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Journal of Virology, August 2000, p. 7064-7071, Vol. 74, No. 15
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Efficient Translation of Rotavirus mRNA Requires Simultaneous
Interaction of NSP3 with the Eukaryotic Translation Initiation
Factor eIF4G and the mRNA 3' End
Patrice
Vende,
Maria
Piron,
Nathalie
Castagné, and
Didier
Poncet*
Laboratoire de Virologie et Immunologie
Moléculaires INRA, C.R.J.J., 78352 Jouy-en-Josas Cedex,
France
Received 17 February 2000/Accepted 5 May 2000
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ABSTRACT |
In contrast to the vast majority of cellular proteins, rotavirus
proteins are translated from capped but nonpolyadenylated mRNAs. The viral nonstructural protein NSP3 specifically
binds the 3'-end consensus sequence of viral mRNAs and
interacts with the eukaryotic translation initiation factor eIF4G. Here
we show that expression of NSP3 in mammalian cells allows the efficient translation of virus-like mRNA. A synergistic effect between
the cap structure and the 3' end of rotavirus mRNA was
observed in NSP3-expressing cells. The enhancement of viral
mRNA translation by NSP3 was also observed in a rabbit
reticulocyte lysate translation system supplemented with recombinant
NSP3. The use of NSP3 mutants indicates that its RNA- and eIF4G-binding
domains are both required to enhance the translation of viral
mRNA. The results reported here show that NSP3 forms a link
between viral mRNA and the cellular translation machinery and
hence is a functional analogue of cellular poly(A)-binding protein.
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INTRODUCTION |
The vast majority of cellular
mRNAs possess a 3' terminal poly(A) sequence. This sequence
plays a major role in many aspects of cellular mRNA
metabolism (11). Together with the 5' cap structure, poly(A)
synergistically enhances the translation of mRNA (8, 12). This effect is mediated by the poly(A)-binding protein (PABP) (20), which interacts with the 3' poly(A) and
the eukaryotic initiation factor eIF4G (14, 23, 33).
eIF4G is a scaffold protein that brings together eIF4E
(cap-binding protein), eIF4A (a helicase), PABP, and eIF3
(5). As a consequence of these multiple interactions, the
40S subunit of the ribosome, loaded with initiator tRNA and
methionine, is brought to the 5' end of a circularized mRNA
and starts scanning the 5' untranslated region (UTR) for the first
initiation codon (21). The circularization of the mRNA
via eIF4E-eIF4G-PABP and mRNA interactions
(34) is thought to enhance the translation of the
mRNA by allowing rapid reinitiation of new rounds of
translation. Circularization of the mRNA seems particularly
important for efficient and accurate initiation when competition exists
between mRNA (27) or when the supply of ribosomes
or initiation factors is limited (28).
Rotaviruses are the major cause of diarrhea in young animals and
children; they are involved in the death of more than 800,000 children
each year worldwide (10). Rotaviruses are members of the
Reoviridae family, and their genome is composed of 11 molecules of double-stranded RNA, which encode six structural
proteins and five or six nonstructural proteins (6, 17). The
virus replication cycle occurs entirely in the cytoplasm. Upon virus
entry, the viral transcriptase synthesizes capped but nonpolyadenylated
mRNAs (13). The viral mRNAs bear 5' and
3' untranslated regions (UTR) of variable length and are flanked by two
different sequences common to all genes. In the group A rotaviruses,
the 3'-end consensus sequence UGACC is highly conserved among the 11 genes.
We have previously shown that rotavirus NSP3 presents several
similarities to PABP; in rotavirus-infected cells, NSP3 can be
cross-linked to the 3' end of rotavirus mRNAs (24,
25) and is coimmunoprecipitated with eIF4G (23). The
binding of NSP3A to eIF4G and its specific interaction with the 3' end
of viral mRNA (24, 25) brings the viral
mRNA and the translation initiation machinery into contact,
thus favoring efficient translation of the viral mRNA. NSP3
interacts with the same region of eIF4G as PABP does (14,
23). As a consequence, during rotavirus infection PABP is evicted
from eIF4G, probably impairing the translation of polyadenylated
mRNA and leading to the shutoff of cellular mRNA
translation observed during rotavirus infection (23).
Here we describe the establishment of a cell line expressing NSP3. We
used this cell line to show that NSP3 and the cap structure synergistically enhance rotavirus-like mRNA translation in
vivo. Then we used an in vitro assay to study the effect of NSP3 on the
translation of reporter mRNAs with poly(A) or rotavirus
3'-end sequences. Using the same in vitro translation assay and NSP3 mutants, we show that the two functional domains of NSP3 are required to enhance rotavirus mRNA translation and that the
eIF4G-binding domain of NSP3 inhibits translation when it is separated
from the RNA-binding domain.
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MATERIALS AND METHODS |
Plasmid construction.
Manipulation of nucleic acids was done
as described by Sambrook et al. (31) unless otherwise
indicated. The cDNA of rotavirus RF gene 7 (RF7) encoding NSP3A has
been described previously (25). It was subcloned into pTEJ4
(15), which allows constitutive expression under the control
of the ubiquitin promoter. To obtain pT7-RF6-F.Luc-RF6, two
complementary synthetic oligonucleotides with the sequence of the T7
promoter fused to the sequence of the 5' UTR of rotavirus RF6 (with a
NcoI site on the ATG) (3) were cloned between the
HindIII and XbaI sites of pUC19 to produce pT7-RF6-5'. The 3' UTR of RF6 bearing a BamHI site at its 5'
end and KspI and EcoRI sites at its 3' end was
amplified by PCR from RF6 cDNA (3) and was cloned between
the BamHI and EcoRI sites of pT7-RF6-5'. The
firefly luciferase (F. Luc) gene (NcoI-BamHI fragment) was purified from pSP-Luc(+) (Promega) and inserted between the 5' and 3' RF6 UTRs to produce pT7-RF6-F.Luc-RF6.
The sequences of the RF6 UTRs of pT7-RF6-F.Luc-RF6 were checked by DNA sequencing. pCMVT7-R.Luc-polyA was obtained by cloning a
synthetic oligonucleotide bearing 25 adenines, followed by a
NsiI restriction site, between the XbaI and
BamHI sites of pRL CMV (Promega). pRF6-F.Luc-polyA was
obtained by subcloning the HindIII-XbaI
fragment from pRF6-F.Luc-RF6 into pCMV-RL-polyA.
In vitro synthesis of mRNAs.
F.Luc-rota and
F.Luc-nonrota mRNAs were synthesized from pT7-RF6-F.Luc-RF6
templates linearized with KspI or EcoRI,
respectively. The pT7-RF6-F.Luc-RF6 linearized with KspI was
treated with T4 polynucleotide kinase in the absence of dCTP to
ensure the production of 3' recessed ends. Renilla
luciferase (R.Luc) poly(A) and F.Luc poly(A) mRNA were
obtained respectively from pCMV-T7-R.Luc-poly(A) and
pRF6-F.Luc-poly(A), linearized with NsiI and treated
with T4 DNA polymerase in the absence of dATP to obtain 3' recessed ends.
T7 polymerase transcription systems supplied or not supplied with cap
analogue (Message Machine [Ambion] and Ribomax [Promega], respectively) were used to synthesize capped and uncapped
mRNA. In all cases, tritiated UTP (800 mCi/mmol [Amersham])
was added in trace amount (40 µCi) to determine the quantity of
mRNA synthesized and to monitor the purification of the
mRNAs. After treatment with RNase-free DNase I for 10 min at
37°C, RNAs were purified from nucleotides and cap analogue by phenol
extraction and repeated ethanol precipitation in the presence of
ammonium acetate or on Sephadex G-10 push columns (Stratagene). In both
cases, measurement of trichloroacetic acid-precipitable radioactivity
by scintillation counting indicated that more than 90% of the
unincorporated nucleoside triphosphates were removed. The purified RNA
was finally analyzed by agarose gel electrophoresis.
DNA and RNA transfections.
ST (swine testis) cells were
cultivated in Earle's minimal essential medium supplemented with 10%
fetal calf serum. They were transfected with pTEJ4 RF7 (10 µg) by
lipofection (Lipofectin; Life Technologies) together with pX343 (1 µg). Stable antibiotic-resistant clones were selected by the presence
of 100 µg of hygromycin per ml for 10 days. Clones were isolated
using cloning cylinders and screened for NSP3 expression by indirect
immunofluorescence with monoclonal antibody ID3 as previously described
(26).
ST or ST-NSP3 cells grown to 70% confluence in 12-well plates were
transfected with cationic liposomes (DRMIE-C; Life Technologies)
with
both 400 ng of F.Luc mRNA and 4 ng of R.Luc mRNA.
After 5
h of lipofection, the RNA mixture was removed and fresh
medium
was added. Cells were lysed 20 h later for the measurement
of
luciferase activities by the dual-luciferase method
(Promega).
For electroporation of RNA, ST-NSP3 cells grown to 70% confluence were
trypsinized and adjusted to 10
7 cells/ml of Earle's
minimal essential medium. The cells were
transferred to a sterile
electroporation cuvette, mixed with 200
ng of F.Luc mRNA and
10 ng of capped-R.Luc-poly(A) mRNA, and electroporated
(Easyject+) at 260 V and 1,050 µF. Immediately after electroporation,
MEM with 10% serum was added and the cells were incubated at 37°C
for 5 to 24
h.
Immunoprecipitation and growth curves.
ST and ST-NSP3 cells
in 100-mm-diameter dishes were labeled for 2 h with 20 µCi of
Tran35S-label (38 TBq/mol; ICN) as previously
described (24). Immunoprecipitation with an anti-NSP3
monoclonal antibody was carried out as described previously
(24), except that cell lysates were precleared by a 1-h
incubation with protein A-Sepharose before being subjected to immunoprecipitation.
Plates (35 mm) were seeded with 6 × 10
5 ST or ST-NSP3
cells, and living cells were counted at different times by trypan blue
exclusion after
trypsination.
Mutagenesis and expression of recombinant NSP3.
Expression
of NSP3 fused to a track of 6 histidine residues in Escherichia
coli using the T7 expression system (32) has been
described previously (22). Two mutations that improve the expression and renaturation of the protein were introduced into NSP3
cDNA by using the Quick-Change (Stratagene) protocol; cysteine 227 was
changed to asparagine to impair the formation of disulfide bonds during
purification of the protein, and methionine 206 was changed to
isoleucine to impair internal translation initiation and copurification
of a truncated C-terminal fragment of NSP3. All the NSP3 mutants used
in this study were obtained by site-directed mutagenesis (Quick-Change)
of this modified cDNA. The resulting plasmids were transfected into
E. coli BL21/DE3, and the expression of NSP3 was induced by
the addition of IPTG (isopropyl-
-D-thiogalactoside; final concentration, 1 mM). Induction and purification of the recombinant protein were conducted as described previously
(22). Renaturation of recombinant proteins eluted from
nickel-chelating Sepharose columns (Pharmacia) was modified to improve
the solubility and stability of the proteins. Wild-type NSP3 and
RNA-binding mutants were renatured by stepwise dialysis with decreasing
concentrations of urea in 10 mM Tris (pH 7.4)-10% glycerol-1 mM
EDTA-10 mM dithiothreitol. NSP3 mutants on the eIF4G-binding domain
were renatured by stepwise dialysis against 10 mM Tris (pH 8)-10%
glycerol-1 mM EDTA-10 mM dithiothreitol.
Analysis of the RNA-binding and eIF4G-binding properties of
recombinant NSP3.
Purified recombinant proteins were tested for
their RNA-binding activity by UV cross-linking and gel retardation as
previously described (22, 25). The eIF4G-binding capacity of
NSP34-290 was tested by the two-hybrid assay after
transfer of the NSP34-290 gene into the yeast vector pGBT9
NS (22, 23). Yeast strain HF7C was transfected with
pGAD424-eIF4GI/+37:174 and pGBT9-NSP34-290 or
pGBT9-NSP34-313 and spread onto medium without tryptophan and leucine to monitor the cotransfection efficiency and onto medium
without tryptophan, leucine, or histidine to assess the interaction
between the two fusion proteins.
Measurement of luciferase activities and synergy.
After
lipofection or electroporation of ST or ST-NSP3 cells with firefly and
Renilla reporter mRNAs, cells were lysed and luciferase activities were measured using the Dual Luciferase kit as
specified by the supplier (Promega) and an automatic luminometer (Berthold). The luciferase activities of cells transfected with the DNA
used as matrices for in vitro RNA transcription were measured and found
to be equal to the luciferase activities of mock-transfected cells.
Luciferase activities were standardized to the protein concentration of
the samples.
The ratio of the capped-Luc-poly(A) mRNA translation to the
sum of capped-Luc-nonrota mRNA and uncapped-Luc poly(A)
mRNA gives
the amount of synergy between cap and poly(A)
(
21). The ratio
of the capped-Luc-rota mRNA
translation to the sum of capped-Luc-nonrota
mRNA and
uncapped-Luc rotavirus mRNA gives the amount of synergy
between cap and rotavirus 3'
end.
In vitro translation.
Nuclease-treated rabbit reticulocyte
lysate (FlexiRRL; Promega) adjusted to 110 mM KCl was used to translate
reporter mRNAs in the presence of recombinant NSP3. Each
translation mixture consisted of 70% of RRL (Promega), 10 U of RNasin
RNase inhibitor per µl, 20 µM amino acid mixture, 5 ng of
mRNA (1 nM), and 100 ng of purified NSP3 (230 nM) or the same
volume of the same batch of NSP3 final dialysis buffer. After a 1-h
incubation at 30°C, the luciferase activity of samples was measured
using the F Luciferase kit as recommended by the supplier (Promega).
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RESULTS |
NSP3 expression enhances the expression of rotavirus-like
mRNA in vivo.
To investigate the role of NSP3 in
rotavirus translation, we first established a stable cell line
expressing NSP3. The cell line generally used to propagate rotaviruses
is MA104. In our hands, MA104 cells age poorly, as seen by reduced
growth capacity and susceptibility to rotavirus infection with
increased passage number. This cell line was therefore not suited for
our purpose; instead, we chose to use the ST cell line, whose growth is
not restricted by passage number and which is fully susceptible to rotavirus infection (19).
ST cells constitutively expressing NSP3 (under the control of the
ubiquitin promoter [
15]) were screened by indirect
immunofluorescence
(Fig.
1A and B) with a
monoclonal antibody directed against NSP3
(
2). Only a few
cell clones were positive for NSP3 expression,
and one (called ST-NSP3)
that synthesizes a detectable amount
of NSP3 by immunoprecipitation
(Fig.
1C) was selected for this
study. The cells were affected by
expression of NSP3; ST-NSP3
cells seemed slightly larger than the
parental ST cell, they were
more robust when electroporated (see
below), and their growth
(doubling time, 48 h) was slower than
that of the parental cell
line (Fig.
1D; doubling time, 35 h).
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FIG. 1.
Expression of NSP3 in ST-NSP3 cells. (A and B)
Expression of NSP3 in ST (A) and ST-NSP3 (B) cells was detected by
indirect immunofluorescence using monoclonal antibody ID3 directed
against NSP3. (C) Radiolabeled proteins from 3 × 106
ST-NSP3 cells and ST cell lysates were immunoprecipitated with
monoclonal antibody ID3 and analyzed by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). An autoradiogram
of the gel is shown. Controls are provided by immunoprecipitation of
radiolabeled proteins from 3 × 105 MA104 cells
infected (MA104-RF) or mock infected (MA104) with rotavirus RF strain.
(D) Growth curves of ST and ST-NSP3 cells.
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NSP3 produced by ST-NSP3 cells had the same molecular weight as NSP3
produced during virus infection (Fig.
1C), was recognized
by the
monoclonal antibody against NSP3, and had an intracytoplasmic
distribution (Fig.
1D) similar to that of NSP3 produced during
rotavirus infection (data not
shown).
Rotavirus-like reporter mRNAs (F.Luc-rota) were obtained by
subcloning the firefly luciferase gene (F.Luc) between the 5'
UTR and
3' UTR of RF6 (
3) (Fig.
2).
The addition of a
KspI
restriction site overlapping the
rotavirus 3' consensus sequence
UGACC allowed mRNAs to be
synthesized, in vitro, with a UGACC
sequence precisely at their 3' end.
In contrast, if the same plasmid
is linearized with
EcoRI
prior to transcription, the UGACC sequence
lies 4 nucleotides upstream
of the AAUU sequence at the 3' end
of the mRNA. Such
mRNAs serve as control reporter (F.Luc-nonrota),
since we
have previously shown that NSP3 recognizes the UGACC
sequence only if
it is located at the 3' end of the RNA (
24,
25). Thus,
F.Luc-rota and F.Luc-nonrota mRNAs differ only in
their very
3' ends. To study the effect of NSP3 on the expression
of poly(A)
mRNA, a 25-base poly(A) sequence was inserted in place
of the
3' RF6 UTR; this mRNA is referred to as F.Luc-poly(A).
To
compare the effect of the 3' ends on the expression of the
various
reporter capped mRNAs, capped F.Luc-rota, F.Luc-nonrota,
and
F.Luc-poly(A) mRNAs were introduced by lipofection into ST
and ST-NSP3 cells. Figure
3 illustrates
the results of such experiments.
Poly(A) mRNAs were
efficiently translated in ST and ST-NSP3 cells.
The
F.Luc-pA translation in ST-NSP3 cells was slightly diminished,
probably reflecting a slight inhibition of poly(A)-dependent
translation
in this cell line. Non-poly(A) mRNA were barely
translated in
ST cells irrespective of their (rota or nonrota) 3' ends.
However,
F.Luc-rota mRNA was efficiently translated in
ST-NSP3 cells (Fig.
3). The major difference in ST-NSP3 cells is the
sevenfold increase
in F.Luc-rota translation efficiency, showing that
the expression
of NSP3 in mammalian cells promotes efficient
translation of rotavirus-like
mRNAs.
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FIG. 2.
Rotavirus-like and poly(A) reporter mRNAs. The
salient features of the DNA matrices used for in vitro synthesis of the
different mRNAs used are shown (not to scale). (Top) The
firefly luciferase gene was cloned between the 5' and 3' UTRs of RF6
downstream of the T7 promoter (T7). F.Luc-rota mRNA was
obtained from KspI (K)-linearized matrix, and F.Luc-nonrota
was obtained from EcoRI (E)-linearized matrix. The DNA
sequences at the junction between the T7 promoter and the 5' UTR (bold
type) and between the 3' UTR (bold type) and the vector are indicated.
The site of cleavage on the top strand of the DNA template and the
restriction enzymes used are indicated. (Middle) The 3' RF6 UTR was
replaced by a poly(A)25 sequence, and F.Luc poly(A)
mRNA was obtained from the NsiI-linearized matrix.
(Bottom) A poly(A)25 sequence was cloned downstream of the
R.Luc gene and RLuc poly(A) mRNA was obtained with
NsiI-linearized matrix. The box labeled T7 indicates the T7
promoter;arrows indicate the T7 transcription start site; AAA
indicates poly(A)25; K indicates the KspI
site; E indicates the EcoRI site; ATG indicates the
initiation codon; STOP indicates the termination codon. Boxes indicate
nonrotavirus sequences, and shaded areas indicate transcribed vector
sequences.
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FIG. 3.
Translation of capped rotavirus-like and poly(A)
mRNA in ST and ST-NSP3 cells. F.Luc activity was
measured 24 h after lipofection of capped F.Luc-rota (black
columns), capped F.Luc-nonrota (hatched columns), and
capped-F.Luc-poly(A) (white columns) mRNAs into ST (left) or
ST-NSP3 (right) cells. The level of translation is expressed in
arbitrary firefly translation units. These units were standardized with
respect to concomitantly transfected R.Luc-poly(A) mRNA so
that variation due to different transfection efficiencies could be
discounted.
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Synergy between the 5' cap and 3' rotavirus ends.
The 5' cap
structures and the 3'-end poly(A) sequences act
synergistically to enhance mRNA translation.
Synergy can be measured by the ratio between the F.Luc
activity obtained with capped F.Luc-poly(A) mRNA and
the sum of F.Luc activities obtained with uncapped F.Luc-poly(A) and
capped F.Luc-nonrota mRNA (20). Measuring synergy
in vivo requires measuring the activity of uncapped mRNA.
When introduced into ST-NSP3 cells by lipofection, uncapped
mRNAs were very poorly translated (data not shown),
precluding the use of this method to study synergy. Alternatively, RNA
can be introduced into cells by electroporation (8).
Electroporation has the advantages over lipofection of being much
quicker and synchronous for all cells and allowing the rapid recovery
of cells after introduction of mRNA.
We set up electroporation conditions for ST-NSP3 cells, but for unknown
reasons, ST cells did not sustain the conditions used
to electroporate
ST-NSP3 cells and could not be used in the following
experiments.
mRNAs with different 3' ends were electroporated
into ST-NSP3
cells and the F.Luc activity was measured 5 h after
electroporation (Fig.
4) (Note that the
F.Luc translation is different
from that in Fig.
2 due to the different
proportions of R.Luc
and F.Luc mRNAs introduced into the cells.)
The F.Luc-rota and
F.Luc-poly(A) mRNAs were translated more
efficiently than the
F.Luc-nonrota mRNA was, irrespective of
their 5'-end structure.
This result showed that F.Luc-rota is
efficiently translated in
ST-NSP3 cells when introduced by
electroporation and confirmed
the results obtained with lipofection.
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FIG. 4.
Translation of capped and uncapped rotavirus-like
and poly(A) mRNA in ST-NSP3 cells. F.Luc activity was
measured 5 h after electroporation of capped (left) or uncapped
(right) F.Luc-rota (black columns), F.Luc-nonrota (hatched columns),
and F.Luc-poly(A) (white columns) mRNAs into ST-NSP3 cells.
The level of translation is expressed by the ratio of F.Luc to R.Luc
activities. The level of translation is expressed in arbitrary firefly
translation units. These units were standardized with respect to
concomitantly transfected R.Luc-poly(A) mRNA so that
variation due to different transfection efficiencies could be
discounted.
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Synergy between mRNA 3' and 5' ends can be
calculated from the results in Fig.
4. Synergy values of 21 (standard deviation
= 1,
n = 2) between cap
and poly(A) were calculated when the luciferase
activity was measured
5 h after the introduction of F.Luc-poly(A)
mRNA into
ST-NSP3 cells. Under the same conditions, a synergy
value of 35 (standard deviation = 5,
n = 2) was calculated for
rotavirus-like mRNA, showing that the UGACC sequence can also
cooperate with the NSP3 5' cap structure to enhance the translation
of
rotavirus
mRNA.
NSP3 stimulates the translation of virus-like mRNA in
vitro when bound to eIF4G.
To examine directly the enhancement of
translation of virus-like mRNA by NSP3, we turned to in vitro
translation systems. Purified recombinant NSP3 expressed in E. coli (NSP34-313) was added to RRL programmed with
uncapped mRNAs with different 3' ends (Fig.
5). The effect of NSP3 (added at a final
concentration of 20 or 200 mM) on translation efficiency was
expressed by the ratio of F.Luc activity obtained in the presence
of NSP3 to the F.Luc activity measured in the presence of NSP3
renaturation buffer (Fig. 5). Adding NSP3 to uncapped-F.Luc-rota
mRNAs increased their translation nearly 100-fold (Fig. 5,
200 mM). At the same concentration, NSP3 increased the translation of
uncapped-F.Luc-nonrota or uncapped-F.Luc-poly(A) mRNA only
15- and 10-fold, respectively. With both concentrations of NSP3,
the ratio of specific to nonspecific enhancement of translation was
nearly 10 (Fig. 5).
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FIG. 5.
Stimulation of translation by recombinant wild-type
NSP3 of rotavirus-like, non-poly(A), and poly(A)
mRNAs in vitro. Wild-type NSP3
(NSP34-313) was added to RRL programmed with
different mRNAs (same abbreviations as in Fig. 3) at a 200- or 20-fold excess over the RNA 3' end. Fold stimulation is expressed by
the ratio of F.Luc activity obtained in the presence of recombinant
protein to the activity obtained in the absence of recombinant protein.
Bars indicate standard deviation calculated from three independent
experiments.
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Addition of a cap to the 5' end of F.Luc mRNAs enhances the
translation (in the absence of NSP3) of any F.Luc mRNA
50-fold
(data not shown). Thus, when the same experiments were
conducted
with capped-F.Luc-rota, only a modest additional twofold
increase
in translation was obtained on addition of NSP3 (data not
shown).
However, a clear effect of NSP3 on translation of
rotavirus-like
mRNA could be observed in vitro with uncapped
mRNAs which were
used for the in vitro experiments described
below.
Next we wanted to study the relative importance of the RNA- and
eIF4G-binding activities of NSP3 in the enhancement of translation
of
rotavirus mRNA. The in vitro assay described above was used
to test NSP3 mutants. A deletion of the last 24 amino acids of
NSP3
abolishes the interaction between NSP3 and eIF4G when tested
by the
two-hybrid assay (
23) but has no effect on the RNA-binding
activity (
22) (see Fig.
7B and C). The same mutant was
produced
in
E. coli, and the purified recombinant
protein (NSP3
3-290)
was used in the in vitro translation
assay. NSP3
3-290 does
not enhance the translation of the
uncapped-F.Luc-rota mRNA (Fig.
6). Even the nonspecific stimulation of
F.Luc-nonrota and F.Luc-poly(A)
translation observed with NSP3 was
abolished. We concluded from
this experiment that an interaction
between NSP3 and eIF4G is
required to enhance the translation of
rotavirus-like mRNA.
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FIG. 6.
Effect of recombinant wild-type NSP3 and the
eIF4G-binding mutant of NSP3 on the translation of
rotavirus-like, non-poly(A), and poly(A) mRNAs in vitro.
Wild-type NSP3 (NSP34-313) (left) or
NSP3 mutated in its eIF4G-binding domain
(NSP34-290) (right) was added to RRL programmed
with different mRNAs (same abbreviations as in Fig. 3). Fold
stimulation is expressed by the ratio of F.Luc activity obtained in the
presence of recombinant protein to the activity obtained in the absence
of recombinant protein.
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Enhanced translation correlates with the RNA-binding capacity of
NSP3.
The RNA-binding domain of NSP3 has been mapped to the
N-terminal half of the protein (22). Two mutations
were introduced into this domain: a conserved tryptophan
(29) at position 87 was changed to alanine to give
NSP3W/A:87, and a deletion of 11 amino acids was
introduced at positions 65 to 75 to give
NSP3
65-75. Figure
7A depicts an example of the purified
NSP3 used in the experiments described below. The RNA-binding
activity of these two mutants was tested in a UV cross-linking assay
with synthetic RNA (Fig. 7A and B) and in a gel retardation assay (Fig.
7C) as described previously (25). The two assays are not
exactly equivalent. Gel retardation is dynamic, so that protein and RNA
can dissociate during the running of the gel, whereas UV cross-linking
creates a covalent link between molecules and even brief interactions occurring during the UV irradiation can be observed.
NSP3W/A:87 could be UV cross-linked to the
rotavirus group A 3'-end sequence (Fig. 7B) but not to the rotavirus
group C 3'-end sequence. However, NSP3W/A:87 was
able to make only an unstable complex with the rotavirus group A 3'-end
RNA sequence, as evidenced by the absence of a neat band in a gel
retardation assay (Fig. 7C). Instead, the presence of a smear on the
autoradiogram reflected the instability of the
RNA-NSP3W/A:87 complexes that dissociated during
electrophoresis (16). The deletion of amino acids 65 to 75 totally abolished the capacity of NSP3 to bind RNA when
tested either by UV cross-linking (Fig. 7B) or by gel retardation (Fig.
7C).
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[in a new window]
|
FIG. 7.
Characterization of the RNA-binding properties of
wild-type NSP3 and NSP3 mutants. Wild-type NSP3
(NSP34-313), and mutants with mutations of the
eIF4G-binding domain (NSP34-290) or the
RNA-binding domains (NSP3W/A:87 and
NSP3 65-75) were expressed in E. coli and purified. Recombinant proteins were incubated with 5'-end
radiolabeled RNA bearing the group A rotavirus 3'-end sequence
(AUAUGACC) (lanes A) or the group C rotavirus 3'-end sequence
(AUAUGGCU) (lanes C). Samples were analyzed, after UV cross-linking, by
SDS-PAGE and Coomassie blue staining (A) followed by autoradiography
(B) (autoradiography of the gel shown in panel A). Alternatively,
samples were directly analyzed in a gel retardation assay by
nondenaturing PAGE and autoradiography (C).
|
|
When NSP3
W/A:87 was used in the in vitro
translation assay, it stimulated rotavirus-like mRNA
translation better than it stimulated
nonrotavirus
mRNA translation but to a much lower extent than
did
wild-type NSP3
4-313 (Fig.
8). With the deletion mutant
NSP3
65-75, a twofold nonspecific stimulation
of translation
was observed with the three kinds of RNA tested. Thus,
the strength
of the interaction of NSP3 with RNA correlates
with its translation
stimulation capacities and specificity.
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 8.
Effect of RNA-binding mutants of NSP3 on the
translation of rotavirus-like non-poly(A) and poly(A) mRNA in
vitro. Wild-type NSP3 (NSP34-313)
(left) or NSP3 mutated in its RNA-binding domain
(NSP3WA:87 [middle] and
NSP365-75 [right]) was added to RRL
programmed with different mRNAs (same abbreviations as in
Fig. 3). Fold stimulation is expressed as the ratio of F.Luc activity
obtained in the presence of recombinant protein to the activity in the
absence of recombinant protein.
|
|
Inhibition of translation by NSP3.
The ability of
NSP3 to bind to eIF4G and to evict PABP from eIF4F is thought
to be the cause of the shutoff of cellular mRNA translation
observed during rotavirus infection (23). If this is the
case, addition of NSP3 to the in vitro translation assay might be expected to inhibit the translation of polyadenylated mRNA. However, the addition of wild-type NSP3 or
the RNA-binding mutant of NSP3 to the in vitro translation
assay mixture programmed with uncapped poly(A) mRNA slightly
stimulated the translation (Fig. 5, 6, and 8). Two possible
explanations for this observation can be provided. First, the
recombinant wild-type and mutant NSP3 have a nonspecific
RNA-binding activity not detected by the RNA-binding assays used here.
This nonspecific RNA-binding activity could be sufficient to
enhance the translation of any mRNA added to the RRL. Second,
this effect could be similar to the trans-activation properties described recently for yeast PABP (20). Otero et al. have shown that addition of PABP, disabled for poly(A) or eIF4G
binding, to yeast cell extract stimulates the translation of
non-poly(A)+ mRNA (20). This property
of PABP has been called trans-activation.
To attempt to discriminate between these two possibilities, two new
NSP3 mutants were designed;
NSP3
163-313 bears an
entire deletion of the
RNA-binding domain but is still able to
bind eIF4G (
22), and
NSP3
163-290 contains an additional
deletion of
the last C-terminal 24 amino acids of NSP3, which
completely
disables its eIF4G-binding domain (
22). The complete
deletion of the RNA-binding domain totally abolished the translation
stimulation properties of NSP3 (Fig.
9) and even induced a 50%
reduction in
the translation of any mRNA. This inhibition of translation
was not the result of the toxicity of
NSP3
163-313 but, rather,
was the result of its
interaction with eIF4G, since the addition
of
NSP3
163-290 prepared under the same conditions
had no
inhibitory effect on translation (Fig.
9).
View larger version (21K):
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[in a new window]
|
FIG. 9.
Effect of the eIF4G-binding domain of NSP3 on
the translation of rotavirus-like and poly(A) mRNAs in vitro.
Recombinant NSP3 from which the entire RNA-binding domain
NSP3163-313) (left) or both the RNA- and
eIF4G-binding domains (NSP3163-290) (right) were
deleted, was added to RRL programmed with different mRNAs
(same abbreviations as in Fig. 3). Fold stimulation is expressed by the
ratio of F.Luc activity in the presence of recombinant protein to the
activity in the absence of recombinant protein.
|
|
| |
DISCUSSION |
NSP3 specifically enhances the translation of rotavirus
mRNA.
The properties of NSP3 described
previously (23, 24) strongly suggest that it is involved in
the translation of rotavirus mRNAs. To definitively establish
the role of NSP3 in translation, we generated a mammalian
cell line expressing NSP3, into which we introduced
mRNA by lipofection or electroporation. To our knowledge, this is the first time that a cell line expressing a rotavirus protein has been described. Unlike poly(A) mRNAs,
rotavirus-like mRNAs are not translated efficiently when introduced
into ordinary cells. However, expression of NSP3, even at a low level,
allowed efficient translation of rotavirus-like mRNA. As
expected from the RNA-binding specificity of NSP3, the
enhancement of translation was specific for mRNAs ending with
the rotavirus 3'-end UGACC sequence whereas mRNAs ending with
AAUU were poorly translated in either normal or
NSP3-expressing cells.
It has been shown that the 3' poly(A) and 5' cap structure act
synergistically to enhance the translation of cellular mRNA
(
8) in vivo. Synergy between poly(A) and cap has also been
observed in yeast cells and in yeast cell extracts (
12,
20),
with which a synergy value of 7 has been measured (
20). For
mammalian CHO cells, a synergy value of 27 (5 h postelectroporation)
can be calculated from Fig. 3 of reference
8 and
synergy value
of 16 and up to 15 has been found with carrot protoplasts
and
Drosophila embryo cells extract, respectively (
9,
18). The
value of 20 for synergy between poly(A) and cap measured
in ST-NSP3
cells (from Fig.
4) is comparable to the value
obtained with CHO
cells. A value of 35 for synergy between the cap and
the rotavirus
3' ends could be measured in vivo in the presence of
NSP3. This
is similar to the synergy between cap and poly(A)
and suggests
that NSP3 and PABP use the same mechanisms to
enhance
translation.
An effect of NSP3 on rotavirus mRNA stability,
similar to the effect of PABP (
4) on poly(A) mRNA,
could not be entirely
excluded. However, the role of NSP3 as
a viral translation factor
was confirmed by using recombinant proteins
and an in vitro translation
assay in which little mRNA
degradation occurs (
7,
8). The
use of NSP3 mutants
in this system allowed the roles of the different
domains identified on
NSP3 to be determined precisely. NSP3 mutants
disabled in their ability to bind either eIF4G or RNA were not
functional. These results strongly suggest that NSP3 acts as
a
physical link between viral mRNA and the cellular
translation
initiation complex. NSP3 brings the viral
mRNA in contact with
the translation initiation complex eIF4F
by interacting simultaneously
with eIF4G and the viral mRNA
3' end. In vitro, these interactions
enhance viral mRNA
translation independently of the 5' end of
the viral mRNA,
but in vivo, a strong synergy occurs between the
5' cap structure and
the 3' end. For poly(A) mRNA, this role is
fulfilled by PABP,
and the results described here demonstrate
that NSP3 is a
functional mimic of
PABP.
Effect of NSP3 on the translation of other mRNAs.
NSP3 has
been suspected to be involved in the shutoff of cellular mRNA
translation (22). Nevertheless, we managed to isolate a cell
line expressing a low level of NSP3. ST-NSP3 cells
present some differences from the parental ST cell line; notably, its growth is slightly slower (Fig. 1D). It is not known if this results from a general decrease in the translation of poly(A) mRNAs
induced by expression of NSP3 or from the specific decrease
in the translation of few cellular mRNAs. An inducible
expression system for NSP3 is needed to precisely answer this question.
A slight decrease of the F.Luc-poly(A) translation in ST-NSP3 cells
compared to ST cells was noted (Fig.
3). This observation
might
reflect an increased competition for poly(A) mRNA translation
in ST-NSP3 cells, induced by the interaction of
NSP3 with eIF4G.
Eviction of PABP from eIF4F (
23)
impairs the use of eIF4G by
PABP, and consequently less eIF4G is
available for the translation
of poly(A) mRNAs. However,
similar results were obtained when
F.Luc poly(A) was normalized to
protein concentration instead
of R.Luc-poly(A) translation. This
suggests that R.Luc poly(A)
mRNAs perform better than
F.Luc-poly(A) mRNAs in ST-NSP3
cells.
Using a deletion mutant of the whole RNA-binding domain, we
showed that NSP3 has the capacity to inhibit translation in vitro.
This inhibition is due to its interaction with eIF4G, as demonstrated
by the lack of effect of NSP3
4-290 and
NSP3
163-290 on translation. The exact
concentration of eIF4G in RRL is not
known and can only be
roughly estimated. The upper estimate of
the eIF4E concentration in RRL
is 400 nM (
30), and the concentration
of eIF4G is estimated
to be 1/10 the concentration of eIF4E (
1).
Therefore, the
concentration of eIF4G in RRL could be around 40
nM. The amount of
NSP3 used in our assay (200 nM) is thus sufficient
to recruit
all the eIF4G and inhibit translation. This observation
indicates that
non-poly(A)- and poly(A)-dependent translations
use the same
translation initiation pathway, which can be blocked
by NSP3.
However, we observed that a fraction of the mRNA was
still
translated in the presence of a vast excess of NSP3. This
residual translation could be due to a fraction of PABP that could
not
be removed from eIF4G or to an alternative translation initiation
pathway not sensitive to
NSP3.
| |
ACKNOWLEDGMENTS |
We are indebted to Katherine Kean (Pasteur Institute) for
critical and constructive reading of the manuscript. We acknowledge stimulating discussions with J. Cohen throughout this work. We acknowledge the skillful technical assistance of C. Jaegger.
M. Piron was supported by a fellowship from the French Ministère
de l'Education Nationale de la Recherche et de la Technologie. This
work was funded in part by "Programme de Recherches Fondamentales en
Microbiologie, Maladies Infectieuses et Parasitologie" of MENRT.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Virologie et Immunologie Moléculaires INRA, C.R.J.J., Domaine de
Vilvert, 78352 Jouy-en-Josas Cedex, France. Phone: 33-(0)1 34 65 26 11. Fax: 33-(0)1 34 65 26 21. E-mail:
poncet{at}biotec.jouy.inra.fr.
Present address: Laboratory of Infectious Diseases, National
Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892.
Present address: Medicina Interna-Hepatologia, Hospitals Generals
Vall d'Hebron, 08035 Barcelona, Spain.
| |
REFERENCES |
| 1.
|
Altmann, M.,
N. Schmitz,
C. Berset, and H. Trachsel.
1997.
A novel inhibitor of cap-dependent translation initiation in yeast: p20 competes with eIF4G for binding to eIF4E.
EMBO J.
16:1114-1121[CrossRef][Medline].
|
| 2.
|
Aponte, C.,
N. M. Mattion,
M. K. Estes,
A. Charpilienne, and J. Cohen.
1993.
Expression of two bovine rotavirus non-structural proteins (NSP2, NSP3) in the baculovirus system and production of monoclonal antibodies directed against the expressed proteins.
Arch. Virol.
133:85-95[CrossRef][Medline].
|
| 3.
|
Cohen, J.,
F. Lefevre,
M. K. Estes, and M. Bremont.
1984.
Cloning of bovine rotavirus (RF strain): nucleotide sequence of the gene coding for the major capsid protein.
Virology
138:1780-1782.
|
| 4.
|
Coller, J. M.,
N. K. Gray, and M. P. Wickens.
1998.
mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation.
Genes Dev.
12:3226-3235[Abstract/Free Full Text].
|
| 5.
|
Dever, T. E.
1999.
Translation initiation: adept at adapting.
Trends Biochem. Sci.
24:398-403[CrossRef][Medline].
|
| 6.
|
Estes, M. K., and J. Cohen.
1989.
Rotavirus gene structure and function.
Microbiol. Rev.
53:410-449[Abstract/Free Full Text].
|
| 7.
|
Furuichi, Y.,
A. LaFiandra, and A. J. Shatkin.
1977.
5'-Terminal structure and mRNA stability.
Nature
266:235-239[CrossRef][Medline].
|
| 8.
|
Gallie, D. R.
1991.
The cap and poly(A) tail function synergistically to regulate mRNA translational efficiency.
Genes Dev.
5:2108-2116[Abstract/Free Full Text].
|
| 9.
|
Gebauer, F.,
D. F. Corona,
T. Preiss,
P. B. Becker, and M. W. Hentze.
1999.
Translational control of dosage compensation in Drosophila by Sex-lethal: cooperative silencing via the 5' and 3' UTRs of msl-2 mRNA is independent of the poly(A) tail.
EMBO J.
18:6146-6154[CrossRef][Medline].
|
| 10.
|
Glass, R. I.,
J. S. Bresee,
U. Parashar,
M. Miller, and J. R. Gentsch.
1997.
Rotavirus vaccines at the threshold.
Nat. Med.
3:1324-1325[CrossRef][Medline].
|
| 11.
|
Gray, N. K., and M. Wickens.
1998.
Control of translation initiation in animals.
Annu. Rev. Cell. Dev. Biol.
14:399-458[CrossRef][Medline].
|
| 12.
|
Iizuka, N.,
L. Najita,
A. Franzusoff, and P. Sarnow.
1994.
Cap-dependent and cap-independent translation by internal initiation of mRNAs in cell extracts prepared from Saccharomyces cerevisiae.
Mol. Cell. Biol.
14:7322-7330[Abstract/Free Full Text].
|
| 13.
|
Imai, M.,
K. Akatani,
N. Ikegami, and Y. Furuichi.
1983.
Capped and conserved terminal structures in human rotavirus genome double-stranded RNA segments.
J. Virol.
47:125-136[Abstract/Free Full Text].
|
| 14.
|
Imataka, H.,
A. Gradi, and N. Sonenberg.
1998.
A newly identified N-terminal amino acid sequence of human eIF4G binds poly(A) binding protein and functions in poly(A)-dependent translation.
EMBO J.
17:7480-7489[CrossRef][Medline].
|
| 15.
|
Johansen, T. E.,
M. S. Scholler,
S. Tolstoy, and T. W. Schwartz.
1990.
Biosynthesis of peptide precursors and protease inhibitors using new constitutive and inducible eukaryotic expression vectors.
FEBS Lett.
267:289-294[CrossRef][Medline].
|
| 16.
|
Lane, D.,
P. Prentki, and M. Chandler.
1992.
Use of gel retardation to analyze protein-nucleic acid interactions.
Microbiol. Rev.
56:509-528[Abstract/Free Full Text].
|
| 17.
|
Mattion, N. M.,
J. Cohen, and M. K. Estes.
1994.
Rotavirus proteins, p. 169-249.
In
A. Kapikian (ed.), Viral infections of the gastrointestinal tract. Marcel Dekker, Inc., New York, N.Y.
|
| 18.
|
Niepel, M.,
J. Ling, and D. R. Gallie.
1999.
Secondary structure in the 5'-leader or 3'-untranslated region reduces protein yield but does not affect the functional interaction between the 5'-cap and the poly(A) tail.
FEBS Lett.
462:79-84[CrossRef][Medline].
|
| 19.
|
Nilsson, M.,
C. H. von Bonsdorff, and L. Svensson.
1993.
Biosynthesis and morphogenesis of group C rotavirus in swine testicular cells.
Arch. Virol.
133:21-37[CrossRef][Medline].
|
| 20.
|
Otero, L. J.,
M. P. Ashe, and A. B. Sachs.
1999.
The yeast poly(A)-binding protein Pab1p stimulates in vitro poly(A)-dependent and cap-dependent translation by distinct mechanisms.
EMBO J.
18:3153-3163[CrossRef][Medline].
|
| 21.
|
Pestova, T. V., and C. U. Hellen.
1999.
Ribosome recruitment and scanning: what's new?
Trends Biochem. Sci.
24:85-87[CrossRef][Medline].
|
| 22.
|
Piron, M.,
T. Delaunay,
J. Grosclaude, and D. Poncet.
1999.
Identification of the RNA-binding, dimerization and eIF4GI-binding domains of rotavirus NSP3.
J. Virol.
73:5411-5421[Abstract/Free Full Text].
|
| 23.
|
Piron, M.,
P. Vende,
J. Cohen, and D. Poncet.
1998.
Rotavirus RNA-binding protein NSP3 interacts with eIF4GI and evicts the poly(A) binding protein from eIF4F.
EMBO J.
17:5811-5821[CrossRef][Medline].
|
| 24.
|
Poncet, D.,
C. Aponte, and J. Cohen.
1993.
Rotavirus protein NSP3 (NS34) is bound to the 3' end consensus sequence of viral mRNAs in infected cells.
J. Virol.
67:3159-3165[Abstract/Free Full Text].
|
| 25.
|
Poncet, D.,
S. Laurent, and J. Cohen.
1994.
Four nucleotides are the minimal requirement for RNA recognition by rotavirus non-structural protein NSP3.
EMBO J.
13:4165-4173[Medline].
|
| 26.
|
Poncet, D.,
P. Lindenbaum,
R. L'Haridon, and J. Cohen.
1997.
In vivo and in vitro phosphorylation of rotavirus NSP5 correlates with its localization in viroplasms.
J. Virol.
71:34-41[Abstract].
|
| 27.
|
Preiss, T., and M. W. Hentze.
1998.
Dual function of the messenger RNA cap structure in poly(A)-tail-promoted translation in yeast.
Nature
392:516-520[CrossRef][Medline].
|
| 28.
|
Proweller, A., and S. Butler.
1997.
Ribosome concentration contributes to discrimination against poly(A) mRNA during translation initiation in Saccharomyces cerevisiae.
J. Biol. Chem.
272:6004-6010[Abstract/Free Full Text].
|
| 29.
|
Rao, C. D.,
M. Das,
P. Ilango,
R. Lalwani,
B. S. Rao, and K. Gowda.
1995.
Comparative nucleotide and amino acid sequence analysis of the sequence-specific RNA-binding rotavirus nonstructural protein NSP3.
Virology
207:327-333[CrossRef][Medline].
|
| 30.
|
Rau, M.,
T. Ohlmann,
S. J. Morley, and V. M. Pain.
1996.
A reevaluation of the cap-binding protein, eIF4E, as a rate-limiting factor for initiation of translation in reticulocyte lysate.
J. Biol. Chem.
271:8983-8990[Abstract/Free Full Text].
|
| 31.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1992.
Molecular cloning: a laboratory manual, 3rd ed.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 32.
|
Studier, F. W.
1991.
Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system.
J. Mol. Biol.
219:37-44[CrossRef][Medline].
|
| 33.
|
Tarun, S. Z., Jr., and A. B. Sachs.
1996.
Association of the yeast poly(A) tail binding protein with translation initiation factor eIF-4G.
EMBO J.
15:7168-7177[Medline].
|
| 34.
|
Wells, S. E.,
P. E. Hillner,
R. D. Vale, and A. B. Sachs.
1998.
Circularization of mRNA by eukaryotic translation initiation factors.
Mol. Cell
2:135-140[CrossRef][Medline].
|
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[Full Text]
-
Imbert, I., Dimitrova, M., Kien, F., Kieny, M. P., Schuster, C.
(2003). Hepatitis C virus IRES efficiency is unaffected by the genomic RNA 3'NTR even in the presence of viral structural or non-structural proteins. J. Gen. Virol.
84: 1549-1557
[Abstract]
[Full Text]
-
Mitzel, D. N., Weisend, C. M., White, M. W., Hardy, M. E.
(2003). Translational regulation of rotavirus gene expression. J. Gen. Virol.
84: 383-391
[Abstract]
[Full Text]
-
Sanchez, R., Marzluff, W. F.
(2002). The Stem-Loop Binding Protein Is Required for Efficient Translation of Histone mRNA In Vivo and In Vitro. Mol. Cell. Biol.
22: 7093-7104
[Abstract]
[Full Text]
-
Bushell, M., Sarnow, P.
(2002). Hijacking the translation apparatus by RNA viruses. JCB
158: 395-399
[Abstract]
[Full Text]
-
Leonard, S., Chisholm, J., Laliberte, J.-F., Sanfacon, H.
(2002). Interaction in vitro between the proteinase of Tomato ringspot virus (genus Nepovirus) and the eukaryotic translation initiation factor iso4E from Arabidopsis thaliana. J. Gen. Virol.
83: 2085-2089
[Abstract]
[Full Text]
-
Mossel, E. C., Ramig, R. F.
(2002). Rotavirus Genome Segment 7 (NSP3) Is a Determinant of Extraintestinal Spread in the Neonatal Mouse. J. Virol.
76: 6502-6509
[Abstract]
[Full Text]
-
Zeenko, V. V., Ryabova, L. A., Spirin, A. S., Rothnie, H. M., Hess, D., Browning, K. S., Hohn, T.
(2002). Eukaryotic Elongation Factor 1A Interacts with the Upstream Pseudoknot Domain in the 3' Untranslated Region of Tobacco Mosaic Virus RNA. J. Virol.
76: 5678-5691
[Abstract]
[Full Text]
-
Neeleman, L., Olsthoorn, R. C. L., Linthorst, H. J. M., Bol, J. F.
(2001). Translation of a nonpolyadenylated viral RNA is enhanced by binding of viral coat protein or polyadenylation of the RNA. Proc. Natl. Acad. Sci. USA
10.1073/pnas.251542798v1
[Abstract]
[Full Text]
-
Patton, J. T., Taraporewala, Z., Chen, D., Chizhikov, V., Jones, M., Elhelu, A., Collins, M., Kearney, K., Wagner, M., Hoshino, Y., Gouvea, V.
(2001). Effect of Intragenic Rearrangement and Changes in the 3' Consensus Sequence on NSP1 Expression and Rotavirus Replication. J. Virol.
75: 2076-2086
[Abstract]
[Full Text]
-
Thole, V., Garcia, M.-L., van Rossum, C. M. A., Neeleman, L., Brederode, F. T., Linthorst, H. J. M., Bol, J. F.
(2001). RNAs 1 and 2 of Alfalfa mosaic virus, expressed in transgenic plants, start to replicate only after infection of the plants with RNA 3. J. Gen. Virol.
82: 25-28
[Abstract]
[Full Text]
-
KAHVEJIAN, A., ROY, G., SONENBERG, N.
(2001). The mRNA Closed-loop Model: The Function of PABP and PABP-interacting Proteins in mRNA Translation. Cold Spring Harb Symp Quant Biol
66: 293-300
[Abstract]
-
Schuck, P., Taraporewala, Z., McPhie, P., Patton, J. T.
(2001). Rotavirus Nonstructural Protein NSP2 Self-assembles into Octamers That Undergo Ligand-induced Conformational Changes. J. Biol. Chem.
276: 9679-9687
[Abstract]
[Full Text]
-
Neeleman, L., Olsthoorn, R. C. L., Linthorst, H. J. M., Bol, J. F.
(2001). Translation of a nonpolyadenylated viral RNA is enhanced by binding of viral coat protein or polyadenylation of the RNA. Proc. Natl. Acad. Sci. USA
98: 14286-14291
[Abstract]
[Full Text]
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Abstract
Introduction
