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Journal of Virology, November 1998, p. 8659-8668, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Multimer Formation Is Not Essential for Nuclear
Export of Human T-Cell Leukemia Virus Type 1 Rex
trans-Activator Protein
Peter
Heger,1
Olaf
Rosorius,1
Claudia
Koch,1
Georg
Casari,2
Ralph
Grassmann,1 and
Joachim
Hauber1,*
Institute for Clinical and Molecular
Virology, University Erlangen-Nürnberg, D-91054
Erlangen,1 and
Lion AG, D-69120
Heidelberg,2 Germany
Received 12 May 1998/Accepted 17 July 1998
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ABSTRACT |
The Rex trans-regulatory protein of human T-cell
leukemia virus type 1 (HTLV-1) is required for the nuclear export of
incompletely spliced and unspliced viral mRNAs and is therefore
essential for virus replication. Rex is a nuclear phosphoprotein that
directly binds to its cis-acting Rex response element RNA
target sequence and constantly shuttles between the nucleus and
cytoplasm. Moreover, Rex induces nuclear accumulation of unspliced
viral RNA. Three protein domains which mediate nuclear import-RNA
binding, nuclear export, and Rex oligomerization have been mapped
within the 189-amino-acid Rex polypeptide. Here we identified a
different region in the carboxy-terminal half of Rex which is also
required for biological activity. In inactive mutants with mutations
that map within this region, as well as in mutants that are deficient
in Rex-specific multimerization, Rex trans activation could
be reconstituted by fusion to a heterologous leucine zipper
dimerization interface. The intracellular trafficking capabilities of
wild-type and mutant Rex proteins reveal that biologically inactive and
multimerization-deficient Rex mutants are still efficiently
translocated from the nucleus to the cytoplasm. This observation
indicates that multimerization is essential for Rex function but is not
required for nuclear export. Finally, we are able to provide an
improved model of the HTLV-1 Rex domain structure.
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INTRODUCTION |
Human T-cell leukemia virus type 1 (HTLV-1) (66) is the causative agent of adult T-cell
leukemia, an aggressive malignancy of human T lymphocytes (38, 67,
96). In addition, HTLV-1 has been associated with a chronic
neurodegenerative disorder termed tropical spastic paraparesis
(28) or HTLV-1-associated myelopathy (61). As for
every replication-competent retrovirus, the HTLV-1 genome contains the
gag, pol, and env genes, which encode
the viral structural proteins and enzymes. In addition to structural
proteins, HTLV-1 encodes at least two trans-regulatory proteins, Tax and Rex, which are essential for virus replication (reviewed in references 18, 29, and
78). The Tax protein activates the viral long
terminal repeat promoter element, resulting in enhanced transcription
of all viral genes (15, 79). Furthermore, Tax also activates
cellular promoters of various genes and stimulates the growth of T
lymphocytes (16, 26, 44, 75). In contrast, the
rex gene product acts at the posttranscriptional level,
permitting the expression of the viral structural proteins (37,
45). In the absence of Rex, the unspliced and incompletely
spliced viral transcripts are retained in the nucleus and either
spliced to completion or subjected to degradation. When Rex is present, however, these transcripts are exported from the nucleus to the cytoplasm, where they are either translated or packaged as genomes into
progeny virions (36).
Previous studies have demonstrated that Rex is a 27-kDa phosphoprotein
that accumulates at steady state in the nucleoli of expressing cells
(1, 39, 50, 60). Rex binds directly and specifically to its
cis-acting RNA target sequence, the Rex response element
(RxRE) (6, 7, 12, 30-32, 90, 91). The RxRE is a highly
stable 255-nucleotide RNA stem-loop structure, which is encoded by
sequences within the retroviral 3' long terminal repeat, thereby making
it an integral sequence element of all viral mRNAs (3, 36, 76,
90). In addition to its role in mediating Rex responsiveness, the
RxRE also plays a role in the 3' processing of viral primary
transcripts. The viral polyadenylation signal is separated from the 3'
cleavage site by the RxRE sequence, a distance that does not allow
processing of the 3' ends of the viral primary transcripts. Formation
of the correct RxRE secondary structure, however, brings the two
elements in close proximity to each other, thereby permitting correct
polyadenylation (2).
So far, mutational analyses have revealed three distinct functional
domains in the rex gene product. A basic domain rich in arginine residues, which maps to amino acids (aa) 1 to 19 in the 189-aa
Rex protein, is critically required for the sequence-specific binding
of the RxRE RNA sequence (12, 30, 83) and also serves as a
nuclear localization signal (13, 51, 60, 71, 77). It has
been suggested that a region which maps to aa 57 to 66 is involved in
Rex oligomer formation (9, 92). Finally, a protein
activation or effector domain, which is required for interaction of Rex
with one or multiple cellular cofactors, is located between aa 79 and
99 (93). In fact, various proteins have been reported to
bind to this domain and/or to mediate Rex effector function. These
include the nucleoporin-like proteins hRIP/Rab (10, 11, 25),
the yeast factor Rip1p (85), and eukaryotic initiation factor 5A (48). An essential feature of the Rex activation
domain is a sequence motif rich in leucine residues (40)
that appears to be a target for the export factor CRM1 (24, 27,
63, 81) and acts as a nuclear export signal (10, 49,
64). As the protein contains both nuclear export and import
signals, Rex is able to constantly shuttle between the nucleus and
cytoplasm of the host cell (64).
Another human pathogenic retrovirus, human immunodeficiency virus
type 1 (HIV-1), encodes a regulatory protein of similar activity,
termed Rev (for a recent review, see reference 68). Like Rex, Rev is a shuttle protein that induces the cytoplasmic accumulation of unspliced and incompletely spliced viral mRNA species.
Similarly, Rev binds to its highly structured cis-acting Rev
response element (RRE) sequence, which is part of the viral unspliced
and incompletely spliced mRNA species. Although HTLV-1 Rex and HIV-1
Rev lack any significant sequence homology, Rex is able to functionally
substitute for Rev in HIV-1, by rescuing replication of a Rev-deficient
HIV-1 provirus (72). The molecular basis for this
cross-activation is the ability of HTLV-1 Rex to bind the heterologous
HIV-1 RRE (3, 12, 21, 36, 46, 80, 91). This finding also
suggests that both Rev and Rex access the same cellular pathway for
nuclear export of their unspliced and incompletely spliced viral mRNAs.
In support of this notion, it has previously been shown that HIV-1 Rev
interacts with the same cellular activation domain binding proteins as
Rex (8, 11, 25, 73, 85). Finally, extensive mutational
analysis of Rex has allowed the identification of mutant proteins that inhibit not only the function of the homologous Rex wild-type protein
on the RxRE but also the function of the heterologous Rev
trans activator on the RRE, in a dominant-negative
(trans-dominant) manner (14, 71). Taken together,
these data led to the general notion that the biological activity and
molecular mode of action of HTLV-1 Rex are identical to those of HIV-1
Rev.
This study was undertaken to characterize in more detail the regions in
the HTLV-1 Rex protein which are required for biological activity. A
series of Rex mutants were characterized according to their potential
to stimulate expression of intron-containing mRNA and nuclear export
function. Our data demonstrate that a previously unrecognized region in
the carboxy-terminal half of Rex is essential for function but not for
the intracellular trafficking of Rex and suggest that this region is
required for protein oligomerization.
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MATERIALS AND METHODS |
Molecular clones.
The expression plasmid pcRex and the
vectors encoding the Rex mutants RexM6, RexM7, RexM13, RexIW18, and
Rex
5 have been described in detail elsewhere (71, 72, 92,
93). The parental vector pBC12/CMV (17) and the
construct pBC12/CMV/
Gal (73) were used in transfection
experiments to maintain constant input DNA levels and for internal
control of transfection efficiencies, respectively. pDM128/CMV/RxRE is
a Rex-responsive reporter construct containing the bacterial
chloramphenicol acetyltransferase (CAT) gene and was constructed by
ligating the 1.7-kb XbaI-BglII fragment from the
Rev-specific reporter construct pDM128 (41) between the
SalI and BglII sites of pgTat-RxRE
(39). Plasmid pRRX is a Rex-responsive reporter construct
that is derived from the 3' half of the HTLV-1 proviral genome and
gives rise to a full-length primary transcript and a spliced derivative
(33).
A bacteriophage M13-based oligonucleotide-directed mutagenesis system
(United States Biochemicals, Cleveland, Ohio) was used to introduce
in-frame NheI restriction sites into the coding region of
pcRex, which permitted the subsequent construction of a series of Rex
internal deletion mutants (RexID1 to RexID6). Vectors expressing leucine zipper-Rex fusion proteins (Zip-Rex) were generated by fusing
synthetic oligonucleotides that encode the GCN4-derived leucine zipper
element
(NH2-MDPKLQRMKQLEDKVEELLSKNYHLENEVARLKKLVGG-COOH) (42) in front of the rex gene in the
respective pcRex vectors. Plasmids expressing glutathione
S-transferase (GST)-Rex fusion proteins (pGEX-Rex and
pGEX-RexM13) were generated by cloning the respective PCR-generated
(59) rex coding regions between the
BamHI and EcoRI sites of the bacterial expression
vector pGEX-3X (Pharmacia Biotech, Freiburg, Germany). Finally, the
coding regions of all constructs generated in the course of this study
were confirmed by DNA sequence analysis.
Cell culture, transfections, and assays.
The cell lines COS
and HeLa were maintained and transfected with either DEAE-dextran and
chloroquine or calcium phosphate, as previously described (89,
97). 293T cells were cultured in Dulbecco modified Eagle medium
with 10% fetal calf serum and transfected by using the Lipofectamine
reagent according to the protocol of the manufacturer (Gibco BRL,
Eggenstein, Germany).
Rex protein expression was evaluated by transfection of 1.5 × 105 COS cells with 300 ng of the various Rex expression
plasmid DNAs. The transfected-cell cultures were radiolabelled with
[35S]cysteine at ~48 h posttransfection, followed by
immunoprecipitation with a polyclonal anti-Rex antibody (14)
and electrophoresis on sodium dodecyl sulfate-polyacrylamide gels as
described previously (14, 39).
Rex
trans activation was investigated by cotransfection of
2.5 × 10
5 COS cells with 250 ng of pDM128/CMV/RxRE
DNA and 250 ng of pBC12/CMV/
Gal
DNA, together with 250 ng of pcRex
(positive control), pBC12/CMV
(negative control), or mutant Rex
expression plasmid. At ~60 h
posttransfection, cell lysates were
prepared and the levels of
-galactosidase activity were measured as
described previously
(
89). These values were subsequently
used to determine the amount
of cell extract to be assayed for CAT by
an enzyme-linked immunosorbent
assay (Boehringer GmbH, Mannheim,
Germany).
The effect of Rex on accumulation of unspliced HTLV-1-derived mRNAs was
evaluated by cotransfection of 6 × 10
5 293T cells
with 1 µg of pRRX DNA together with 3 µg of pBC12/CMV
(negative
control), pcRex (positive control), or mutant Rex expression
plasmid.
At ~48 h posttransfection, total cellular RNA was isolated
and
subjected to Northern analysis by using an intron-specific
hybridization probe as previously described (
33).
A total of 2.5 × 10
5 HeLa cells (HeLaneoRRE)
(
97), grown on glass coverslips, were transfected with 5 µg of expression plasmid
to determine the subcellular localization of
Rex proteins by indirect
immunofluorescence.
Purification of GST-Rex fusion proteins.
Wild-type Rex and
RexM13 were expressed as carboxy-terminal fusions to GST in
Escherichia coli BL21. The fusion proteins were purified
from crude lysates by affinity chromatography with
glutathione-Sepharose 4B according to the specifications of the
manufacturer (Pharmacia Biotech). Eluted proteins were analyzed by
Rex-specific Western analysis, pooled, concentrated by ultrafiltration
with a PM10 filter device (Amicon Inc., Beverly, Mass.), and stored at
70°C.
Immunofluorescence studies and microinjection.
The
nucleocytoplasmic shuttling capabilities of Rex proteins were
investigated with transfected HeLa cells. At ~40 h posttransfection, cell cultures were supplemented with 50 µg of cycloheximide (Sigma, Deisenhofen, Germany) per ml to inhibit protein synthesis. After 30 min, 5 µg of actinomycin D (Sigma) per ml was added in order to
inhibit gene transcription. After a further incubation for 2.5 h,
cells were fixed with paraformaldehyde and subcellular localization of
Rex proteins was determined by indirect immunofluorescence as described
previously (54). The primary rabbit anti-Rex polyclonal antibody (14) was used at a 1:250 dilution. The second
antibody, rhodamine-conjugated goat anti-rabbit immunoglobulin G (IgG), was used at a 1:200 dilution.
HeLa cells (HeLaneoRRE) constitutively expressing the HIV-1 RRE
sequence (
97) were comicroinjected into the nucleus with
GST-Rex (1.5 mg/ml) or GST-RexM13 and rabbit IgG (1.0 mg/ml) (injection
control) by using a CompiC INJECT computer-assisted injection
system
(Cellbiology Trading, Hamburg, Germany). Cells were fixed
at 30 min
postinjection with paraformaldehyde, and the injected
proteins were
visualized by indirect immunofluorescence analysis
as described
previously (
8). The primary mouse anti-GST monoclonal
antibody (Serotec, Oxford, United Kingdom) was used at a 1:50
dilution.
The secondary antibodies, fluorescein isothiocyanate-conjugated
goat
anti-rabbit IgG and Texas red-conjugated goat anti-mouse
IgG, were used
at a 1:100 dilution.
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RESULTS |
Functional analysis of the HTLV-1 Rex trans-activator
protein.
Previous studies in which various Rex regions were
deleted in order to identify distinct protein domains indicated that a functionally critical region in the 189-aa Rex protein might be located
somewhere between the carboxy-terminal border of the activation domain
at aa 99 and aa 132 (39, 93). To test this hypothesis and to
investigate the function of this protein region, we generated a series
of mutants that are characterized by internal deletions between aa 99 and 133 in the carboxy-terminal half of the Rex protein (RexID1 to
RexID6) (Fig. 1). In addition, various
prototypic Rex control mutants which have been previously described as
trans-activation negative (Table
1) were included in the study. In
particular, the missense substitutions in RexM6 and RexM7
(71) and the deletion in Rex5 (92) have been
speculated to inhibit Rex oligomer formation (9, 92). The
RexIW18 protein is nonfunctional due to complete deletion of the Rex
activation domain (10, 40, 49, 93). Finally, RexM13 has been
described as a dominant-negative point mutant that binds the RxRE RNA
with wild-type efficiency but has a negative effect on the Rex effector
domain (9, 12, 71). As it has been suggested that some of
these mutations impair the capacity of Rex to form multimers, we
also engineered variants of all mutants in which a dimerization
interface was provided by a heterologous sequence. For this, the
leucine zipper domain of the yeast transcription factor GCN4, which has
previously been shown to be a protein motif that forms stable dimers
(20, 42, 62), was fused in front of the various
rex genes, creating in-frame leucine zipper-Rex (Zip-Rex)
fusion proteins. Protein expression was then confirmed for all
constructs by Rex-specific immunoprecipitation analysis (Fig.
2A) with radiolabelled protein extracts
of transiently transfected COS cells and a polyclonal anti-Rex
antiserum directed against the Rex carboxy terminus (14). As
noted previously (14, 71), even small alterations to the Rex
amino acid sequence affected the electrophoretic mobility of some of
the mutant proteins (e.g., RexM6 versus RexM7; Fig. 2A, lanes 13 and
14). This apparent difference in molecular weight probably reflects
altered posttranslational modification (e.g., phosphorylation)
(1) of these Rex mutant proteins. In general, however, all
rex expression vectors appeared to produce comparable levels
of protein.
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FIG. 1.
Localization of HTLV-1 Rex internal deletion mutants. A
series of small internal deletion mutants, designated Rex ID1 to Rex
ID6, with mutations between aa 99 and 133 in the carboxy-terminal half
of the 189-aa Rex protein were generated. This amino acid sequence is
shown in the expanded section. Residues mutated in the previously
published sequence of the biologically inactive RexM13 protein
(71) are underlined. Deleted protein regions are indicated
by bars.
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FIG. 2.
Functional characterization of HTLV-1 Rex mutant
proteins. (A) Radioimmunoprecipitation of wild-type (WT) and mutant Rex
proteins. COS cell monolayers were transiently transfected with either
a negative control plasmid (neg.) (lane 1), pcRex (lane 2), or the
mutant rex expression vectors indicated (see also Table 1).
At ~48 h posttransfection, cultures were radiolabelled with
[35S]cysteine and subjected to immunoprecipitation with a
polyclonal anti-Rex antibody (14). Precipitated proteins
were resolved on sodium dodecyl sulfate-14% polyacrylamide gels and
visualized by autoradiography. Molecular mass standards (in
kilodaltons) are at the left. (B) Rex trans-activation
capacity was determined by cotransfection of COS cell monolayers with
the Rex-responsive reporter plasmid pDM128/CMV/RxRE, the mutant Rex
expression plasmids indicated, and the constitutive internal control
vector pBC12/CMV/Gal. Data are expressed as percentages of wild-type
Rex activity (set to 100%), and the error bars represent the standard
deviations from three independent experiments. All CAT values were
adjusted for transfection efficiency by determining the level of
-galactosidase in each culture and were corrected for background
(mock) activity.
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Next we tested the biological activities of these Rex mutant proteins
in
trans-activation assays. For this, we employed the
Rex-responsive reporter construct pDM128/CMV/RxRE. This plasmid
contains the CAT indicator gene and the RxRE target sequence of
Rex.
These elements are positioned between HIV-1 splice sites
and are under
control of the cytomegalovirus immediate-early promoter
(
41,
56). As shown in previous studies (
10,
32,
40,
49),
RNA produced from this type of Rex reporter construct contains
a single
intron containing the CAT gene and RxRE sequence, which
is removed when
the RNA is spliced. However, in the presence of
a functional Rex
protein, unspliced message is exported to the
cytoplasm, resulting in
high levels of CAT protein and thereby
providing an assay system for
Rex functionality. Figure
2B summarizes
the activities of the various
Rex mutants in this reporter system.
These experiments were internally
controlled for varying transfection
efficiencies by inclusion of the
constitutive control vector pBC12/CMV/
Gal
(
73). All CAT
values are expressed as a percentage of wild-type
Rex activity (set
arbitrarily to 100%; Fig.
2B, bar 2) and have
been corrected for
background (mock) activity. The controls RexM6,
RexM7, Rex
5,
RexIW18, and RexM13 (Fig.
2B, bars 12 to 16) were,
as reported
previously (
71,
92,
93), inactive in this assay.
The
trans-activation phenotypes of our newly generated mutants
revealed wild-type activities for RexID1 and RexID6 (Fig.
2B,
bars 17 and 22). In contrast, RexID2 to RexID5 were characterized
by complete
nonfunctionality (Fig.
2B, bars 18 to 21). Importantly,
in-frame fusion
of the GCN4 leucine zipper dimerization domain
to RexID2 to RexID5
restored Rex function to a maximal level of
~80% of wild-type
activity (Fig.
2B, bars 8 to 11). The reconstitution
of biological
activity was also seen in the case of RexM13 (Fig.
2B, bar 7), which
contains a point mutation that maps to the protein
region that is
covered by the overlapping internal deletions in
RexID2 to RexID5
(Table
1). Furthermore, the leucine zipper element
also rescued Rex
activity to a similar extent in the mutants RexM6,
RexM7, and Rex
5
(Fig.
2B, bars 3 to 5), which have previously
been shown to be
important for Rex oligomerization (
9,
92).
In contrast, Rex
activity could not be reconstituted in the activation
domain mutant
RexIW18 (Fig.
2B, bar 6).
In a previous study we were able to show that HTLV-1 Rex activity not
only affects the nucleocytoplasmic transport of viral
RNA, as
demonstrated in the pDM128/CMV/RxRE-based
trans-activation
assay, but also interferes with intron excision, thereby inducing
nuclear accumulation of unspliced viral RNAs (
33). As a
second
indicator of Rex function, we also tested selected prototypic
Rex deletion mutants for their capacity to stimulate the intracellular
accumulation of intron-containing HTLV-1-derived mRNAs. For this,
293T
cells were cotransfected with the HTLV-1-derived Rex-responsive
reporter construct pRRX (
33) and various Rex expression
plasmids.
Total cellular RNAs were isolated at 48 h
posttransfection, separated
by gel electrophoresis, and subjected to
Northern analysis. The
pRRX plasmid contains sequences derived from the
3' half of the
proviral genome, including genuine HTLV-1 splice donor
and splice
acceptor sites and the full-length RxRE sequence. Since the
pRRX
plasmid alone does not express Rex, cotransfection with a Rex
expression vector allowed the effect of Rex on the accumulation
of
unspliced RNAs to be monitored by using an intron-specific
hybridization probe. As shown in Fig.
3,
coexpression of an unrelated
control vector (lane 1) or the
trans-activation-negative mutant
RexID2, RexID3, or RexID4
(lanes 3, 5 and 7, respectively) failed
to induce significant levels of
unspliced HTLV-1-specific mRNAs
in the transfected cell cultures. In
agreement with previous data
(
33), coexpression of the Rex
wild-type protein resulted in
a marked increase in unspliced RNA (Fig.
3, lane 2). As in the
CAT indicator assay, the leucine zipper-Rex
fusion proteins Zip-RexID2,
Zip-RexID3, and Zip-RexID4 displayed
activities which were comparable
to that of the wild-type Rex protein
(Fig.
3, lanes 4, 6, and
8).
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FIG. 3.
Effect of HTLV-1 Rex on viral mRNA splicing. The mutant
rex genes were cotransfected together with pRRX into 293T
cells. The plasmid pRRX is derived from the 3' half of the HTLV-1
provirus and produces spliced and unspliced HTLV-1 RNAs, whose amounts
can be regulated by Rex. Total cellular RNA from transfected cells was
separated on a 1% formaldehyde agarose gel, blotted onto a nylon
membrane, and hybridized to an intron-specific radioactive probe. Lane
1, pBC12/CMV (unrelated control vector), lane 2, pcRex, lanes 3 to 8, plasmids expressing the indicated Rex mutants. WT, wild type; neg.,
negative control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
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Taken together, these data suggest that a region in HTLV-1 Rex, located
between aa 106 and 124, is essential for Rex-specific
trans
activation. Furthermore, the consistent reconstitution of
Rex
biological activity by a heterologous dimerization domain
in otherwise
inactive and multimerization-deficient mutants (e.g.,
RexM6, RexM7, and
RexM13) (
9) suggests that this region of
Rex is involved in
the formation of Rex oligomeric complexes.
Intracellular trafficking of HTLV-1 Rex mutants.
As noted
above, nuclear export of HTLV-1 Rex protein is mediated by the protein
activation domain. The mutation of critical amino acid residues in the
activation domain results in Rex mutant proteins that are nuclear
export deficient and also inactive when tested in
trans-activation assays (10, 49, 64). These data indicate that the nuclear export of Rex is essential for its biological activity. Therefore, in this study, we also evaluated the
nucleocytoplasmic trafficking capabilities of wild-type and mutant Rex
proteins in HeLa cells. Although Rex is a protein that constantly
shuttles between the nucleus and cytoplasm, previous studies have shown that Rex accumulates at steady state in the nuclei and nucleoli of
transfected cells (1, 39, 50, 60). However, inhibition of
RNA synthesis by actinomycin D induces the relocation of Rex from the
nucleus to the cytoplasm (19, 64, 82). The molecular basis
for this effect appears to be that nuclear import of shuttle proteins
depends on continuous transcription. This was shown originally for the
hnRNP A1 protein (65), a factor which appears to be involved
in the transport of mRNA, and subsequently for the HIV-1 Rev
trans activator (47, 57, 70, 82, 95).
Figure
4 shows the subcellular
distribution of the various Rex proteins in transfected HeLa cells, as
determined by indirect
immunofluorescence. Clearly, wild-type Rex
displayed its predominantly
nuclear-nucleolar localization in untreated
cells (Fig.
4A). In
contrast, and as expected, significant amounts of
the protein
were found in the cytoplasm in cells exposed to 5 µg of
actinomycin
D per ml and 50 µg of cycloheximide per ml, which was
added in
order to inhibit de novo protein synthesis. This actinomycin
D-dependent
cytoplasmic accumulation of Rex was not observed in the
activation-deletion
mutants RexIW18 (Fig.
4G and H) and Zip-RexIW18
(Fig.
4I and K),
which served as negative controls in this assay.
However, the
mutants Rex
5, RexID5, and RexM13 relocated to the
cytoplasm in
the presence of actinomycin D (Fig.
4C, L, and P versus
Fig.
4D,
M, and Q). This was also seen in experiments using the
respective
leucine zipper-Rex variants (Fig.
4E, N, and R versus Fig.
4F,
O, and S) and was comparable to the actinomycin D-induced
cytoplasmic
accumulation of the wild-type Rex protein (Fig.
4B). These
data
suggest that
trans-activation-negative mutants, such as
Rex
5,
RexID5, and RexM13, are still capable of translocating from
the
nucleus to the cytoplasm.
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FIG. 4.
Subcellular localization of Rex mutant proteins in
transfected HeLa cells. Cells transfected with the indicated Rex
mutants were either incubated in medium alone (Act.D) (A, C, E, G, I,
L, N, P, and R) or exposed to 50 µg of cycloheximide per ml plus 5 µg of the transcription inhibitor actinomycin D per ml (+Act. D) (B,
D, F, H, K, M, O, Q, and S). After fixation, the subcellular locations
of the various Rex proteins were determined by indirect
immunofluorescence with a rabbit polyclonal anti-Rex antibody
(14) and rhodamine-conjugated secondary antibody. WT, wild
type. Bar, 20 µm.
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We next wanted to investigate Rex nuclear export more directly.
Experimentally this can be carried out independent of nuclear
import,
by microinjection of GST fusion proteins into the nuclei
of human
somatic cells. As demonstrated previously for GST-Rev
proteins, active
export occurs within minutes after microinjection
and can be easily
visualized by indirect immunofluorescence (
8,
74). This is
possible because nuclear export of Rev has been
shown to be
significantly more efficient than its reimport into
the nucleus
(
84).
As shown clearly in Fig.
5B,
microinjection of GST-Rex together with rabbit IgG, which was included
to establish the site
of injection, into the nuclei of HeLa cells
resulted in nucleocytoplasmic
translocation of GST-Rex. In contrast,
the coinjected rabbit IgG
remained in the cell nucleus (Fig.
5A). Next
we investigated the
export capacity of a biologically inactive Rex
mutant, which is
representative of the type of mutants described in
this study.
GST-RexM13 protein was injected together with rabbit IgG
into
HeLa cell nuclei as before. Indirect immunofluorescence analysis
clearly demonstrated translocation of the GST-RexM13 mutant protein
from the nucleus to the cytoplasm (Fig.
5C and D). These experiments
confirm that nonfunctional Rex molecules can be exported from
the
nucleus.
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FIG. 5.
Nuclear export of HTLV-1 Rex in human somatic cells. The
nuclei of HeLa cells were microinjected with wild-type GST-Rex (A and
B) or GST-RexM13 (C and D) fusion proteins together with rabbit IgG. At
30 min after microinjection, the cells were fixed and subjected to
double-label indirect immunofluorescence analysis. The GST-Rex proteins
were visualized by using a mouse monoclonal anti-GST antibody followed
by Texas red-conjugated goat anti-mouse IgG (B and D). The
microinjected rabbit IgG, which served to control the site of
injection, was detected with a fluorescein isothiocyanate-conjugated
goat anti-rabbit antibody (A and C). WT, wild type. Bar, 20 µm.
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DISCUSSION |
By constructing small internal deletions, in this study we have
identified a region of 19 amino acid residues (aa 106 to 124) (Fig.
6) in HTLV-1 Rex that is required for
biological activity. Interestingly, we were able to rescue at least
partial Rex activity by fusing our inactive mutants in frame to the
heterologous GCN4 leucine zipper element. This effect was detectable in
a standard Rex trans-activation assay, which measures the
cytoplasmic expression of RxRE-containing mRNAs (Fig. 2B), as well as
in an independent assay that measures the Rex-dependent increase of
unspliced RNA (Fig. 3). Furthermore, the finding that the leucine
zipper element also reconstituted the function of mutants that were
previously reported to be defective in their ability to form Rex
homomultimers (RexM6, RexM7, and RexM13) (9) suggests that
the region between aa 106 and 124 in Rex functions as a protein
oligomerization domain. However, whether a homodimer is sufficient to
create a fully functional Rex complex cannot be concluded from our
experiments using the leucine zipper element. It is indeed conceivable
that multiple Rex dimers are able to interact with the RxRE sequence,
thereby creating a higher-order oligomeric complex. Unfortunately, it was not possible to investigate the multimerization status of our Rex
deletion mutants in vitro by RNA gel retardation analysis, because
these proteins repeatedly proved to be unstable when expressed as
recombinant proteins in E. coli.
View larger version (22K):
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|
FIG. 6.
Domain structure of the HTLV-1 Rex
trans-activator protein. The four distinct functional
regions located within the 189-aa Rex protein are indicated by boxes
(see text for details). Interaction of Rex with its RxRE target
sequence and nuclear-nucleolar localization of the protein are mediated
by the Rex amino terminus (aa 1 to 19; hatched box). The protein
activation domain, which contains a leucine-rich peptide core motif
that serves as a nuclear export signal, is located in the center of the
protein (aa 79 to 99; cross-hatched box). Regions involved in the
multimerization of Rex are localized in both the amino-terminal and
carboxy-terminal halves of the protein (aa 57 to 66 and aa 106 to 124;
filled boxes). The amino acid sequences of these multimerization
domains are shown as expanded sections. The Rex sequences indicated by
grey boxes are dispensable for in vivo Rex function (39) and
appear to serve a structural rather than a directly functional role in
Rex. NLS, nuclear localization signal; NES, nuclear export signal.
|
|
Nevertheless, we are now able to suggest an improved model of the Rex
domain structure (Fig. 6). The Rex amino terminus (aa 1 to 19) is
required for both the nuclear accumulation of Rex (13, 60, 71,
77) and its direct binding to the RxRE RNA (12, 30,
83). Therefore, this region can be considered the Rex RNA binding
and nuclear localization domain. The activation domain, required for
the interaction of Rex with cellular cofactors, is located in the
center of the protein (aa 79 to 99) (93) and is
characterized by hydrophobic residues (commonly leucine)
(40) that constitute a nuclear export signal (10, 49,
64). So far, two regions have been identified that appear to be
involved in Rex multimer formation and are therefore operationally
referred to as multimerization domains. The more amino-terminal region stretches from aa 57 to 66. The notion that this region constitutes a
multimerization domain originated from the finding that this sequence
can be functionally replaced by a region of HIV-1 Rev that has been
implicated in the formation of Rev homomultimers (53, 55,
92). Point mutants with mutations that map within this Rex region
(e.g., RexM6 and RexM7) (Table 1) failed to form homomultimeric
complexes in a mammalian cell-based two-hybrid assay (9). In
contrast, the second multimerization region, identified in this study,
localizes to the carboxy-terminal half of Rex and maps to aa 106 to
124. The only inactive point mutant described so far with a mutation
that maps within this region, namely, RexM13 (Table 1) (71),
has also been reported to lack the intrinsic capacity of wild-type Rex
for protein-protein interaction (9). Obviously, our findings
with the leucine zipper dimerization interface further support the
notion that both regions, which are separated by the protein activation
domain, participate in Rex oligomerization. It should be noted that a
similar domain arrangement also occurs in the HIV-1 Rev
trans-activator protein. Two multimerization domains are
separated by the RNA binding-nuclear localization domain in the
amino-terminal half of Rev (35, 53, 55, 86, 88), which forms
a helix-loop-helix motif (4), thereby permitting the
creation of a single exposed hydrophobic oligomerization interface
(87, 88). It remains to be seen whether biophysical
measurements are able to confirm a similar structural organization of
the multimerization interface in HTLV-1 Rex.
Random mutational analysis of the rex gene allowed the
identification of dominant-negative inhibitors for both HTLV-1 Rex and
HIV-1 Rev function (14, 71). By comparing the positions of
the mutations introduced within the Rex domain structure (Fig. 6), it
is evident that all of the mutations that gave rise to trans-dominant Rex repressors targeted residues within
either the activation domain or the multimerization domains (14,
71). These data suggest that both the interaction of Rex with
cellular cofactors via its activation domain and the formation of
oligomeric Rex complexes via the multimerization domains are equally
required for full Rex biological activity.
Inspection of the amino acid sequence of the Rex multimerization
domains reveals no known or apparent motifs that indicate the
structural basis of this protein-protein interaction. The high proline
content of the multimerization domains does not contrast with the
overall composition of Rex and suggests a mechanically rather rigid
protein chain that does not allow formation of any of the known protein
contact structural motifs. The multimerization domains may be close
together in the functional fold of the protein, thereby forming an
interaction surface. It is most likely that the hydrophobic residues
(Y59, I60, Y64, W65, L109, L114, and F120) (Fig. 6) play a role in
contact formation. A histidine residue at position 121 could also be
involved in specific hydrogen bonds between the interacting proteins.
The reconstitution of function by chimeric constructs with
amino-terminal leucine zipper elements is remarkable, as it
demonstrates the modular architecture of Rex, whereby multimerization
functionality can be swapped to other regions of the protein. It is
conceivable that the function of such multimerization mutants can
usually be restored only when the structure has been destabilized
rather than irreversibly disrupted. In this case the zipper element can
support the protein-protein interactions that result in the formation
of multimers and that would otherwise be too weak.
As noted before, Rex mutants that are deficient in nuclear export due
to disruption of the protein activation domain also lack
trans-activation capacity (10, 49, 64). As this
correlated with data generated for the HIV-1 Rev protein (22, 54,
56, 58, 94), and as Rev-mediated viral mRNA transport is known to
occur independent of any pre-mRNA splicing events (23), it seemed likely that Rex, like Rev, acts primarily at the level of
nuclear export. However, evaluation of the nuclear export capacity of
biologically inactive Rex mutants in this study (Fig. 4 and 5)
suggested that, although it is required, the nuclear export activity is
not sufficient for Rex-mediated trans activation. For
example, the RexM13 protein is multimerization deficient (9) and biologically inactive in trans-activation assays (Fig.
2) (71). Despite this, RexM13 still binds to its RxRE RNA
target sequence (12) and is also exported from the nucleus
(Fig. 4Q and Fig. 5D) with wild-type efficiency. Thus, the recruitment of multiple Rex monomers appears to be required for an activity other
than the one seen in nuclear export. This notion is directly supported
by a recent study in which conditional Rex-human estrogen receptor (ER)
fusion molecules were investigated (69): in the absence of
hormone, Rex-ER protein remained in the cytoplasm but was relocated
into the nucleus, and particularly to the nuclear pore complex (NPC),
in a hormone-dependent manner; this localization also correlated with
Rex trans activation. Importantly, the biologically inactive
RexM7-ER chimera exhibited NPC colocalization comparable to that of the
wild-type protein in these experiments, providing evidence that
intranuclear translocation of Rex to the NPC is independent of the
oligomeric status of Rex.
Although our study does not directly address the question of which
functions other than nuclear export are executed by the HTLV-1 Rex
protein, it is likely that these activities take place at the level of
viral pre-mRNA splicing for a number of reasons. For example, it has
been shown that unspliced HTLV-1 RNA accumulates in the nuclei and
cytoplasm of transiently transfected cells in the presence of Rex
(43). Furthermore, we have been able to provide evidence
that expression of Rex increases the levels of unspliced viral RNA by
reducing the rates of intron excision and degradation in the nucleus
(33). The most direct confirmation of this idea, however,
comes from a recent study in which it was demonstrated that the Rex
protein of HTLV-2 acts as a potent inhibitor of in vitro splicing
reactions by interfering with an early step in spliceosome assembly
(5). It is important that HTLV-1 Rex has previously been
reported to bind a protein that is associated with the splicing factor
ASF/SF2 (52). Finally, indirect evidence that Rex has
functions other than the one linked to its activation domain (e.g.,
nuclear export) originates from a study in which the biological
activities of HIV-1 Rev and HTLV-1 Rex in Jurkat T cells were compared
(34). It was shown that although the activation domain
function was intact, Rex appeared to be biologically inactive in these
cells.
Taken together, these various lines of evidence suggest that HTLV-1 Rex
trans activation is the sum of multiple Rex activities, including those at the level of nuclear export and, presumably, splicing of viral RNAs. While HTLV-1 Rex nuclear export, and therefore cofactor interaction via its activation domain, apparently takes place
in the absence of Rex multimerization, the formation of Rex oligomers
appears to be required for full biological activity. It is hoped that
mapping of the regions in HTLV-1 Rex that mediate these homomultimeric
protein-protein interactions will provide the basis to allow
elucidation of the Rex protein structure in greater detail.
| |
ACKNOWLEDGMENTS |
We thank Warner C. Greene for the RexM6, RexM7, and RexM13
constructs and Nicole Hirschmann and Lotte Hofer for excellent technical assistance.
This work was supported by the Deutsche Forschungsgemeinschaft (SFB466)
and Johannes and Frieda Marohn-Stiftung.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute for
Clinical and Molecular Virology, University Erlangen-Nürnberg,
Schlossgarten 4, D-91054 Erlangen, Germany. Phone: 49-9131-852 6182. Fax: 49-9131-852 2101. E-mail:
jmhauber{at}viro.med.uni-erlangen.de.
| |
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Journal of Virology, November 1998, p. 8659-8668, Vol. 72, No. 11
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
