Previous Article | Next Article 
Journal of Virology, July 2001, p. 6517-6526, Vol. 75, No. 14
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.14.6517-6526.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The Reovirus S4 Gene 3' Nontranslated Region
Contains a Translational Operator Sequence
Michelle
Mochow-Grundy1,2 and
Terence S.
Dermody1,2,3,*
Departments of Microbiology and
Immunology1 and
Pediatrics3 and Elizabeth B. Lamb Center for Pediatric Research,2 Vanderbilt
University School of Medicine, Nashville, Tennessee 37232
Received 21 June 2000/Accepted 25 April 2001
 |
ABSTRACT |
Reovirus mRNAs are efficiently translated within host cells despite
the absence of 3' polyadenylated tails. The 3' nontranslated regions
(3'NTRs) of reovirus mRNAs contain sequences that exhibit a high degree
of gene-segment-specific conservation. To determine whether the 3'NTRs
of reovirus mRNAs serve to facilitate efficient translation of viral
transcripts, we used T7 RNA polymerase to express constructs engineered
with full-length S4 gene cDNA or truncation mutants lacking sequences
in the 3'NTR. Full-length and truncated s4 mRNAs were translated using
rabbit reticulocyte lysates, and translation product 
3 was
quantitated by phosphorimager analysis. In comparison to full-length s4
mRNA, translation of the s4 mRNA lacking the 3'NTR resulted in a 20 to
50% decrease in 3 produced. Addition to translation reactions of an
RNA oligonucleotide corresponding to the S4 3'NTR significantly
enhanced translation of full-length s4 mRNA but had no effect on s4
mRNA lacking 3'NTR sequences. Translation of s4 mRNAs with smaller
deletions within the 3'NTR identified a discrete region capable of
translational enhancement and a second region capable of translational
repression. Differences in translational efficiency of full-length and
deletion-mutant mRNAs were independent of RNA stability. Protein
complexes in reticulocyte lysates that specifically interact with the
S4 3'NTR were identified by RNA mobility shift assays. RNA
oligonucleotides lacking either enhancer or repressor sequences did not
efficiently compete the binding of these complexes to full-length
3'NTR. These results indicate that the reovirus S4 gene 3'NTR contains
a translational operator sequence that serves to regulate translational
efficiency of the s4 mRNA. Moreover, these findings suggest that
cellular proteins interact with reovirus 3'NTR sequences to regulate
translation of the nonpolyadenylated reovirus mRNAs.
| |
INTRODUCTION |
All viruses must compete for
cellular factors to efficiently express and replicate their genomes.
Transcription and translation of the mammalian reovirus genome occurs
within the cytoplasm of infected cells. The viral transcriptional
machinery is contained within the viral core (5, 32);
however, translation of viral mRNAs requires host cell factors and
occurs in an environment replete with translationally functional
cellular transcripts. Reovirus mRNAs lack polyadenylated tails, which
serve to promote translation of many cellular mRNAs (10, 24,
30). Therefore, reovirus mRNAs must use alternative strategies
to usurp the translational machinery. Viral sequences that enable
reovirus mRNAs to achieve translational competence in a milieu of
cellular mRNAs are not well understood.
Mammalian reoviruses are nonenveloped, icosahedral viruses formed from
two concentric protein shells, termed outer capsid and core (reviewed
in reference 26). The reovirus genome consists of 10 discrete segments of double-stranded RNA. After removal of the viral
outer capsid during entry of the virus into cells, the core becomes
enzymatically active and catalyzes transcription of 10 species of viral
mRNA (3, 20, 33). Capped, nonpolyadenylated messages are
extruded from the viral core into the cytoplasm, where they are
efficiently translated despite an abundance of polyadenylated
cellular mRNAs. The s4 mRNA, which encodes the 3 protein
(22, 25), is the most efficiently translated viral transcript in both in vitro translation reactions and reovirus-infected cells (1, 21, 42). Specific nucleotides at the 
3 and +4 positions relative to the AUG initiation codon contribute to
translational efficiency of some mRNAs (19). However, for
the reovirus s4 mRNA, nucleotide polymorphism at these positions does
not significantly influence the amount of translation product produced
(23), indicating that this region is not the primary
determinant of translational efficiency. Sequence elements that
influence translational efficiency of the s4 mRNA have not been defined.
Sequences in the 3' nontranslated regions (3'NTRs) of many viral and
cellular transcripts have been shown to regulate translational efficiency. The poly(A) tail augments translation by enhancing mRNA
stability and by facilitating ribosome reinitiation through indirect
interactions with the 5'NTR via poly(A)-binding protein and translation
factor elF-4G (reviewed in reference 29). Specific 3'NTR
sequences upstream of the poly(A) tail that influence stability of
mRNAs or interact with viral or cellular proteins to either enhance or
repress protein synthesis also have been identified. In addition, the
3'NTRs of nonpolyadenylated transcripts also contain sequences that
influence translation outcome for this class of mRNAs. For example,
deletion of the 3'NTR of the nonpolyadenylated coat protein mRNA of
alfalfa mosaic virus decreases the efficiency with which the coat
protein transcript is translated when translation factors are limiting
(14). This observation suggests that the 3'NTRs of
nonpolyadenylated viral mRNAs facilitate efficient translation when
competing with polyadenylated mRNAs for the translational machinery.
The 3'NTRs of reovirus gene segments range from 35 to 80 nucleotides in
length (9, 15). Analysis of sequence diversity of the
reovirus S-class gene segments indicates that sequences at the 5' and
3' termini are highly conserved (6, 8, 13, 18). Conserved
sequences at the termini of each of these gene segments are not limited
to the NTRs and extend into the open reading frames. These observations
suggest that there is selection pressure to maintain conservation of
these sequences at the nucleotide level. Similar levels of conservation
have been observed at the gene-segment termini of other double-stranded
RNA viruses, and these conserved sequences have been implicated in
regulation of translation and genome replication (27, 28, 38,
40).
To test the hypothesis that conserved sequences in the 3'NTRs
of reovirus mRNAs influence translation, we generated full-length and 3'NTR-truncated (3'
) s4 mRNAs and introduced these mRNAs into
translation reactions using rabbit reticulocyte lysates. Our results
indicate that discrete sequences within the S4 3'NTR serve to either
enhance or repress translation of the s4 transcript. Additionally, we
demonstrate that an RNA oligonucleotide corresponding to the S4 gene
3'NTR forms stable complexes with proteins contained in reticulocyte
lysates. These findings indicate that the reovirus S4 3'NTR contains
sequences that regulate translational efficiency of the s4 mRNA and
suggest that cellular components interact with these regulatory
sequences to influence translation.
| |
MATERIALS AND METHODS |
Cells and viruses.
Mouse L929 cells were grown in either
suspension or monolayer cultures in Joklik's modified Eagle's minimal
essential medium (Irvine Scientific, Santa Ana, Calif.) that was
supplemented to contain 5% fetal calf serum (Intergen Co., Purchase,
N.Y.), 2 mM L-glutamine, 100 U of penicillin G per ml, 100 µg of streptomycin per ml, and 250 ng of amphotericin B per ml
(Irvine Scientific). Reovirus strain type 3 Dearing (T3D) is a
laboratory stock. A second-passage L-cell lysate stock of
twice-plaque-purified T3D was used for the experiments described
(36).
Generation of reovirus mRNA expression constructs.
A cDNA
corresponding to the reovirus T3D S4 gene was amplified by reverse
transcription (RT) and PCR using avian myeloblastosis virus (AMV)
reverse transcriptase and Pfu DNA polymerase (Promega Corp.,
Madison, Wis.) with primers a and b (Table
1). The S4 gene cDNA was ligated into
plasmid pCR2.1 (Invitrogen, Carlsbad, Calif.) and then transferred to
T7 RNA polymerase expression vector pGEM3z/f± (Promega) following
digestion with EcoRI and PstI (New England
Biolabs, Beverly, Mass.). Sequences between the T7 RNA polymerase
promoter and the 5' nucleotide of the S4 gene cDNA were deleted by PCR
mutagenesis (Exsite; Stratagene, La Jolla, Calif.) with
5'-phosphorylated primers c and d (Table 1) to generate plasmid
construct pS4.
Deletions within the S4 gene 3'NTR were generated by PCR mutagenesis
using pS4 and the phosphorylated primer sets shown in
Table
1:
pS4-3'
(e and f), pS4-
1134-1150 (g and h), pS4-
1140-1159
(i
and j), pS4-
1162-1181 (k and l), pS4-
1169-1189 (m and n),
and
pS4-
1181-1195 (o and p). The pS4-3'NTR construct was generated
by
PCR mutagenesis using pS4 and phosphorylated primers d and
q.
Mutagenesis was performed according to the manufacturer's instructions
as follows. Sixty micrograms of plasmid template per ml, 20 mM
Tris-HCl
(pH 8.8), 10 mM KCl, 10 mM
(NH
4)
2SO
4, 2 mM MgSO
4,
0.1%
Triton X-100, 100 µg of bovine serum albumin per ml, and 5 U of
a
Pfu/
Taq polymerase blend (Pfu Turbo;
Stratagene) were combined
in a volume of 25 µl. Reaction temperatures
were used as recommended
except that annealing temperatures of 52°C
were used during the
initial cycle and increased to 58°C in the
subsequent eight cycles.
The 5' and 3' termini of the PCR products were
ligated, and the
resulting plasmids were used to transform XL-1 Blue
supercompetent
Escherichia coli (Epicurian coli;
Stratagene). Plasmids were isolated
by Wizard Mini-prep column
purification (Promega) and sequenced
using T4 DNA polymerase and
dideoxy-chain termination (Sequenase;
U.S. Biochemical, Cleveland,
Ohio).
A cDNA corresponding to the reovirus T3D S1 gene was amplified by RT
and PCR using AMV reverse transcriptase and
Pfu/
Taq DNA
polymerase blend with primers aa and
bb (Table
2). The S1 gene
cDNA was
ligated into plasmid pCR2.1 and then transferred to pGEM3z/f±
following digestion with
EcoRI and
PstI.
Sequences between the
T7 RNA polymerase promoter and the 5' nucleotide
of the S1 gene
cDNA were deleted using PCR mutagenesis with
5'-phosphorylated
primers cc and dd (Table
2) to generate plasmid
construct pS1.
The S1 gene 3'NTR was deleted by PCR mutagenesis using
pS1 and
the phosphorylated primers ee and ff (Table
2). S1 gene
sequences
were confirmed prior to use.
Generation of reovirus mRNA transcripts.
Plasmids were
linearized by digestion with 1 U of PstI per µl, and 3'
overhang sequences were removed by incubation with 1 to 2 U of T4 DNA
polymerase (Promega) per µl at 11°C for 20 min in the presence of 1 mM Tris-HCl (pH 7.9), 1 mM MgCl2, 5 mM NaCl, 0.1 mM
dithiothreitol (DTT), 10 µM deoxynucleoside triphosphate, and 50 µg
of bovine serum albumin per in a final volume of 20 µl. Linearized
plasmids were gel purified and resuspended in nuclease-free water.
In vitro transcription of capped reovirus mRNAs was performed using
linearized plasmid as template and the mMessage mMachine
T7 RNA
polymerase transcription system (Ambion, Austin, Tex.).
Transcription
reactions were incubated with or without 1 µl of
[
-
32P]UTP (3,000 Ci per mmol; Dupont NEN, Wilmington,
Del.) at 37°C
for 2 h followed by digestion of input plasmid
with 250 U of DNase
1 per ml at 37°C for 15 min. Prior to addition of
T7 RNA polymerase,
linearized plasmid and transcription reagents were
incubated with
RNase inhibitor RNAsecure (Ambion) at 60°C for 20 min.
RNA oligonucleotides
were similarly transcribed from plasmid linearized
with
PstI using
the megaShortscript T7 RNA polymerase
transcription system
(Ambion).
RNA transcripts and oligonucleotides were purified from transcription
reactions and rabbit reticulocyte lysates using Tri-reagent
(Molecular
Research, Cincinnati, Ohio). Transcription reactions
were mixed with 3 volumes of Tri-reagent and incubated at room
temperature for 5 min. The
mixture was vortexed vigorously with
1.5 volumes of chloroform followed
by incubation at room temperature
for 15 min. Following centrifugation
at 12,000 ×
g, RNA was precipitated
in 2.5 volumes of
isopropanol and 0.75 volumes of NC buffer (0.8
M sodium citrate, 1.2 M
NaCl). RNA concentration was determined
using optical density at 260 nm
and confirmed by electrophoresis
in 5% polyacrylamide-urea gels
followed by ethidium bromide staining
and densitometry (Eagle Eye
software;
Stratagene).
Sequencing 5' and 3' termini of reovirus RNAs.
Sequences of
s4 and s1 mRNA 5' termini were determined by dideoxy-chain termination
using reverse transcriptase and primers 5'-ACATACCATATCTGG-3'
and 5'-CTCAAGAGCGATGATTCG-3', respectively (6). Sequences of 3' termini were determined by rapid
amplification of cDNA ends (RACE)-PCR. Adenosine bases were appended to
the 3' terminus of transcribed RNA by incubation with dATP and terminal transferase (Roche Molecular Biochemicals, Indianapolis, Ind.). RNA was
purified by Tri-reagent and subjected to RT using AMV reverse
transcriptase and oligo(dT) primer
5'-GACCACGCGTATCGATGTCGAC(T)16N-3'. The
resultant cDNA was amplified in a primary round of PCR using Pfu DNA polymerase with oligo(dT) primer and either
S4-specific primer 5'-GCTATTTTTGCCTCTTCC-3' or S1-specific
primer 5'-GCTATTGGTCGGATGG-3'. A secondary round of PCR
amplification was performed using Taq DNA polymerase with a
primer corresponding to a sequence 5' to the oligo(dT) primer,
5'-GACCACGCGTATCGATGTCGAC-3', and either S4-specific primer
5'-CGTCGTTTGCATGCATTG-3' or S1-specific primer 5'-GTCAGTGATGCTCAACTTGCAATCTCC-3'. PCR product from the
secondary amplification was ligated into pCR2.1, and sequences were
determined by dideoxy-chain termination.
Translation of reovirus mRNAs in rabbit reticulocyte
lysates.
Full-length and mutant reovirus mRNAs were translated by
incubation with rabbit reticulocyte lysates (Promega) at 30°C for various intervals. Translation reactions were supplemented with 2 to 3 U of RNasin RNase inhibitor (Promega) per µl, 2.25 mM DTT, 0.05 M
KCl, 1 mM magnesium acetate 0.2 mM amino acid mixture minus methionine,
and 0.5 mCi of Easy Tag Express -[35S] Protein Labeling
Mix (Dupont NEN) per ml at 1,175 Ci per mmol in a final volume of 3.12 µl. Reactions were incubated in the presence or absence of various
concentrations of globin mRNA (Gibco BRL, Rockville, Md.).
SDS-PAGE and phosphorimager analysis of translation
products.
Equal volumes of translation reactions were added to 2×
sample buffer (125 mM Tris, 10% 
-mercaptoethanol, 4% sodium
dodecyl sulfate [SDS], 0.02% bromophenol blue). Samples were boiled
for 5 min, loaded into lanes of 12% polyacrylamide gels, and subjected to polyacrylamide gel electrophoresis (PAGE) at 200-V constant voltage
for 1 h (2). Following electrophoresis, gels were
dried and exposed either to film (Eastman Kodak Company, Rochester, N.Y.) or an imaging plate (Fuji Medical Systems, Inc., Stamford, Conn.)
for phosphorimager analysis. Band intensity was quantitated by
determining photostimulus luminescence (PSL) units, using a Fuji2000
phosphorimager (Fuji Medical Systems, Inc.).
Statistical analysis.
Differences in translational
efficiency of full-length and s4 3' mRNAs in the presence or absence
of S4 3'NTR RNA oligonucleotides were analyzed by calculating the area
under the curve and performing an equal-variance t test. The
panel of mutants was analyzed statistically using a single-factor
analysis of variance followed by a pairwise comparison of individual
mutants with the full-length s4 mRNA, using a one-tailed t
test assuming unequal variance. Statistical analysis was performed
using Microsoft Excel 95 (Microsoft, Redmond, Wash.).
RNA gel shift analysis of S4 3'NTR-binding complexes.
Radiolabeled probes for electrophoretic mobility shift assays (EMSAs)
were generated using in vitro transcription with the pS4-3'NTR
construct as template and T7 RNA polymerase in the presence of 0.0011 mM [-32P]UTP (3,000 Ci per mmol) with 7.5 mM ATP, CTP,
and GTP and 0.0275 mM UTP. Reactions were incubated at 37°C for
1 h, followed by addition of 2 µl of T7 RNA polymerase enzyme
mix (MegaShortscript; Ambion). Reactions were incubated at 37°C
for an additional 1 h and then terminated by plasmid digestion with
DNase I. The 3'NTR probe was purified using Tri-reagent as described
above and analyzed for purity by electrophoresis in a 5%
polyacrylamide-urea gel and autoradiography.
RNA EMSAs were performed according to previously described techniques
(
4), with minor modifications. Rabbit reticulocyte
lysate
at a concentration of 12.5 mg of protein per ml was incubated
in the
presence or absence of increasing concentrations of unlabeled
competitor RNA in 20 mM HEPES-5% glycerol-1 mM EDTA-0.1 mg of
yeast
tRNA per ml-60 mM KCl-1 mM DTT in a volume of 20 µl. Either
5S rRNA
(Gibco BRL) or poly(A) RNA (400 to 600 bp) (Sigma) was
added to the
lysates as nonspecific competitor RNA. Following
incubation on ice for
20 min, radiolabeled probe (10
5 cpm, 1 ng of probe) was
added to the lysates and incubated at
room temperature for 20 min.
Competitor probes were added in molar
excess to the radiolabeled probe.
Reactions were incubated in
the presence of 10 mg of heparin per ml at
room temperature for
10 min, followed by the addition of gel shift
loading buffer (0.25%
bromophenol blue, 0.25% xylene cyanol, 15%
Ficoll 400). Protein-RNA
complexes were electrophoresed in 30% TBE (89 mM Tris, 89 mM boric
acid, 2 mM EDTA)-acrylamide (29:1) nondenaturing
gels at 180-V
constant voltage for 2 h. Following electrophoresis,
gels were
dried and exposed to
film.
| |
RESULTS |
Deletion of the reovirus S4 gene 3'NTR reduces
translational efficiency of the s4 mRNA.
To determine whether the
3'NTR is required for efficient translation of reovirus mRNAs, we
generated a cDNA corresponding to the reovirus S4 gene, which encodes
the 3 protein (12, 22, 25). The S4 gene cDNA was
inserted into a plasmid containing a T7 RNA polymerase promoter such
that transcription initiated with a 5'-terminal guanosine and
terminated with a 3'-terminal cytosine, which correspond to the
terminal nucleotides of the S4 gene (12) (Fig.
1A). Plasmid constructs containing the S4 gene cDNA were transcribed in the presence of m7GTP to
generate capped transcripts with 5'- and 3'-terminal sequences identical to native reovirus s4 mRNAs. To confirm authenticity of the
in vitro-generated s4 transcripts, nucleotide sequences at the 5'
termini of these transcripts were determined by direct RNA sequence
analysis, and sequences at the 3' termini were determined following
RACE-PCR. Analysis of these sequences demonstrated that the 5' and 3'
termini of in vitro-generated s4 mRNAs are identical to native s4 mRNAs
(data not shown).
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 1.
Diagram of templates used for transcription of
full-length and deletion-mutant s4 mRNAs. (A) The relevant region of
plasmid pGEM3z/f± used for cloning a full-length cDNA corresponding to
the S4 gene of reovirus strain T3D. Indicated are a T7 RNA polymerase
promoter (T7), which was cloned adjacent to the 5'-terminal
guanosine of the S4 gene cDNA, the 3 open reading frame (ORF),
and a PstI site, which was engineered adjacent to the 3'
terminus of the S4 gene cDNA. Digestion of plasmid constructs with
PstI followed by incubation with T4 DNA polymerase resulted
in templates for transcription that initiate with the 5' guanosine and
terminate with the 3' cytosine of the S4 gene. Capped s4 mRNAs and s4
3' mRNAs were transcribed in vitro and used in
translation studies. Nucleotide lengths are indicated on the right. The
s4 3' mRNA terminates with UAAC, corresponding to the
stop codon of the transcript and the 3'-terminal cytosine of the
full-length S4 gene. (B) Schematic of S4 gene 3'NTR deletion constructs
generated from cDNA clones by PCR mutagenesis. The viral sequences
deleted in each of the mutant S4 gene cDNAs are indicated on the left;
the length of each mRNA transcript is indicated on the right.
|
|
We examined whether sequences in the 3'NTR influence the translation of
the reovirus s4 mRNA under conditions in which components
of
translation were either in excess or limiting. Full-length
s4 mRNAs or
s4 mRNAs lacking the 3'NTR (s4 3'
) at a concentration
of 10 nM were
incubated in rabbit reticulocyte lysates in the
presence of increasing
concentrations of polyadenylated globin
mRNA from 0 to 100 nM.
Translation product
3 was resolved by
SDS-PAGE and quantitated by
phosphorimager analysis (Fig.
2).
In
reticulocyte lysates lacking globin mRNA, full-length s4 and
s4 3'
mRNAs were translated equivalently. However, as the concentration
of
globin mRNA added to the translation reactions was increased,
3
protein produced during translation of the full-length s4 mRNA
was
greater than that produced during translation of s4-3'
mRNA.
We
found that at concentrations of globin mRNA greater than 1
nM,
full-length s4 mRNA was translated 20% more efficiently than
s4-3'
mRNA (
P < 0.05). These findings suggest that the S4
3'NTR
confers a translational advantage to the reovirus s4 mRNA under
conditions in which components of the translational machinery
are
limiting.
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 2.
Translational efficiency of full-length s4 and s4 3'
mRNAs. Reovirus s4 and s4 3' mRNAs at a concentration of 10 nM were
added to rabbit reticulocyte lysates containing increasing
concentrations of globin mRNA from 0 to 100 nM. Following incubation at
30°C for 15 min, translation products were resolved by SDS-PAGE (A)
and quantitated by phosphorimager analysis (B). Translation units are
defined as the PSL units of translation product at the indicated
concentration of globin mRNA divided by the PSL units for the s4 mRNA
translation product in the absence of globin mRNA. The results are
expressed as the mean translation units (×100) determined from three
independent experiments performed in triplicate. Error bars indicate
standard errors of the means.
|
|
To determine whether the kinetics of translation of full-length s4 mRNA
differ from those of s4 3'
mRNA, transcripts at a
concentration of
40 nM were incubated in reticulocyte lysates
in the presence of 10 nM
globin mRNA for intervals from 5 to 60
min (Fig.
3). After 5 min of incubation, no protein
was detected
by autoradiography, and very little was detected by
phosphorimager
analysis. However, after 10 min of incubation,
significantly more
3 was produced following translation of
full-length s4 mRNA than
following translation of s4 3'
mRNA. The
magnitude of this difference
in translation efficiency remained
constant at each interval tested
and was statistically significant
(
P < 0.05). Translation products
of both transcripts
approached a plateau by 30 min of incubation,
with little change in
steady-state levels of
3 produced after
that time. This result
indicates that full-length s4 mRNAs are
translated at an increased rate
relative to s4 mRNAs lacking the
3'NTR, resulting in synthesis of
significantly greater protein
product.
View larger version (20K):
[in this window]
[in a new window]
|
FIG. 3.
Translation kinetics of full-length s4 and s4 3'
mRNAs. Reovirus s4 and s4 3' mRNAs at a concentration of 40 nM were
added to rabbit reticulocyte lysates containing 10 nM globin mRNA.
Reactions were incubated at 30°C for the times shown followed by
SDS-PAGE (A) and phosphorimager analysis (B). Translation units are
defined as the PSL units of translation product at the indicated time
divided by the PSL units for the s4 mRNA translation product at 60 min.
The results are expressed as the mean translation units (×100)
determined from two independent experiments performed in triplicate.
Error bars indicate standard errors of the means.
|
|
The S4 gene 3'NTR alters translational efficiency when provided in
trans.
To test whether translational enhancement of
the s4 mRNA provided by the 3'NTR could be altered by supplying S4
3'NTR sequences in trans, we incubated full-length s4 mRNA
and s4 3' mRNA in reticulocyte lysates containing RNA
oligonucleotides corresponding to the 3'-terminal 100 nucleotides of
the S4 gene (Fig. 4). In an analysis of
S4 gene nucleotide sequences of 16 reovirus strains, sequence
variability within this region is restricted to only six nucleotides
(18). We observed no significant change in basal levels of
translation of s4 3' mRNA in the presence of increasing concentrations of exogenous S4 3'NTR RNA oligonucleotide. In contrast, translation of full-length s4 mRNA was significantly enhanced in the
presence of the S4 3'NTR RNA oligonucleotide. At a 100-fold molar
excess of 3'NTR oligonucleotide to s4 mRNA, translation of the
full-length s4 mRNA was increased by approximately 60% in comparison
to mRNA translated in the absence of exogenous S4 3'NTR
oligonucleotide. This finding indicates that when provided in
trans, the S4 gene 3'NTR blocks inhibition of translation of the full-length s4 mRNA, which suggests that the S4 3'NTR contains sequences that confer translational repression.
View larger version (21K):
[in this window]
[in a new window]
|
FIG. 4.
Translation of full-length s4 and s4 3' mRNAs in the
presence of an RNA oligonucleotide corresponding to the S4 3'NTR
supplied in trans. Reovirus s4 and s4 3' mRNAs at a
concentration of 10 nM were incubated at 30°C for 15 min in rabbit
reticulocyte lysates containing 10 nM globin mRNA. An RNA
oligonucleotide corresponding to the 3'-terminal 100 nucleotides of the
s4 mRNA was added at the concentrations shown prior to incubation.
Translation products were resolved by SDS-PAGE (A) and quantitated by
phosphorimager analysis (B). Translation units are defined as the PSL
units of translation product at the indicated S4 3'NTR oligonucleotide
concentration divided by the PSL units of either the s4 or s4 3'
mRNA translation product in the absence of exogenous 3'NTR. The results
are expressed as the mean translation units (×100) determined from a
representative experiment performed in triplicate. Error bars indicate
standard deviations of the mean.
|
|
To determine the specificity of the S4 3'NTR RNA oligonucleotide on
translational efficiency, we generated plasmid constructs
to facilitate
transcription of full-length s1 and 3'
s1 mRNAs.
Sequence analysis
confirmed that the 5' and 3' termini of in vitro-generated
s1 mRNAs are
identical to those of native s1 mRNAs (data not shown).
Reovirus s1
mRNAs were translated in reticulocyte lysates in the
presence of 10 nM
globin mRNA and increasing concentrations of
the S4 3'NTR RNA
oligonucleotide. We found no effect on translation
of either the s1 or
s1-3'
mRNA with increasing concentrations
of oligonucleotide (data
not shown). In addition, we observed
no translational enhancement when
nonviral, polyadenylated globin
mRNA was incubated in reticulocyte
lysates containing the S4 3'NTR
RNA oligonucleotide (data not shown).
These results suggest that
the translational enhancement provided by
addition of the S4 3'NTR
to translation reactions is specific to the s4
mRNA.
Discrete regions of the S4 gene 3'NTR differentially influence
translational efficiency.
Our data show that deletion of the 3'NTR
reduces translational efficiency of the reovirus s4 mRNA, yet addition
of these sequences in trans augments translation of the same
transcript. These results suggest that the S4 3'NTR has independent
sequence elements that differentially influence translation of the s4
mRNA. We examined whether such functions could be genetically dissected
by using s4 mRNAs lacking small regions of sequence within the 3'NTR
(Fig. 1B). Translation of full-length s4 and s4 3' mRNAs was
compared to five additional s4 mRNAs with 3'NTR deletions ranging from 15 to 21 nucleotides in length (Fig. 5).
Deletion of nucleotides 1140 to 1159 reduced translational efficiency
approximately 40% relative to full-length s4 mRNA (P < 0.05), which suggests that this sequence region contains an
enhancer of translation. In contrast, deletion of nucleotides 1162 to
1181 enhanced translational efficiency greater than 50% relative to
full-length s4 mRNA (P < 0.05). Deletion of
nucleotides 1169 to 1189 also enhanced translational efficiency, but
the effect was more modest, approximately 10 to 15% (P < 0.05). These findings demonstrate that sequences encompassing
nucleotides 1162 to 1181 contain a repressor of translation.
View larger version (51K):
[in this window]
[in a new window]
|
FIG. 5.
Effect of deletions within the S4 3'NTR on translation
of the s4 mRNA. Full-length and deletion-mutant s4 mRNAs at a
concentration of 10 nM were incubated in rabbit reticulocyte lysates
containing 10 nM globin mRNA at 30°C for 15 min. Translation products
were resolved by SDS-PAGE (A) and quantitated by phosphorimager
analysis (B). Translation units are defined as the PSL units of
translation product of the indicated s4 mRNA divided by the PSL units
of translation product of full-length s4 mRNA. The results are
expressed as the mean translation units (×100) determined from three
independent experiments performed in triplicate. Error bars indicate
standard errors of the means.
|
|
To test whether deletion of the S4 3'NTR sequences that mediate
enhancer and repressor effects alters the enhancement activity
observed
when an oligonucleotide corresponding to the S4 3'NTR
is added in
trans, we generated RNA oligonucleotides corresponding
to
the 3'NTRs of s4 mRNA deletion mutants
1140-1159 and
1162-1181
and determined their effects on translational outcome. Full-length
s4
mRNA was incubated in reticulocyte lysates with oligonucleotides
corresponding to the full-length S4 3'NTR, the
1140-1159 3'NTR,
or
the
1162-1181 3'NTR, and translation product
3 was resolved
by
SDS-PAGE and quantitated by phosphorimager analysis (Fig.
6).
As shown previously, translation of
s4 mRNA in reticulocyte lysates
in the presence of a 100-fold molar
excess of the S4 3'NTR RNA
oligonucleotide resulted in a 50% increase
in
3 protein produced
in comparison to translation of s4 mRNA alone
(
P < 0.05). When
s4 transcripts were translated in
reticulocyte lysates in the
presence of increasing concentrations of
the RNA oligonucleotide
corresponding to the
1140-1159 3'NTR,
production of
3 was similarly
enhanced in the presence of both 10- and 100-fold molar excess
oligonucleotide, reaching an approximately
50% increase in comparison
to
3 produced from s4 mRNA alone
(
P < 0.05). However, when s4
mRNA was translated in
reticulocyte lysates in the presence of
an RNA oligonucleotide
corresponding to the
1162-1181 3'NTR,
no significant enhancement of
3 synthesis was observed. This
finding indicates that an S4 3'NTR
oligonucleotide with a deletion
of nucleotides 1162 to 1181 does not
enhance translation of the
s4 mRNA in
trans, which suggests
that nucleotides 1162 to 1181
of the 3'NTR titrate a translational
repressor activity.
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 6.
Translation of full-length s4 mRNA in the presence of
full-length and internally truncated S4 3'NTR RNA oligonucleotides
supplied in trans. Reovirus s4 mRNA at a concentration of 1 nM was incubated at 30°C for 15 min in rabbit reticulocyte lysates
containing 1 nM globin mRNA. Increasing concentrations (×1 nM) of RNA
oligonucleotides corresponding to the 3'-terminal 100 nucleotides of
the s4 mRNA (S4) or mutant RNA oligonucleotides corresponding to the
3'-terminal 100 nucleotides of mutant s4 mRNAs 1140-1159 and
1162-1181 were added prior to incubation. Translation products were
resolved by SDS-PAGE (A) and quantitated using phosphorimager analysis
(B). Translation units are defined as the PSL units of translation
product of s4 mRNA in the presence of 3'NTR RNA oligonucleotide divided
by the PSL units of translation product in the absence of 3'NTR RNA
oligonucleotide. The results are expressed as the mean translation
units (×100) determined from a representative experiment performed in
triplicate. Error bars indicate standard deviations of the mean. C,
control without oligonucleotide.
|
|
Deletion of the reovirus S4 gene 3'NTR does not alter s4 mRNA
stability.
To determine whether deletion of the 3'NTR affects
stability of the s4 mRNA, full-length s4 and s4 3' mRNAs were
quantitated following incubation in reticulocyte lysates. Radiolabeled
transcripts were incubated in reticulocyte lysates from 5 to 60 min,
isolated by Tri-reagent extraction, and subjected to electrophoresis in denaturing polyacrylamide gels. The relative amounts of mRNA present were determined by phosphorimager analysis (Fig.
7 and data not shown). Full-length s4 and
s4 3' mRNAs were equivalently stable in reticulocyte lysates, with
greater than 60% of input RNA lost by 30 min of incubation and greater
than 80% of input mRNA lost by 60 min of incubation. The kinetics of
degradation of the full-length s4 mRNA and the s4 3' mRNA were
identical (data not shown). Therefore, differences in the translational
efficiency of these mRNAs are not attributable to differences in
relative stability.
View larger version (28K):
[in this window]
[in a new window]
|
FIG. 7.
Effect of deletions within the s4 3'NTR on RNA
stability. Radiolabeled full-length s4 mRNA (s4), s4 3' mRNA (s4
3'), and 1140-1159 and 1162-1181 mRNAs were incubated in
rabbit reticulocyte lysates and purified at the times shown. Purified
transcripts were analyzed by electrophoresis in denaturing
polyacrylamide gels and quantitated by phosphorimager analysis. RNA
units are defined as the PSL units of mRNA at the indicated time
divided by the PSL units of the homologous s4 mRNA at time zero. The
results are expressed as the mean RNA units (×100) determined from two
independent experiments performed in triplicate. Error bars indicate
standard errors of the means.
|
|
Although full-length and 3'
s4 mRNAs showed identical degradation
kinetics, we thought it possible that independent regions
within the
3'NTR might differentially regulate transcript stability.
To test this
hypothesis, we compared the stability of mutant s4
reovirus mRNAs
1162-1181 and
1140-1159 with the stability of
the full-length and
s4-3'
mRNAs. We found that smaller deletions
within the 3'NTR were
not associated with alterations in the kinetics
of mRNA decay (Fig.
7).
These findings indicate that alterations
in translational efficiency
observed for s4 mRNAs containing internal
deletions in the 3'NTR occur
independently of RNA stability, which
suggests that the S4 3'NTR
mediates translational enhancement
by an alternative
mechanism.
The reovirus S4 gene 3'NTR forms complexes with proteins in
reticulocyte lysates.
To determine whether proteins contained in
rabbit reticulocyte lysates bind specifically to the S4 3'NTR, RNA
EMSAs were performed with 32P-labeled S4 3'NTR as probe
(Fig. 8A). Following incubation of reticulocyte lysate with the S4 3'NTR probe, three probe-containing protein complexes were observed after electrophoresis in nondenaturing polyacrylamide gels. Specificity of the interactions was determined by
competition analysis using unlabeled S4 3'NTR as specific competitor and unlabeled 5S rRNA, a 120-nucleotide RNA oligonucleotide, as nonspecific competitor. Band intensity of each of the three
3'NTR-containing complexes was diminished in the presence of excess
unlabeled 3'NTR, with competition of each complex occurring with
different efficiencies. In contrast, no change was observed in the
intensity of the protein-RNA complexes in the presence of excess
unlabeled 5S rRNA. As an additional test of specificity, an unlabeled
poly(A) oligonucleotide was assessed for the capacity to compete the
binding of the labeled S4 3'NTR to the reticulocyte protein complexes.
Similar to the results for 5S rRNA, poly(A) did not compete the binding
of the S4 3'NTR to these complexes (data not shown). We next tested
whether the mutant S4 3'NTR oligonucleotides 1140-1159 and
1162-1181 could compete the binding of the S4 3'NTR to protein
complexes in reticulocyte lysates (Fig. 8B). Both of the mutant
oligonucleotides were substantially less efficient than the full-length
S4 3'NTR in competing for reticulocyte components that bind the S4
3'NTR. We found no change in the band intensity of the
slowest-migrating complex and only a modest decrease in band
intensities of the two faster-migrating complexes, even at the highest
concentration of competitor used (250-fold). These findings demonstrate
that cellular proteins in rabbit reticulocyte lysates bind specifically to the reovirus S4 3'NTR.
View larger version (72K):
[in this window]
[in a new window]
|
FIG. 8.
The reovirus S4 3'NTR is bound by cellular proteins. (A)
EMSA of rabbit reticulocyte lysate (RRL) using reovirus S4 3'NTR as
probe. Lysates were incubated with 32P-labeled S4 3'NTR RNA
oligonucleotide, and incubation mixtures were resolved by acrylamide
gel electrophoresis, dried, and exposed to film. S4 3'NTR-binding
complexes are indicated. Specificity is shown by incubation with 1-, 10-, or 100-fold, excess unlabeled 3'NTR as specific competitor and
10-, 100-, or 250-fold excess 5S rRNA as nonspecific competitor.
Competitor RNAs were incubated with reticulocyte lysates on ice for 20 min prior to addition of radiolabeled S4 3'NTR probe. (B) Competition
analysis of S4 3'NTR-binding complexes using unlabeled mutant S4 3'NTR
RNA oligonucleotides. Lysates were incubated with either 10- or
100-fold excess unlabeled 3'NTR, 1140-1159 3'NTR, or 1162-1181
3'NTR, and EMSAs were performed as for panel A.
|
|
| |
DISCUSSION |
The 5' and 3'NTRs of reovirus gene segments are highly conserved
(6, 8, 13, 18, 34, 39). This level of sequence conservation suggests that the NTRs contain important cis
regulatory elements that determine the fate of nascent viral RNA. We
used full-length and truncated mRNA transcripts to determine the role of the 3'NTR in translation of the reovirus s4 mRNA. Our results indicate that the S4 gene 3'NTR contains discrete regions of sequence that differentially influence translational efficiency. A region of
sequence including nucleotides 1140 to 1159 enhances translation, while
a second region including nucleotides 1162 to 1181 represses translation. Furthermore, the S4 gene 3'NTR interacts with protein components of reticulocyte lysates, which suggests that cellular proteins mediate the regulatory properties of the S4 3'NTR.
Differences in translational efficiency of full-length and 3' s4
mRNAs were determined in vitro using rabbit reticulocyte lysates in the
presence and absence of polyadenylated globin mRNA as competitor. We
found that the S4 3'NTR facilitates efficient translation of reovirus
transcripts in an environment in which reovirus mRNAs must compete with
cellular mRNAs for components of the translational machinery. Although
the effects observed in the in vitro assays used were modest, the
translational enhancement provided by the S4 3'NTR is likely to be
substantial during the many cycles of viral replication that occur in
an infected host. Importantly, the observed differences in
translational efficiency were found to be independent of RNA stability,
thus excluding the possibility that the S4 3'NTR regulates translation
via preservation of RNA structural integrity. A similar finding has
been made in studies of alfalfa mosaic virus. Deletion of the coat
protein mRNA 3'NTR results in approximately 50% less translation
product in comparison to that produced following translation of a
full-length transcript when translated in the presence of an equimolar
ratio of globin mRNA (14). However, equivalent yields of
coat protein are synthesized from full-length and 3'NTR-truncated mRNAs
in the absence of competitor mRNA (14). Thus, our studies
of reovirus s4 mRNA in conjunction with studies of alfalfa mosaic virus
translational control define a paradigm in which 3'NTRs contain
sequences that enhance translation in an environment where cellular
transcripts are in excess and components of the translational machinery
are limited.
Sequences in the 3'NTRs of many transcripts have been shown to enhance
translation via interactions with the translational machinery
(29). We hypothesized that if a component of the
translational machinery bound the S4 3'NTR to augment translation,
addition of exogenous 3'NTR might compete for interactions with such
components and reduce translational efficiency of the s4 mRNA.
Surprisingly, we found that addition to translation reactions of an RNA
oligonucleotide corresponding to the S4 3'NTR augmented translation of
the full-length s4 mRNA and had no effect on translation of the s4
3' mRNA. Additionally, the S4 3'NTR does not alter translational
efficiency of other RNAs, including the reovirus s1 mRNA, indicating
that the observed effects are specific to the s4 transcript. These
findings suggest that the S4 3'NTR contains a repressor sequence that
when added in trans is capable of relieving translational
repression by titrating a repressor activity, resulting in
translational enhancement. We postulate that the S4 3'NTR also contains
an enhancer sequence, but the effect of the repressor sequence is
dominant when the 3'NTR is added to translation reactions in
trans. Such a scenario would be possible if either the
translational enhancer activity was in molar excess of repressor
activity or translational enhancement required sequences elsewhere in
the transcript in addition to sequences in the 3'NTR.
We directly tested whether enhancer and repressor sequences are present
in the S4 3'NTR by assessing translational efficiency of s4 mRNAs
containing small deletions in the 3'NTR. Findings from these
experiments confirm the existence of distinct sequences in the 3'NTR
that differentially regulate translation. Nucleotides 1142 to 1159 are
required for translational enhancement, whereas nucleotides 1162 to
1181 are required for translational repression. We also observed a
modest increase in translation of an mRNA lacking nucleotides 1169 to
1189, which suggests that the repressor region may include those
sequences as well. Opposing enhancer and repressor regions in the
3'NTRs of eukaryotic mRNAs have been previously described. The 3'NTR of
the -F1-ATPase mRNA contains a translational repressor that is bound
by an inhibitory protein found in fetal rat liver extracts. The 3'NTR
of this transcript also contains a translational enhancer that is
capable of titrating enhancement activity when added to translation
reactions in trans (17), a situation similar in
some respects to the translational regulatory region in the reovirus s4 mRNA.
As an additional confirmatory test of whether the reovirus S4 gene
3'NTR contains discrete enhancer and repressor elements, we tested the
effect of RNA oligonucleotides with deletions of either enhancer or
repressor sequences on translational efficiency of the full-length s4
mRNA when added to translation reactions in trans. We
hypothesized that if enhancer sequences were deleted from an S4 3'NTR
RNA oligonucleotide, only proteins that repress translation would be
capable of binding the NTR oligonucleotide, and translational
inhibition of the s4 mRNA would be relieved, similar to the effect
observed with the intact S4 3'NTR. As anticipated, we found that
addition to translation reactions of an RNA oligonucleotide lacking
3'NTR nucleotides 1140 to 1159 (the enhancer region) enhanced translation. However, we found that addition to translation reactions of an RNA oligonucleotide lacking 3'NTR nucleotides 1162 to 1181 (the
repressor region) did not diminish translation of the s4 mRNA. It is
possible that proteins mediating the enhancer function are in vast
excess of the RNA oligonucleotide. Alternatively, sequences within the
enhancer region may be involved in intramolecular interactions, with
sequences elsewhere in the s4 mRNA required for efficient assembly of
the translational machinery. Consistent with this idea, RNA secondary
structure algorithms suggest that the full-length s4 mRNA folds such
that the 5' and 3' termini base pair to form an extended region of
duplex RNA (reference (18) and data not shown).
Sequences in the 3'NTRs of many viral and cellular transcripts have
been shown to interact with components of the translational machinery
to influence translational efficiency (11, 16, 28, 37). We
performed RNA EMSAs to test whether cellular proteins are capable of
binding the S4 3'NTR. The results show that three complexes contained
in reticulocyte lysates bind specifically to a probe corresponding to
the S4 3'NTR. The binding of the labeled S4 3'NTR to each of these
complexes was inhibited by preincubation with unlabeled S4 3'NTR but
not with nonspecific competitors or mutant S4 3'NTRs. The competition
by the unlabeled S4 3'NTR does not follow first-order kinetics, which
is what would be expected if multiple complexes are bound by the
labeled S4 3'NTR. It is not apparent from our studies whether these
protein complexes serve to enhance or repress translation, but these
results are consistent with a model in which cellular proteins are
important for mediating translational control. Studies are now in
progress to identify the cellular proteins that interact with the S4
3'NTR and determine whether these interactions occur in cultured cells.
Regulation of gene expression for mammalian reoviruses is not well
understood. Since there is little evidence to support control of gene
expression at the level of transcription or mRNA stability, the primary
opportunity for regulation occurs during translation. The existence of
a translational operator sequence in the S4 gene 3'NTR suggests that
translation of the s4 mRNA is a tightly controlled process. We
hypothesize that this region determines appropriate expression of the
3 protein by enhancing or repressing translation at optimal times
during infection or in appropriate cell types. Careful assessment of
this hypothesis awaits development of a means to introduce targeted
mutations into the reovirus genome. In addition, our results suggest
that cellular proteins serve both regulatory and catalytic functions
during translation of reovirus transcripts. It will be interesting to
explore the precise biochemical mechanisms and biological roles of the
translational program of reovirus. These studies will contribute to an
enhanced understanding of protein-RNA interactions and mechanisms of
translational control in eukaryotic cells.
| |
ACKNOWLEDGMENTS |
We are grateful to Margo Brinton, Susan Low, and Erica White for
expert advice. We thank Christopher Coffey for assistance with
statistical analysis and Michelle Becker, Jim Chappell, Ron Emeson,
Neil Green, Jacek Hawiger, and Jim Patton for careful review of the manuscript.
This work was supported by Public Health Service awards F31 GM17208
from the National Institute of General Medical Sciences (M.M.-G.) and
AI32539 from the National Institute of Allergy and Infectious Diseases,
the Turner Scholars Program (T.S.D.), and the Elizabeth B. Lamb Center
for Pediatric Research. Additional support was provided by Public
Health Service awards CA68485 for the Vanderbilt Cancer Center and
DK20593 for the Vanderbilt Diabetes Research and Training Center.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Lamb Center for
Pediatric Research, D7235 MCN, Vanderbilt University School of
Medicine, Nashville, TN 37232. Phone: (615) 343-9943. Fax: (615)
343-9723. E-mail: terry.dermody{at}mcmail.vanderbilt.edu.
Dedicated to the memory of George C. Lamb, Jr.
| |
REFERENCES |
| 1.
|
Atwater, J. A.,
S. M. Munemitsu, and C. E. Samuel.
1987.
Biosynthesis of reovirus-specified polypeptides. Efficiency of expression of cDNAs of the reovirus S1 and S4 genes in transfected animal cells differs at the level of translation.
Virology
159:350-357[CrossRef][Medline].
|
| 2.
|
Baer, G. S., and T. S. Dermody.
1997.
Mutations in reovirus outer-capsid protein 3 selected during persistent infections of L cells confer resistance to protease inhibitor E64.
J. Virol.
71:4921-4928[Abstract].
|
| 3.
|
Banerjee, A. K., and A. J. Shatkin.
1970.
Transcription in vitro by reovirus-associated ribonucleic acid-dependent polymerase.
J. Virol.
6:1-11[Abstract/Free Full Text].
|
| 4.
|
Blackwell, J. L., and M. A. Brinton.
1997.
Translation elongation factor-1 alpha interacts with the 3' stem-loop region of West Nile virus genomic RNA.
J. Virol.
71:6433-6444[Abstract].
|
| 5.
|
Borsa, J., and A. F. Graham.
1968.
Reovirus RNA polymerase activity in purified virions.
Biochem. Biophys. Res. Commun.
33:895-901[CrossRef][Medline].
|
| 6.
|
Chappell, J. D.,
M. I. Goral,
S. E. Rodgers,
C. W. dePamphilis, and T. S. Dermody.
1994.
Sequence diversity within the reovirus S2 gene: reovirus genes reassort in nature, and their termini are predicted to form a panhandle motif.
J. Virol.
68:750-756[Abstract/Free Full Text].
|
| 7.
|
Constable, A.,
S. Quick,
N. K. Gray, and M. W. Hentze.
1992.
Modulation of the RNA-binding activity of a regulatory protein by iron in vitro: switching between enzymatic and genetic function?
Proc. Natl. Acad. Sci. USA
89:4554-4558[Abstract/Free Full Text].
|
| 8.
|
Dermody, T. S.,
M. L. Nibert,
R. Bassel-Duby, and B. N. Fields.
1990.
Sequence diversity in S1 genes and S1 translation products of eleven type 3 reovirus strains.
J. Virol.
64:4842-4850[Abstract/Free Full Text].
|
| 9.
|
Dermody, T. S.,
L. A. Schiff,
M. L. Nibert,
K. M. Coombs, and B. N. Fields.
1991.
The S2 gene nucleotide sequences of prototype strains of the three reovirus serotypes: characterization of reovirus core protein 2.
J. Virol.
65:5721-5731[Abstract/Free Full Text].
|
| 10.
|
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].
|
| 11.
|
Gallie, D. R.,
N. J. Lewis, and W. F. Marzluff.
1996.
The histone 3'-terminal stem-loop is necessary for translation in Chinese hamster ovary cells.
Nucleic Acids Res.
24:1954-1962[Abstract/Free Full Text].
|
| 12.
|
Giantini, M.,
L. S. Seliger,
Y. Furuichi, and A. J. Shatkin.
1984.
Reovirus type 3 genome segment S4: nucleotide sequence of the gene encoding a major virion surface protein.
J. Virol.
52:984-987[Abstract/Free Full Text].
|
| 13.
|
Goral, M. I.,
M. Mochow-Grundy, and T. S. Dermody.
1996.
Sequence diversity within the reovirus S3 gene: reoviruses evolve independently of host species, geographic locale, and date of isolation.
Virology
216:265-271[CrossRef][Medline].
|
| 14.
|
Hann, L. E.,
A. C. Webb,
J. M. Cai, and L. Gehrke.
1997.
Identification of a competitive translation determinant in the 3' untranslated region of alfalfa mosaic virus coat protein mRNA.
Mol. Cell. Biol.
17:2005-2013[Abstract].
|
| 15.
|
Harrison, S. J.,
D. L. Farsetta,
J. Kim,
S. Noble,
T. J. Broering, and M. L. Nibert.
1999.
Mammalian reovirus L3 gene sequences and evidence for a distinct amino-terminal region of the  1 protein.
Virology
258:54-64[CrossRef][Medline].
|
| 16.
|
Ito, T.,
S. M. Tahara, and M. M. Lai.
1998.
The 3'-untranslated region of hepatitis C virus RNA enhances translation from an internal ribosomal entry site.
J. Virol.
72:8789-8796[Abstract/Free Full Text].
|
| 17.
|
Izquierdo, J. M., and J. M. Cuezva.
1997.
Control of the translational efficiency of F1-ATPase mRNA depends on the regulation of a protein that binds the 3' untranslated region of the mRNA.
Mol. Cell. Biol.
17:5255-5268[Abstract].
|
| 18.
|
Kedl, R.,
S. Schmechel, and L. Schiff.
1995.
Comparative sequence analysis of the reovirus S4 genes from 13 serotype 1 and serotype 3 field isolates.
J. Virol.
69:552-559[Abstract].
|
| 19.
|
Kozak, M.
1981.
Possible role of flanking nucleotides in recognition of the AUG initiator codon by eukaryotic ribosomes.
Nucleic Acids Res.
9:5233-5252[Abstract/Free Full Text].
|
| 20.
|
Levin, D. H.,
N. Mendelsohn,
M. Schonberg,
H. Klett,
S. Silverstein, and A. M. Kapuler.
1970.
Properties of RNA transcriptase in reovirus subviral particles.
Proc. Natl. Acad. Sci. USA
66:890-897[Abstract/Free Full Text].
|
| 21.
|
Levin, K. H., and C. E. Samuel.
1980.
Biosynthesis of reovirus-specified polypeptides: purification and characterization of the small-sized class mRNAs of reovirus type 3. Coding assignments and translational efficiencies.
Virology
106:1-13[CrossRef][Medline].
|
| 22.
|
McCrae, M. A., and W. K. Joklik.
1978.
The nature of the polypeptide encoded by each of the ten double-stranded RNA segments of reovirus type 3.
Virology
89:578-593[CrossRef][Medline].
|
| 23.
|
Munemitsu, S. M., and C. E. Samuel.
1988.
Biosynthesis of reovirus-specified polypeptides: effect of point mutation of the sequences flanking the 5'-proximal AUG initiator codons of the reovirus S1 and S4 genes on the efficiency of messenger RNA translation.
Virology
163:643-646[CrossRef][Medline].
|
| 24.
|
Munroe, D., and A. Jacobson.
1990.
mRNA poly(A) tail, a 3' enhancer of translational initiation.
Mol. Cell. Biol.
10:3441-3455[Abstract/Free Full Text].
|
| 25.
|
Mustoe, T. A.,
R. F. Ramig,
A. H. Sharpe, and B. N. Fields.
1978.
Genetics of reovirus: identification of the dsRNA segments encoding the polypeptides of the µ and size classes.
Virology
89:594-604[CrossRef][Medline].
|
| 26.
|
Nibert, M. L.,
L. A. Schiff, and B. N. Fields.
1996.
Reoviruses and their replication, p. 1557-1596.
In
B. N. Fields, D. M. Knipe, and P. M. Howley (ed.), Fields virology, 3rd ed. Lippincott-Raven, Philadelphia, Pa.
|
| 27.
|
Patton, J. T.,
M. Wentz,
J. Xiaobo, and R. F. Ramig.
1996.
cis-acting signals that promote genome replication in rotavirus mRNA.
J. Virol.
70:3961-3971[Abstract].
|
| 28.
|
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].
|
| 29.
|
Sachs, A., and E. Wahle.
1993.
Poly(A) tail metabolism and function in eucaryotes.
J. Biol. Chem.
268:22955-22958[Free Full Text].
|
| 30.
|
Sachs, A. B., and R. W. Davis.
1989.
The poly(A) binding protein is required for poly(A) shortening and 60S ribosomal subunit-dependent translation initiation.
Cell
58:857-867[CrossRef][Medline].
|
| 31.
|
Schlegl, J.,
V. Gegout,
B. Schlager,
M. W. Hentze,
E. Westhof,
C. Ehresmann,
B. Ehresmann, and P. Romby.
1997.
Probing the structure of the regulatory region of human transferrin receptor messenger RNA and its interaction with iron regulatory protein-1.
RNA
3:1159-1172[Abstract].
|
| 32.
|
Shatkin, A. J., and J. D. Sipe.
1968.
RNA polymerase activity in purified reoviruses.
Proc. Natl. Acad. Sci. USA
61:1462-1469[Free Full Text].
|
| 33.
|
Skehel, J. J., and W. K. Joklik.
1969.
Studies on the in vitro transcription of reovirus RNA catalyzed by reovirus cores.
Virology
39:822-831[CrossRef][Medline].
|
| 34.
|
Tarlow, O.,
J. G. McCorquodale, and M. A. McCrae.
1988.
Molecular cloning and sequencing of the gene (M2) encoding the major virion structural protein µ1-µ1C of serotypes 1 and 3 of mammalian reovirus.
Virology
164:141-146[CrossRef][Medline].
|
| 35.
|
Thomson, A. M.,
J. T. Rogers, and P. J. Leedman.
1999.
Iron-regulatory proteins, iron-responsive elements and ferritin mRNA translation.
Int. J. Biochem. Cell Biol.
31:1139-1152[CrossRef][Medline].
|
| 36.
|
Virgin, H. W., IV,
R. Bassel-Duby,
B. N. Fields, and K. L. Tyler.
1988.
Antibody protects against lethal infection with the neurally spreading reovirus type 3 (Dearing).
J. Virol.
62:4594-4604[Abstract/Free Full Text].
|
| 37.
|
Wang, Z. F.,
T. C. Ingledue,
Z. Dominski,
R. Sanchez, and W. F. Marzluff.
1999.
Two Xenopus proteins that bind the 3' end of histone mRNA: implications for translational control of histone synthesis during oogenesis.
Mol. Cell. Biol.
19:835-845[Abstract/Free Full Text].
|
| 38.
|
Wentz, M. J.,
J. T. Patton, and R. F. Ramig.
1996.
The 3'-terminal consensus sequence of rotavirus mRNA is the minimal promoter of negative-strand RNA synthesis.
J. Virol.
70:7833-7841[Abstract].
|
| 39.
|
Wiener, J. R., and W. K. Joklik.
1989.
The sequences of the reovirus serotype 1, 2, and 3 L1 genome segments and analysis of the mode of divergence of the reovirus serotypes.
Virology
169:194-203[CrossRef][Medline].
|
| 40.
|
Xu, Z. K.,
J. V. Anzola,
C. M. Nalin, and D. L. Nuss.
1989.
The 3'-terminal sequence of a wound tumor virus transcript can influence conformational and functional properties associated with the 5'-terminus.
Virology
170:511-522[CrossRef][Medline].
|
| 41.
|
Zhou, Q.,
Y. Guo, and Y. Liu.
1998.
Regulation of the stability of heat-stable antigen mRNA by interplay between two novel cis elements in the 3' untranslated region.
Mol. Cell. Biol.
18:815-826[Abstract/Free Full Text].
|
| 42.
|
Zweerink, H. J., and W. K. Joklik.
1970.
Studies on the intracellular synthesis of reovirus-specified proteins.
Virology
41:501-518[CrossRef][Medline].
|
Journal of Virology, July 2001, p. 6517-6526, Vol. 75, No. 14
0022-538X/01/$04.00+0 DOI: 10.1128/JVI.75.14.6517-6526.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Top
Abstract
Introduction
