of a patient chronically
infected with HCV genotype 1a and its corresponding translational
activity. Sequence heterogeneity between IRES elements led to important
changes in their translation efficiency both in vitro and in different
cell cultures lines, implying that interactions of RNA with related
transacting factors may vary according to cell type. These data suggest
that variants occasionally carried by the serum prior to reinfection
could be selected toward different compartments of the same infected
organism, thus favoring the hypothesis of HCV multiple tropism.
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TEXT |
The hepatitis C virus (HCV) 5'
untranslated region (5' UTR), is 341 bases long and is the most highly
conserved region of the virus genome among various genotypes
(26), suggesting that it plays a key role in the viral cycle
and may be a potent target for antiviral agents. It is now clear that
initiation of translation of HCV RNA occurs by a cap-independent
mechanism mediated by an internal ribosome entry site (IRES) (29,
30) that comprises most of the 5' UTR and extends at least to the
first 12 to 30 nucleotides (nt) of the coding sequence (14,
19). Most of the studies based on the model of the secondary
structure of HCV 5' UTR that was first proposed by Honda et al.
(8) and recently refined (7) have demonstrated
that the highly ordered structures within IRES elements are absolutely
required for IRES activity (reviewed in reference
20).
Data have accumulated from mutation-deletion analyses (3, 7, 9,
18, 27) and in vitro reconstitution of IRES-mediated initiation
complexes (17, 25) that were performed to gain insight into
the control of viral translation initiation. Reports on comparisons
between IRES efficiencies from different HCV genotypes are conflicting
(2, 4, 11, 22). Like many other RNA viruses, HCV has a very
high mutation rate and circulates as a population of closely related
genomes, referred to as quasispecies (5). At present, little
is known about 5' UTR diversity in a viral population and its dynamics
toward viral multiplication.
In this work, we studied authentic, biologically derived HCV 5' UTRs
isolated from human serum to assess whether sequence heterogeneity
between IRES elements can be linked to changes in their function. Our
data indicate that the HCV 5' UTR, even if it is the most highly
conserved part of the viral genome, has a quasispecies distribution
with minor modifications in its sequence. These modifications result in
dramatic changes of the IRES activity depending on the cell type used
for transfection.
Construction of the pIRF bicistronic vector.
HCV IRES activity
was monitored with the aid of a bicistronic reporter vector, pIRF,
under the control of a T7 promoter, composed of firefly luciferase
(FLuc) followed by the HCV 1a 5' UTR sequence and then by
Renilla luciferase (RLuc) (Fig.
1). In such a system, the upstream
(control) reporter FLuc is translated in a cap-dependent fashion
whereas the downstream (assay) reporter RLuc is under the control of
HCV IRES. The primers used for constructions are detailed in Table
1. We designed three artificial mutants
of the wild-type 5' UTR aimed at testing the accuracy of our
bicistronic system since they were expected to modify HCV IRES
activity. These constructs, displayed in Fig. 1, were named pIRF
20
(lacking the first 20 nt of the HCV sequence), pIRFC (lacking the 30 nt coding for the capsid), and pIRF+8 (containing the additive 8-nt
sequence recently found in an Australian isolate) (28).
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FIG. 1.
Structures of the pIRF bicistronic reporter plasmid and
its variant constructs. Hatched boxes indicate the two luciferase
genes: RLuc, Renilla luciferase; FLuc, firefly luciferase.
The reference HCV sequence is shown in black boxes, with the AUG
initiator codon represented as a small empty box; lines indicate
deletions, and the empty box upstream of nt +1 in the pIRF+8 construct
denotes the extra 8-nt sequence (28). Nucleotide positions
referring to HCV 1a (13) are shown below the constructs. T7,
T7 promoter sequence. The relative in vitro translation efficiency of
each bicistronic RNA variant, shown on the right, was expressed as the
ratio of RLuc to FLuc enzymatic activities, normalized to that of pIRF
reference, which is defined as 100%.
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We next examined in vitro IRES translational efficiency toward the RLuc
gene in different constructs in the rabbit reticulocyte
lysate (RRL)
system, using the TNT coupled reticulocyte lysate
system kit (Promega)
for transcription-translation with T7 RNA
polymerase. When analyzed by
sodium dodecyl sulfate-polyacrylamide
gel electrophoresis,
35S-labeled FLuc and HCV capsid-RLuc fusion proteins
migrated to
the expected sizes of 62 and 37 kDa, respectively (data not
shown).
The enzymatic activities of the luciferases were measured by
using
the dual luciferase kit assay (Promega). By means of this assay
procedure, HCV IRES activity can be analyzed in vitro and in vivo,
with
both reporter enzymes being assessed in the same preparation.
Another
important advantage of such a bicistronic construct is
that during the
transfection procedure the upstream translation
Fluc product appears as
an internal control, which bypasses the
possible differences in
transfection efficiency. IRES relative
translation efficiency was
calculated as the ratio of two luciferase
activities (RLuc/FLuc), and
the relative activities of mutated
constructs were compared to that of
the parental HCV 5' UTR, which
was arbitrarily taken as 100% (Fig.
1).
As expected, deletion
of the first 30 nt from the HCV ORF profoundly
impaired IRES activity
(by 87%). Although conflicting, the hypothesis
that IRES activity
requires the extreme 5' capsid coding sequence was
verified in
our system, in agreement with previous studies (
14,
16,
19).
Surprisingly, in contrast to some other reports
(reviewed in reference
20) except for one earlier
study (
6), the pIRF
20 construct
appeared less effective
than the parental construct (it had 57%
of the parental activity).
Such a discrepancy could be explained
by a possible intervening effect
of the 3' end of the Fluc coding
sequence upstream of the HCV 5' UTR.
Interestingly, the pIRF+8
construct had a slightly higher efficiency
than pIRF. Although
it has not been shown to be a prerequisite for the
full-length
HCV RNA to be functional in order to initiate infection in
the
chimpanzee (
13) and it is not considered part of the HCV
IRES,
the extra 8-nt sequence might be involved by means of structural
interaction with the 20-nt stem-loop I in HCV translation modulation.
We have observed identical stabilities of bicistronic RNA templates
in
RRL after a 30-min reaction by Northern blot analysis with
the IRES 3'
oligonucleotide as a probe, ruling out a significant
variability of
these RNAs that could account for the observed
differences in RLuc
expression (data not
shown).
Characterization of HCV 5' UTR quasispecies.
Heterogeneity
within HCV 5' UTR was investigated by using a pretreatment serum sample
from a 46 year-old man with chronic hepatitis C related to HCV 1a
infection. RNA was extracted from 140 µl of serum using the QIAamp
viral RNA kit (Qiagen). Then reverse transcription-PCR (RT-PCR) was
performed with the Access RT-PCR kit (Promega). Briefly, HCV RNA was
first reverse transcribed at 46°C for 60 min with antisense primer
Quasi 3', and cDNA fragments were further amplified by seminested PCR,
including the high-fidelity Arrow Taq DNA polymerase
(Stratagene), using primers Quasi 5' and Quasi 3' for the first round
and primers IRES 5' and IRES 3' for the second round. The PCR
amplifications involved 30 cycles of 94°C for 20 s, 50°C for 1 min, and 72°C for 1 min, followed by a final elongation step at
72°C for 7 min. PCR products were cloned in place of the parental 5'
UTR into the pIRF vector. A total of 43 clones were generated and
sequenced in both directions on an ABI 377 automated DNA sequencer
(Applied Biosystems, Foster City, Calif.) using the Dye Dideoxy
Terminator cycle-sequencing kit (Applied Biosystems). Despite the
well-known genetic stability of this region, 15 different variants were
found to coexist in the serum sample studied (Fig.
2). When comparing nucleotide sequences to that of pIRF (HCV 1a), we found two common differences. First, GA
residues at positions 34 and 35 (specific to genotype 1a sequence) were
observed whereas AG residues (specific to genotype 1b sequence) were
found in the pIRF reference. Second, a change of A (present at position
204 in the pIRF IRES sequence obtained from chimpanzee liver biopsy
specimens) (C. Daener, C. Wychowski, and S. Feinstone, unpublished
results) to C (detected in quasispecies isolated from a human serum
sample) was observed, in agreement with a previous report
(10) which suggested that this position was representative of the tissue distribution. Since Q7 was the prevalent quasispecies detected, it was chosen as the reference variant throughout our study.
Most of the sequences presented in Fig. 2 differed from Q7 by 1 nt,
although changes of 2 nt (Q2, Q20, Q27, and Q31), 3 nt (Q8, and Q24),
and up to 4 nt (Q1) changes can also be noted. Of the 22 nt changes,
most were substitutions, except for 2 deletions (nt 55, Q1; nt 126, Q15) and a common C insertion (between nt 126 and 127) for Q24 and Q27.
Moreover, all mutations were located within the 5' UTR, except for the
mutation at nt 348 (Q32), which occurred in the coding sequence known
to be part of the IRES (19) (Fig.
3). The diversity observed among 43 clones was assumed to reflect the quasispecies distribution of 5' UTR
in the serum sample studied. The hypothesis that this variability might
be a consequence of nucleotide misincorporation during RT-PCR has been
considered but did not seem valid for several reasons. First, unlike
many studies on quasispecies, we took advantage during seminested PCR of a proofreading activity of Taq polymerase, which is
expected to reduce the misincorporation rate. Second, if incorporation errors from polymerase were implicated, we should have observed an
increment in mutations from a single mutant to multiple mutants, which
was not the case. Finally, we can state that in another study in
progress (data not shown) with the same system, almost the same
mutations have been detected in two independent experiments, each one
using a new RNA template. All these points argue in favor of the
reliability of our sequence data and naturally occurring variability
and quasispecies balance coevolving in the patient at the sampling time
point.
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FIG. 2.
Alignment of nucleotide sequences of the IRES region in
the quasispecies characterized in the serum of a patient infected with
the HCV 1a genotype relative to plasmid pIRF, whose complete sequence
is given in the first line; only differences in nucleotide composition
are indicated. Nucleotide numbers refer to HCV 1a (13); X,
deletion from 1 nt;  , C insertion; brackets, frequency of the
clone among the whole population of 43 clones sequenced.
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FIG. 3.
Scheme of the secondary structure of the HCV IRES,
showing the locations of the nucleotide mutations in IRES elements of
sequenced quasispecies studied. The secondary structure prediction and
loop numbering are based on those of Honda et al. (7); the
initiator AUG codon in stem-loop IV is circled, and the coding sequence
is represented by a dotted line. All quasispecies mutations are
depicted by arrows preceded by  for substitution,   for deletion,
and for insertion.
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In vitro translational efficiency of 5' UTR quasispecies.
To
analyze a possible correlation between the 5' UTR sequence diversity
observed in the patient's serum and IRES activity, each of the
isolated quasispecies was first subjected to in vitro translation as
described above. As shown in Fig. 4, a
great heterogeneity was observed in translation efficiencies, varying
from 3.4 to 93.6% relative to pIRF. Q7 was the most efficient, but on
the whole, the activity was independent of the number of additional mutations detected. Indeed, Q1 (four mutations) was more effective than
Q12 and Q22 (one mutation), suggesting a more critical role for the
nucleotide location (nt 301 and 266) for IRES activity than for the
number of mutations. Base-pairing disruption resulting from a
substitution at nt 301 within the pseudo-knot organization of domain
IIIe might explain the severe loss of IRES function. Interestingly, the
G-to-A substitution at nt 266 (Q22), located within the loop of domain
IIId, had the most drastic effect on RLuc expression. This observation
fits the results of a recent study highlighting the importance of
nucleotides contained in the hairpin structure of stem-loop IIId in
modulating the HCV 5' UTR tertiary folding structure required for
functioning (12). Despite the presence of three changes, Q7
exhibited a marginal reduction compared to pIRF in its capacity of
translation (93.6%), indicating that mutation from AG to GA at
positions 34 and 35 had no influence on translation efficiency. Other
changes resulting in intermediate RLuc expression either were located
in loops or did not strongly affect base-pairings within the
corresponding stem structures, as was the case for deletion at nt 126 (Q15) and insertion between nt 126 and 127 (Q24 and Q27) in the
pyrimidine tract (nt 120 to 130) with relative efficiencies of 79.8, 41, and 62.5%, respectively. In agreement with a previous report
(31), these mutations, while inducing a slight change in the
surrounding sequences, were not sufficient to impair IRES function.
This could imply, as mentioned above, that the secondary structure of
IRES is not the only parameter for optimal activity but that tertiary folding must also be considered. The only mutation observed in the
capsid coding sequence, located at nt 348 (Q32), resulted in an A-to-G
substitution, which was predicted to induce a less stable base-pairing
from UA to UG within domain IV (Fig. 3). Rather than the expected
enhancement of IRES efficiency (8), a significant decrease
was observed, which emphasizes the requirement of the first capsid
coding nucleotides to ensure HCV IRES function (19).
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FIG. 4.
Relative efficiencies of IRES elements of different
quasispecies in vitro. Bicistronic plasmids corresponding to the
indicated quasispecies were used to program in vitro
transcription-translation in the TNT reticulocyte lysate system. IRES
relative activity was then assessed by measuring the ratio of the
expressed RLuc to FLuc by using the dual luciferase kit assay. Relative
activities were normalized to the pIRF reference construct, whose ratio
was arbitrarily taken as 100%. Data shown represent means from two
independent experiments with a variation among the different
experiments of less than 10%.
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In vivo translational efficiency of 5' UTR quasispecies.
Our
constructs were then tested in vivo using three different cell lines to
confirm our in vitro findings in a system having conditions closer to
those existing during viral replication. Indeed, the HCV IRES has been
extensively reported to bind a variety of cellular factors that might
be absent from the RRL system. For that purpose, we selected the
following: pIRF as a reference; Q7 representing the consensus
quasispecies; and Q2, Q12, Q22, and Q31 representative of IRES
quasispecies impaired in in vitro activity. Three cell lines were used:
Vero cells (kidney cells derived from the African green monkey), HepG2
cells (human cells of liver carcinoma origin), and Jurkat cells (human
cells of lymphocyte origin). For transfections, an appropriate number
of cells were seeded in 24-well plates. The next day, the cells were
infected with the vTF7-3 recombinant vaccinia virus expressing T7 RNA
polymerase (kindly provided by B. Moss, National Institutes of Health,
Bethesda, Md.) at 5 PFU/cell in 300 µl of serum-free Opti-MEM medium
(Gibco-BRL) for 1 h at 37°C. The cells were then transfected
with 1 µg of plasmid DNA mixed with 5 µg of DAC-30 (Eurogentec) in
300 µl of Opti-MEM medium. At 16 to 18 h posttransfection, the
cells were harvested and lysates were assayed for luciferase activities
as described above. The results of these experiments are summarized in
Fig. 5A. As in the RRL assay, the IRES
activity of Q12 and Q22 was dramatically reduced in the three types of
cells tested, confirming the crucial role of nt 266 and 301 for this
function. Surprisingly, a considerable disparity of IRES efficiency was found for the four other clones tested with regard to the cell line
used. These observations were highly reproducible and argue in favor of
a biological difference linked to the nature of the IRES sequence and
its capacity to promote internal initiation of translation in vivo,
depending on the cell line used for transfection (1, 4, 22).
To verify that variations in RLuc expression observed with IRES
variants in transfected cells actually reflect different translational
capacities, we investigated the stability of corresponding transcripts
in different cell lines by Northern blot analysis (Fig. 5B). The
results indicate a comparable amount of RNA in transfected HepG2 and
Jurkat cells, ruling out the notion that the observed variations in
RLuc expression could be due to differences in transcription or
stability of the RNA transcripts. Nor could they be attributed to
differences in transfectability of cells, this factor being abrogated
by the use of a bicistronic system. Our results support the emerging
view that HCV translation might be dependent on the interaction with
cellular factors distributed differently among cell types. A noteworthy
observation was the evidence of opposite patterns of IRES activity
depending on the cell type used for the RNA transfection assay.
Although showing a high efficiency in HepG2 and Vero cells, pIRF and Q7
5' UTRs were much less active in Jurkat cells. The opposite was
observed for Q2, Q22, and Q31 (the low level of activity of Q12 did not permit any interpretation in that context). In light of these data, it
is tempting to distinguish between nonlymphoid (pIRF and Q7) and
lymphoid (Q2, Q22, and Q31) optimal IRESs. These observations strongly
indicate that the contribution of the nucleotide sequence of the HCV 5'
UTR relative to IRES function differs according to the cellular system
used, suggesting that the interactions between the highly ordered HCV
IRES structure and related host factors are cell type specific.
Moreover, we have noted that for the former, the activity ratio between
Vero and HepG2 cells was always low (ratio, <1), whereas for the
latter, the activity was always higher in Vero cells (ratio, ca. 2).
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FIG. 5.
Relative translational efficiencies of IRES elements of
different quasispecies in vivo. (A) Relative levels of RLuc expression
from various quasispecies tested. Three different cell lines were used
for transfection: HepG2, Vero, and Jurkat. Cell lysates were prepared
16 to 18 h posttransfection and assayed for FLuc and RLuc
activities as described for the in vitro experiment. Relative
efficiencies of different IRES quasispecies were measured by the
RLuc/FLuc ratio. For each construct, experiments were performed in
triplicate wells, and standard deviations were calculated from the data
obtained for these wells. (B) Northern blot analysis of bicistronic
RNAs extracted from HepG2 or Jurkat cells transfected for 18 h
with the indicated constructs: pIRF, Q7, and Q12. IRES 3'
oligonucleotide was used as a probe. As controls, untransfected Jurkat
cells (MOCK) and pIRF in vitro transcript (WT) (indicated by an arrow)
were used.
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In conclusion, we have shown that HCV 5' UTR has a quasispecies
distribution in a given infected individual and we have been
able, to
our knowledge for the first time, to demonstrate that
the naturally
occurring sequence diversity of IRES elements leads
to important
changes in their ability to direct cap-independent
translation. Such
differences observed in vitro were confirmed
although differently
modulated in in vivo assays, depending on
the transfected cell type.
Because viral particles in serum are
thought to be released from the
liver but also from other compartments
of the organism such as
peripheral blood mononuclear cells (
21,
23,
24), the
observed diversity within 5' UTR might reflect
the existence of various
HCV IRES sequences targeting RNA translation
specifically to the liver
or extrahepatic compartments. In accordance
with that view, initiation
of protein translation may appear as
one rate-limiting factor for viral
replication. It will be of
interest to assess HCV 5'UTR polymorphism
due to viral tropism
in different parts of the organism and its impact
on the selection
of replicative variants. Moreover, the bicistronic
vector designed
in this work presents a unique feature in addition to
its accuracy
and reproducibility. Indeed, in contrast to several
reports on
IRES efficiency, we have conserved the entire HCV 5'UTR in
our
construct in order to preserve a possible influence of
cis non-IRES
elements on the final secondary and/or tertiary
structures of
IRES. In the perspective of correlating differences in
the activity
of IRES sequences with pathogenesis of HCV infection, the
system
used in our study provides a useful tool for structure-function
analyses of HCV IRES. So far, most of the studies on the dynamics
of
HCV quasispecies have been conducted on variable regions of
the HCV
genome such as E2 hypervariable region 1 or parts of the
NS5A protein
and thus have been limited to sequence comparison
without any
functional investigation. To date, although little
work has been
undertaken in that direction, the HCV 5'UTR appears
to be an element of
choice for such an approach. Contradictory
conclusions have been
proposed concerning a possible correlation
between the sequence
variability found for HCV IRESs of different
genotypes and a response
to interferon therapy (
15,
22,
32).
Currently, work is under
way to study HCV 5' UTR diversity displayed
in a viral quasispecies
population and its dynamics toward the
viral life cycle under different
biological
pressures.
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ACKNOWLEDGMENTS |
We thank J. P. Lagarde for his help in sequencing the HCV IRES
of different bicistronic plasmids studied and G. Inchauspé for
helpful discussions.
This work was supported in part by the Ministère de l'Education
Nationale de la Recherche et de la Technologie (MENRT), programme de
Recherche Fondamentale en Microbiologie et Maladies Infectieuses et
Parasitaires (réseau Hépatite C), the Association pour la Recherche contre le Cancer, and the Association Claude Bernard. J.L. is
supported by doctoral grant 99623 from the MENRT.
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FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Virologie, C.E.R.V.I., UPRES EA 2387, Hôpital
Pitié-Salpêtrière, 83 Bd de l'hôpital, 75651 Paris Cedex 13, France. Phone: 33.1.45.82.62.98. Fax: 33.1.45.82.63.14. E-mail: cahour{at}idf.ext.jussieu.fr.
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Journal of Virology, November 2000, p. 10827-10833, Vol. 74, No. 22
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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