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Journal of Virology, November 1998, p. 9374-9379, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
RNA-Binding Activity of the E1B 55-Kilodalton
Protein from Human Adenovirus Type 5
Jackie J.
Horridge and
Keith N.
Leppard*
Department of Biological Sciences, University
of Warwick, Coventry CV4 7AL, United Kingdom
Received 1 June 1998/Accepted 13 August 1998
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ABSTRACT |
The human adenovirus 5 E1B 55-kDa protein is required for efficient
nucleocytoplasmic transport of late viral mRNAs. This protein is shown
to have RNA-binding activity which maps to a region of the protein with
homology to a family of RNA-binding proteins and which has been shown
previously to be essential for functionality of the protein in vivo.
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TEXT |
The expression of human adenovirus
(Ad) genes during lytic infection is carefully regulated from the
transcription of the earliest mRNAs right through to the final
formation of progeny virions. Regulation occurs at all levels of the
gene expression pathway, but posttranscriptional regulation is of
particular importance since most Ad primary transcripts are
differentially spliced to produce an array of distinct mRNA molecules
(reviewed in references 16 and
21). Products of early genes E1B and E4 have been
ascribed roles in regulating Ad late protein synthesis by modulating
mRNA transport. Mutant viruses lacking either the E1B 55-kilodalton (55K) or the E4 Orf6 protein are deficient in transport of their late
mRNAs from the nucleus to the cytoplasm, resulting in poor late protein
synthesis and virus production (2, 12, 28, 36). The
phenotypes of E1B 55K-E4 Orf6 double mutant viruses (5), as
well as biochemical evidence (31), demonstrate that the 55K
and Orf6 proteins interact and that complex formation is vital for
efficient late viral mRNA transport. Deletion of a third protein, E4
Orf3, further exacerbates this defect (4, 15), but the exact
role that the Orf3 protein plays in mRNA transport is still unclear
(20). In addition to their role in mRNA transport, both the
E1B 55K and E4 Orf6 proteins promote cellular transformation by binding
to and inactivating the tumor suppressor protein p53 (8,
39).
Not all Ad mRNAs require 55K/Orf6 for export. Early RNAs are
independent of 55K/Orf6 function, and the level of dependence of the
late RNAs varies considerably, correlating with the number of unused
splice sites or other potential intron sequences which they retain
(22). Eukaryotic cells are thought to possess mechanisms for
retaining RNA within the nucleus until processing is complete; this may
be achieved by association with proteins, such as hnRNP-C1, which carry
specific nuclear retention sequences (26). It has been
suggested that 55K/Orf6 may act to block nuclear retention of
susceptible late viral mRNAs and to divert them into an export pathway
(6, 22), fulfilling a role in Ad infection similar to that
of the Rev and Rex proteins in the life cycles of human immunodeficiency virus and human T-cell lymphotropic virus,
respectively (reviewed in reference 13). Some
evidence that 55K/Orf6 functions similarly to human immunodeficiency
virus Rev has emerged from studies of Ad variants deficient in E1B 55K
but producing instead the Rev protein and containing a specific
Rev-binding site (RRE) within their major late transcription unit. The
Rev/RRE system rescued, albeit to a modest extent, the cytoplasmic
accumulation of viral mRNA (37). More recently, it was
demonstrated that the 55K/Orf6 complex could shuttle between the
nucleus and the cytoplasm (7). The Orf6 protein also
inhibited Rev-mediated export of RRE-containing RNA, which implies that
the 55K/Orf6 complex utilizes at least part of the same nuclear export
pathway used by the retrovirus protein.
Similarity between the mechanism of action of the Ad E1B 55K/E4 Orf6
complex and those of the retroviral Rev and Rex proteins would suggest
that the Ad proteins might perform specific interactions with their
target mRNAs. No specific RRE-like sequences have been identified in Ad
mRNAs, and there is no sequence that is common to all
55K/Orf6-dependent mRNAs; however, it is possible that the complex
interacts with RNA via some feature of the unused splice sites or
intron sequences or via a secondary-structure element. To begin to test
for such interactions, the E1B 55K protein was examined for RNA-binding
activity by using a series of RNA probes.
Preparation and purification of proteins.
E1B 55K was
expressed in Escherichia coli as a glutathione
S-transferase (GST) fusion protein from a plasmid kindly
donated by Thomas Dobner, University of Regensberg, Regensberg, Germany (30); GST was expressed from pGEX2T (Pharmacia). Since the
fusion protein was found to be very unstable, a range of strains was tested to find conditions which minimized degradation. Experiments described here used the GST-E1B 55K (GST-E1B) fusion protein from either XL1-Blue, TOPP 3, TOPP 5, or TOPP 6 cells (Stratagene), although
TOPP 3 produced the highest-quality protein. GST was expressed in BL21
(Novagen) or XL1-Blue. Protein expression was induced in 1.0-liter
cultures grown at 37°C to an optical density at 600 nm of 0.5 by the
addition of IPTG (isopropyl-
-D-thiogalactopyranoside) at
a final concentration of 0.1 mM, and incubation was continued at 30°C
for a further 1 to 2 h (GST-E1B) or 4 h (GST). Cells were washed in 50 ml of ice-cold phosphate-buffered saline (PBS; 140 mM
NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM
KH2PO4 [pH 7.2]) containing inhibitors
(PBS/I; PBS, 500 µM dithiothreitol, 500 µM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 2 µM pepstatin A, 10 mM EDTA and then resuspended in 10 ml of PBS/I and snap-frozen at 
70°C. Cells were
lysed with a French press at 1,010 lb/in2 (American
Instruments Company), and cellular debris was removed by centrifugation
at 32,500 × g for 60 min at 4°C. The lysate was
diluted to 100 ml in PBS-1% Triton X-100 and incubated with 2 ml of a
50% suspension of glutathione-Sepharose 4B beads (Pharmacia) for
1 h at room temperature. The beads were washed four times in PBS,
and protein was eluted with 50 mM Tris (pH 8)-10 mM reduced glutathione. Protein concentrations in fractions were determined by
using the Bio-Rad protein assay, and samples were analyzed by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with
either Coomassie blue staining or Western blotting. Fractions containing the highest concentrations of protein (equivalent also to
those with the most full-length protein) were pooled and stored at
70°C in 50-µl aliquots. Since the proportion of full-length protein in GST-E1B preparations was small, care was taken to analyze each preparation by SDS-PAGE and Coomassie blue staining and to use
preparations with similar full-length protein contents for all
experiments.
Selection and synthesis of RNA probes.
RNA probes P1 to P8
represent adenovirus type 5 (Ad5) genomic sequences (5'-3') of
positions 35617 to 35357 (P1), 35357 to 35617 (P2), 35617 to 35469 (P3), 35357 to 35464 (P4), 35356 to 35094 (P5), 34935 to 34635 (P6),
12039 to 12357 (P7), and 16369 to 16593 (P8). These were selected from
Ad E4, L1, and L2 mRNAs that are known to be highly dependent on
55K/Orf6 for efficient transport in late Ad-infected cells (6,
22) and were designed to contain splice sites or intron sequences
that might act as targets for the action of the 55K/Orf6 complex in
vivo. E4 mRNA A, which relies on the E1B 55K protein for export
(6), encodes the Orf1 protein and differs from the other
late E4 RNAs in that the 5' intron is retained as coding sequence and
the splice donor site D1 and acceptor sites A1a and A1b, etc., remain
unused; probes P1, P3, P5, and P6 together cover the majority of this
region, while P2 and P4 are antisense transcripts. Probes P7 and P8 are sections of major late transcription unit mRNAs for L1 52/55K and L2
III/pVII, respectively, each containing an unused splice acceptor site
and part of the sequence to either side. T7 or SP6 RNA polymerase
(Gibco BRL) was used in accordance with the manufacturer's instructions to transcribe linearized cloned Ad5 genomic DNA in pGEM
vectors (Promega) in the presence of 4 mCi of
[
-32P]CTP (Amersham; 800 Ci/mmol) per ml, 500 µM
each unlabelled GTP, ATP, and UTP, and 100 µM CTP. Unincorporated
nucleotides were removed by gel filtration, and radioactivity
incorporation was quantified by acid precipitation and scintillation
counting. The length and quality of probes were verified by
electrophoresis on 5% acrylamide-urea gels.
The GST-E1B fusion protein has RNA-binding activity.
The
RNA-binding activity of GST-E1B fusion protein was initially detected
by gel mobility shift assays with probe P1. RNA was denatured by
heating to 85°C for 10 min and then cooled on ice for 15 to 30 min
before being mixed with purified GST-E1B or GST in binding buffer (10 mM HEPES [pH 7.6], 20 mM NaCl, 150 mM KCl, 0.5 mM EGTA, 10%
glycerol, 1 mM dithiothreitol, 7.5 µg of bovine serum albumin per ml,
100 U of human placental RNase inhibitor per ml [adapted from
reference 23]). Typically, 200 ng to 8 µg of
protein was incubated with 2.5 fmol of 32P-RNA in a
reaction volume of 25 µl for approximately 20 min on ice and
protein-RNA complexes were resolved by electrophoresis through 4%
polyacrylamide gels (acrylamide-to-bisacrylamide ratio, 79:1) in 0.5×
Tris-borate-EDTA buffer at 4°C and visualized by autoradiography.
GST-E1B caused a marked reduction in the level of unbound probe with
increasing protein concentration, which was not observed for the
equivalent GST samples, although protein-RNA complexes could not be
visualized as specific shifted bands (data not shown). The possibility
that the loss of the unshifted probe was due to RNA degradation was
discounted by recovering RNA from binding reaction mixtures and
analyzing it by denaturing gel electrophoresis. The presence of
labelled probe retained in the wells with high inputs of GST-E1B
suggested that complexes were being formed that were too large to enter
the gel. Since GST is known to dimerize (17),
protein-protein interactions via the GST domain probably contributed to
this phenomenon. However, binding of multiple protein molecules to the
RNA would also affect complex size. To minimize such effects and
produce complexes better able to enter the gel, a shorter probe, P3,
which consisted of the 5'-end 180 nucleotides (nt) of P1 (full length,
325 nt), was tested in similar assays with GST-E1B and GST. A
short-exposure autoradiograph of the gel (Fig.
1A) showed the same reduction in the
amount of unbound probe with increasing amounts of GST-E1B, as was seen
for P1. In addition, small protein-RNA complexes were detected just
above the unbound probe band. Upon longer exposure of the gel (Fig.
1B), these small complexes were obscured but larger GST-E1B-specific
complexes were revealed migrating closer to the wells and, less
clearly, as smears between these large complexes and the unbound probe. The various mobilities of the complexes observed might have been due to
different fragments of GST-E1B since, despite optimization of
expression conditions, GST-E1B preparations always contained mostly
degraded protein (for example, see Fig. 4A). However, in comparisons of
several independent preparations, the amount of full-length GST-E1B
correlated positively with the observation of bands with decreased
mobility of all types and not just with the observation of bands from
the larger complexes (data not shown). An alternative explanation for
the presence of different mobility-retarded bands is that they are due
to protein binding to a different position(s) on the RNA to create
complexes of differing shape and/or to the binding of multiple protein
molecules to an RNA.
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FIG. 1.
GST-E1B RNA binding and competition assays. (A) A short
exposure of the gel shown in panel B, lanes 1 to 8. (B) Assays
comprising 32P-probe RNA and various amounts of either
GST-E1B or GST protein were analyzed on nondenaturing 4%
polyacrylamide gels. The input protein is indicated above the lanes;
, no protein. Protein inputs were 0.1 (lanes 2 and 9), 0.2 (lanes 3 and 10), 0.5 (lanes 4 and 11), 1.0 (lanes 5 and 12), 1.5 (lanes 6 and
13), 2.0 (lanes 7 and 14), or 3.0 (lanes 8 and 15) µg. (C) Assays
using 5 µg of GST-E1B protein and a constant amount of
32P-probe 3 together with either homologous (probe 3, unlabelled) or heterologous (probe 4, unlabelled) competitor RNA were
analyzed on nondenaturing 4% polyacrylamide gels. Competitor-to-probe
molar ratios were 0.0 (lanes 3 and 10), 1.0 (lanes 4 and 11), 2.0 (lanes 5 and 12), 5.0 (lanes 6 and 13), 10 (lanes 7 and 14), 100 (lanes
8 and 15), or 500 (lanes 9 and 16). Lanes 1 and 2 show probe 3 plus
homologous and heterologous competitor RNAs, respectively, at molar
ratios of 500 in the absence of GST-E1B protein.
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Since GST-E1B is a much larger protein than the GST control, it was
possible that it might display different aggregation properties
because
of its size. An unrelated GST fusion protein of similar
size to
GST-E1B, carrying the L1 52/55K sequence of Ad5 (kindly
provided by A. Arslanoglu), was therefore tested in a mobility
shift assay with probes
P3 and P4. No alteration to the mobility
of either probe was observed
in this experiment (data not shown),
further confirming that the
complexes formed by GST-E1B with RNA
were specifically due to the
presence of E1B protein sequences.
The quality of the protein
preparation used in this experiment
is shown below (see Fig.
4A, lane
8) together with an example
of a GST-E1B preparation which showed
RNA-binding activity (see
Fig.
4A, lane 7); these preparations show
comparable levels of
fragmentation. Thus, the RNA-binding activity of
the GST-E1B protein
preparation is unlikely to be a nonspecific
property of partially
folded protein fragments.
Sequence specificity of GST-E1B binding.
To determine whether
the binding of GST-E1B to P1 and P3 represented a sequence-specific
interaction, gel mobility shift assays were conducted with the
antisense mRNA probe P2 (326 nt) and its shorter derivative, P4 (159 nt). Although transcribed from Ad DNA, these probes do not correspond
to true Ad mRNA sequences. In analyses similar to those shown in Fig.
1A, GST-E1B bound to P2 and P4, causing probe shifts comparable to
those of P1 and P3 at similar protein inputs while GST alone showed no
binding (data not shown), suggesting that GST-E1B was binding RNA
nonspecifically. In reciprocal competitor binding assays, unlabelled
versions of P3 and P4 (C3 and C4), produced by using the Ampliscribe
kit (Epicentre Technologies), were preincubated with GST-E1B for 20 min
and then either the homologous or the heterologous probe was added to
determine whether the protein displayed any binding preference for
either of the two RNA sequences. As shown in Fig. 1C, C3 and C4
competed equally with P3 for GST-E1B binding, indicating that the
RNA-binding activity of GST-E1B was not specific for the E4 mRNA
sequence represented in P3. In the equivalent experiment, C3 and C4
also competed equally well for binding to P4 (data not shown).
Although there was no specific affinity of GST-E1B for P3, it was
possible that a high-affinity specific binding site for
the protein
existed elsewhere in E4 mRNA A or in another Ad transcript.
To assess
this, a filter binding assay which measured the total
RNA bound by
protein in each reaction was used to compare the
binding levels of
GST-E1B to a number of RNA probes within a single
experiment. The
binding conditions were similar to those used
for mobility shift
assays, but the incubation times were slightly
increased.
Nitrocellulose filters (Hybond-C; Amersham) were equilibrated
in wash
buffer (10 mM HEPES [pH 7.6], 20 mM NaCl, 150 mM KCl,
0.5 mM EGTA),
and products of binding reactions were collected
under gentle vacuum
pressure by using a 96-well dot blot manifold.
Filters were washed
three times in wash buffer and air dried,
and bound probe was
visualized by phosphorimaging (Molecular Dynamics
Corp.). Computer
analysis of data was performed by using ImageQuant
software. By using
this technique, the binding of GST-E1B to probes
P3 to P8 was tested
(Fig.
2). The molar concentration of
probe
used in each case was the same. RNA-binding activity extended
to
all of the probes chosen, and the binding titration curves
were similar
for all cases. These data confirm the finding that
GST-E1B shows
non-sequence-specific binding to RNA of complex
sequence.
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FIG. 2.
GST-E1B protein binding to a range of RNA probes.
Binding assays comprised various amounts of GST-E1B protein or GST
protein and one of the 32P-labelled RNA probes 3 to 8. RNA-protein complexes were collected on nitrocellulose filters and
detected by phosphorimaging. The amounts of radioactivity bound were
quantified by using ImageQuant software and corrected for differences
in length and percentage of C residues. Results are indicated in
arbitrary units as the mean of three determinations ± standard
deviations. Closed symbols, GST-E1B protein; open symbols, GST
protein.
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The E1B 55K protein has homology with a large number of RNA-binding
proteins.
RNA-binding proteins have been classified by the nature
of their RNA-binding domain(s). One large family of RNA-binding
proteins with diverse origins and functions is defined by possession of the ribonucleoprotein (RNP) motif; family members can display various
degrees of specificity in their RNA binding (14, 18). The
RNP motif is composed of 80 to 100 residues and is characterized by two
highly conserved sequences, RNP 1 and RNP 2, and a number of other,
mostly hydrophobic, residues interspersed throughout the domain
(3, 10). Folding of the RNP domain creates a -sheet with
the RNP 1 and RNP 2 consensus sequences lying on its two central
strands (25, 38). This sheet forms a shallow platform for
nonspecific RNA binding, whereas other parts of the domain, particularly the loops between -strands, act to stabilize the interaction and are responsible for specific sequence recognition (reviewed in references 9 and
24).
Analysis of the E1B 55K amino acid sequence revealed a region that had
homology with the RNP consensus sequence. Stronger
homology was evident
when E1B 55K was compared with selected individual
RNP family members
(Fig.
3). Based on this alignment, a
series
of mutations was introduced into the potential RNP motif of E1B
55K. Three of the mutations were single amino acid substitutions,
A284S
and C288A within RNP 1 and W289F adjacent to RNP 1, and
the fourth
consisted of the substitution of one amino acid and
the deletion of a
second within the RNP 1 sequence, designated
F285L-del287.
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FIG. 3.
Protein sequence alignment between the Ad5 E1B 55K
protein and selected members of the RNP family of proteins. The RNP 1 and RNP 2 consensus elements are boxed and the secondary-structure
elements of the RNP domain are indicated above the alignment. The
consensus sequence is taken from the work of Bandziulis et al.
(3). Residues matching the consensus sequence are shown in
boldface type; nonconsensus residues in consensus positions of E1B 55K
which match those found at that position in at least one member of the
RNP family are underlined. The mutations in E1B 55K described in this
paper are indicated below the alignment at their respective positions
by the amino acid substitution or deletion () in each case. Mutations
were named by using the single-letter amino acid code and the residue
positions in the 496-residue E1B 55K protein, the wild-type residue
being listed first. The sequences for the following proteins are from
the references indicated: U1A #1 (33), U1 70K
(29), hnRNP C1 (34), ASF (11), tra-2
(1).
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The RNP motif of GST-E1B is responsible for binding activity in
vitro.
Mutated E1B sequences were expressed as GST fusions to
assess the effects of the mutations on RNA-binding activity. As
previously discussed in relation to Fig. 1, wild-type GST-E1B protein
was very unstable when expressed in bacteria. Each mutated GST-E1B protein also showed great instability during purification; however, as
shown in Fig. 4A, lanes 1 to 6, this
pattern of degradation was both qualitatively and quantitatively
similar for all four mutant protein preparations and the two wild-type
protein preparations analyzed in parallel. Since, as already discussed,
the RNA-binding activity of GST-E1B wild-type preparations correlated
with the amount of full-length protein present, stained bands of
full-length fusion protein were quantified by scanning densitometry in
comparison with known amounts of a standard protein to provide
estimates of the full-length protein concentration in each preparation. These data were then used to standardize protein inputs to binding assays.
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FIG. 4.
(A) GST-fusion proteins analyzed by SDS-PAGE and
visualized by Coomassie blue staining. Lane 1, GST-E1B A284S (9.6 µg); lane 2, GST-E1B F285L-del287 (20.8 µg); lane 3, GST-E1B W289F
(16.8 µg); lane 4, GST-E1B C288A (27.6 µg); lanes 5 to 7, independent wild-type GST-E1B preparations (34.4, 26.4, and 20.0 µg);
lane 8, GST-L1 52/55K (20.0 µg). (B) Binding of wild-type and mutated
forms of GST-E1B to RNA probe 3. Binding assay mixtures comprised
various amounts of wild-type or mutated GST-E1B protein (panel A, lanes
1 to 6) or GST protein with 32P-labelled RNA probe 3. RNA-protein complexes were collected on nitrocellulose filters and
detected by phosphorimaging. The amounts of radioactivity bound were
quantified by using ImageQuant software and are indicated in arbitrary
units as the mean of three determinations (two wild-type protein
preparations were each analyzed in triplicate) ± standard deviations.
Protein inputs for GST-E1B preparations are indicated as the amounts of
full-length protein added. Actual protein inputs were significantly
higher, due to the presence of degraded fragments (see panel A). To set
the protein inputs for the GST control, the mean total GST-E1B
(wild-type or mutated) protein input used to provide a given amount of
full-length protein was calculated and this series of GST inputs was
used for the experiment. Symbols:  , wild-type GST-E1B;
 , GST-E1B A284S;  ,
GST-E1B F285L-del287;  , GST-E1B W289F;  , GST-E1B C288A;  ,
GST.
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Figure
4B shows the results of a filter binding assay comparing the
abilities of wild-type GST-E1B and the four mutant proteins
shown in
Fig.
4A, lanes 1 to 6, to bind to probe P3. The two mutations
affecting
the central part of RNP 1 (A284S and F285L-del287) had
the most severe
effect on RNA binding activity, reducing binding
to a level only
marginally higher than the GST baseline binding.
In contrast, mutating
the last residue in the RNP 1 sequence (C288A)
caused only a slight
reduction in binding as compared with that
of the wild-type protein and
mutating the first residue outside
the RNP 1 sequence (W289F) caused an
increase in binding at low
protein concentrations, while at high
concentrations the binding
activity was similar to that of wild-type
GST-E1B. The fact that
a single amino acid substitution in the E1B
portion of GST-E1B
could essentially destroy its RNA-binding activity
suggested strongly
that this protein, rather than any background
contaminant present
specifically in GST-E1B preparations, was
responsible for the
activity. However, since full-length protein
comprised only a
small part of the total protein input in these assays,
it was
important to consider whether the standardization of inputs
based
on concentrations of full-length protein might bias the results
by ignoring the contribution of truncated proteins. An alternative
representation of the data from Fig.
4B based on the total protein
input to each assay did not change the relative positions of the
curves
significantly and did not affect any of the conclusions
drawn (data not
shown). Thus, these results demonstrate that the
integrity of the RNP 1 consensus within the candidate RNP motif
of the GST-E1B fusion protein
is essential for this protein to
display RNA-binding activity in vitro,
although this does not
mean that the domain necessarily adopts the RNP
fold.
The region of the E1B 55K protein sequence implicated here in RNA
binding, which includes residues 284, 285, and 287, has
been shown
previously to be crucial for normal virus growth (
40).
However, the lack of apparent specificity in this RNA-binding
activity
is at odds with the highly specific nature of the RNA
export function
which the protein provides in vivo. Many RNA-binding
proteins,
including members of the RNP family, display low-affinity
nonspecific
binding in addition to higher-affinity binding to
a particular RNA
sequence or secondary-structure element, so it
is quite possible that
such high-affinity, specific binding sites
exist for E1B 55K in RNA
sequences not tested in this study. Alternatively,
it is possible that
conditions within the infected cell nucleus
may promote more specific
interactions with RNA than those used
in these experiments. First, the
protein used in these assays,
being expressed in bacteria, lacks the
posttranslational modifications
such as phosphorylation
(
32), which the E1B 55K protein normally
sustains, and this
may have affected its activity. However, the
major sites of
phosphorylation in E1B 55K map to its N- and C-terminal
domains, away
from the region implicated here in RNA binding (
35).
Second,
the structure of RNA can be crucial for its correct recognition
and
might be an important factor in determining the specificity
of
interaction between E1B 55K and mRNA. This structure will in
turn be
affected by the association of RNA with protein to form
RNP complexes,
the natural form for all RNA in vivo and hence
the true substrate for
interaction with E1B 55K. Third, the E1B
55K protein is known to
interact with both viral and cellular
proteins, at least one of which,
E4 Orf6, is also involved in
the RNA export function, and some or all
of these interactions
may be required to impart specificity to RNA
recognition. Alternative
experimental approaches in which the E1B 55K
protein is derived
from eukaryotic expression systems, perhaps in
complex with its
in vivo partner, E4 Orf6, could be used to address
these possibilities.
The secondary structure of RNA is difficult to control experimentally.
For the binding assays described here, probe RNAs were
heat denatured
prior to incubation with the E1B 55K protein, and
although cooling of
the RNA before binding may have allowed some
secondary structure to
reform, this reformed structure cannot
be assumed to be equivalent to
the normal structure of the viral
mRNAs from which the probes were
derived. Furthermore, RNA inside
the cell is always associated with
protein; this association begins
even before transcription is complete,
acting to stabilize the
nascent molecules and to direct processing and
localization of
the RNA within the nucleus (
9). Such
interactions alter RNA
conformation and may affect binding of other
proteins such as
E1B 55K. Since all the probes used here were
synthesized by in
vitro transcription, these proteins were not present
and this
may have prevented the formation of the RNA secondary
structure
necessary for specific recognition by E1B 55K. In support of
the
relevance of RNA secondary structure to 55K protein-RNA
interaction,
we observed that the ability of tRNA to compete with
labelled
probe RNA for binding to E1B 55K varied between experiments
(data
not shown). Unlike the other probes used, tRNA has a defined and
stable secondary structure to which it can refold after denaturation
(
19). Although all probes and competitor RNAs were heat
denatured
prior to their use in binding reactions, cooling of the tRNA
before
binding might have allowed the secondary structure to reform and
this might have variably inhibited its binding to GST-E1B. While
the
secondary structure of tRNA may have inhibited binding to
E1B 55K, the
secondary structure of other types of RNA may of
course promote
binding.
The lack of specificity of E1B 55K RNA binding may indicate that the
formation of the E1B 55K/E4 Orf6 complex is necessary
for specific
binding to occur. It is known that both proteins
are necessary for
efficient regulation of RNA transport and for
the proteins to shuttle
between nucleus and cytoplasm, so it follows
that the Orf6 protein may
be an important factor in E1B 55K-RNA
interactions. Orf6 may be needed
either to alter the conformation
of E1B 55K to promote specific
recognition of RNA or to make direct
interactions with RNA itself. In
preliminary experiments, Orf6
also displays RNA-binding activity
(unpublished data). However,
this could be related either to the RNA
transport function of
the protein (
12) or to its role as a
splicing factor (
27).
It will be important therefore to test
whether formation of the
55K/Orf6 complex can cause selective targeting
of mRNA in vitro
that would correlate with the properties of these
proteins in
vivo. Finally, it is possible that other proteins, possibly
host
cell RNA-binding proteins or viral proteins such as the E4 Orf3
protein, might be important in generating the expected specific
recognition of late viral RNA. The finding that E1B 55K has RNA-binding
activity fits with the idea that it functions to facilitate RNA
export
by accompanying RNA out of the nucleus and suggests several
lines of
experiment which should lead to a full definition of
the RNA-protein
interactions involved in this mechanism.
| |
ACKNOWLEDGMENTS |
We gratefully acknowledge the gift of a plasmid expressing the
GST-E1B 55K fusion protein from Thomas Dobner, University of Regensberg, that was crucial to this work and for his advice on protein
expression from this plasmid. We are also grateful to Alper Arslanoglu
for the gift of negative-control GST L1 fusion protein and to Elizabeth
Harfst for helpful discussions. The mutations in the E1B 55K protein
tested here were originally isolated by Laurie Eden. J.J.H. was
supported by a research studentship from the Medical Research Council.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biological Sciences, University of Warwick, Coventry CV4 7AL, United Kingdom. Phone: (44) 1203 523579. Fax: (44) 1203 523701. E-mail: Keith.Leppard{at}warwick.ac.uk.
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Journal of Virology, November 1998, p. 9374-9379, Vol. 72, No. 11
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