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Journal of Virology, January 1999, p. 120-127, Vol. 73, No. 1
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Nucleoporins Nup98 and Nup214 Participate in
Nuclear Export of Human Immunodeficiency Virus Type 1 Rev
Andrei S.
Zolotukhin and
Barbara K.
Felber*
Human Retrovirus Pathogenesis Group,
ABL-Basic Research Program, National Cancer Institute-Frederick Cancer
Research and Development Center, Frederick, Maryland 21702-1201
Received 22 July 1998/Accepted 9 October 1998
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ABSTRACT |
Human immunodeficiency virus type 1 (HIV-1) Rev contains a
leucine-rich nuclear export signal that is essential for its
nucleocytoplasmic export mediated by hCRM1. We examined the role of
selected nucleoporins, which are located in peripheral structures of
the nuclear pore complex and are thought to be involved in export, in
Rev function in human cells. First, we found that upon actinomycin D
treatment, Nup98, but not Nup214 or Nup153, is able to translocate to
the cytoplasm of HeLa cells, demonstrating that Nup98 may act as a soluble factor. We further showed that Rev can recruit Nup98 and Nup214, but not Nup153, to the nucleolus. We also found that the isolated FG-containing repeat domains of Nup98 and Nup214, but not
those of Nup153, competitively inhibit the Rev/RRE-mediated expression
of HIV. Taken together, the recruitment of Nup98 and Nup214 by Rev and
the competitive inhibition exhibited by their NP domains demonstrate
direct participation of Nup98 and Nup214 in the Rev-hCRM1-mediated export.
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INTRODUCTION |
The active export of macromolecules
from the nucleus is mediated by short peptide signals that act as
ligands for nuclear export factors. The best-studied example is human
immunodeficiency virus type 1 (HIV-1) Rev, which is responsible for the
export of the Rev-responsive element containing viral RNA. The export of Rev of HIV-1 and other lentiviruses is mediated via its leucine-rich nuclear export signals (NES) (13, 29). Functional Rev-like NES have also been identified in several other proteins (for a recent
review, see reference 23). Several NES-containing
proteins such as Rev have been shown to interact directly with the
human protein hCRM1 (14, 18, 42). This interaction is
inhibited by the antibiotic leptomycin B (LMB) (51). hCRM1
belongs to the importin 
protein superfamily (15, 21).
Some of the importin -like proteins have been shown to be involved
in the nucleocytoplasmic transport. The known and predicted
characteristics of the family include (i) preferential binding to Ran
GTPase in its GTP-bound form and (ii) binding to repeated FG-containing
motifs characteristic of some nucleoporins (NP repeats). hCRM1 and a
related protein, CAS (26), have been implicated as direct
receptors mediating the nuclear export of NES-containing proteins and
importin 
, respectively, and hence define a subset of proteins in
the importin superfamily termed exportins. The key feature of
exportins is their ability to cooperatively bind their export substrate
and GTP-bound Ran. The ternary complex is then believed to interact with some components of the nuclear pore complex (NPC) via an exportin,
thus delivering the export cargo to the site of translocation. Upon
translocation of the ternary complex through the NPC, GTP hydrolysis
occurs on Ran, resulting in the dissociation of the complex and release
of the export substrate into the cytoplasm. In this model, exportins
act as transient linkers between the export cargo and the NPC, which
are regulated by the nucleotide-bound status of Ran (14,
26).
The NPC possesses extensive peripheral structures extending into the
nuclear interior (for recent reviews, see references 32 and 33) including filamentous
basket-like structures that have been proposed to provide docking sites
for nuclear export intermediates (2). Vertebrate
NP-repeat-containing nucleoporins Nup98, Nup153, and Nup214 have been
assigned to peripheral NPC structures (4, 8, 37) and have
been implicated in the nuclear export (2, 3, 35, 36, 48). In
the Saccharomyces cerevisiae two-hybrid system, it has been
shown that FG-containing repeat domains of different nucleoporins
interact with the Rev NES via CRM1 with various efficiencies,
suggesting that nucleoporins play a role in NES-mediated export
(5, 16, 17, 31, 45).
In this report, we addressed the roles of Nup98, Nup153, and Nup214 for
Rev function in human cells. We show that Nup98 and Nup214 are not
solely stationary components of the NPC since (i) the presence of
actinomycin D leads to cytoplasmic translocation of Nup98 and (ii) Rev
can associate with Nup98 and Nup214, but not Nup153, outside the NPC.
The codistribution of Rev with Nup98 and Nup214 as well as with hCRM1
and the competitive inhibition of Rev function by the isolated
nucleoporin repeat domains of Nup98 and Nup214 suggest that Nup98 and
Nup214, but not Nup153, are the major downstream partners of hCRM1 and
play a direct role in the export of Rev from the nucleolus to the cytoplasm.
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MATERIALS AND METHODS |
Recombinant DNA.
The molecular clones of HIV-1 pRev(
)HIV
(pNL4-3fB [12, 40]) and pRev()RRE().CTE
[previously named pNL43Rev()RRE().S (53)]
as well as the expression vectors for the tagged green (green
fluorescent protein [GFP]) and blue (blue variant of GFP [BFP])
variants of Rev and NES()Rev (43) have been described elsewhere. The plasmids expressing GFP-tagged proteins were prepared by
insertion of the PCR-amplified coding sequences (CDS) into the
NheI site of pCMV-GFPsg25 (44). The hybrid genes
encode the N-terminal tripeptide Met-Ala-Ser followed by the respective protein starting at its second amino acid residue and the complete GFP.
The untagged versions of these proteins were produced by providing a
stop codon after their CDS in the above-described constructs,
generating proteins with authentic C termini. The GFP-tagged and
untagged NP domains of Nup98 and Nup153 contain the CDS from amino
acids (aa) 2 to 494 of the human Nup98 protein (6), and aa
894 to 1475 of Nup153. The hemagglutinin (HA)-tagged Nup98 plasmid was
based on p37R vector (46, 52) and included an N-terminal HA
epitope (Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala) inserted between aa 1 and
2 of these proteins. HA-tagged Nup214-NP expression plasmid was a gift
from J. Hauber. HA-tagged Nup153 plasmid (2) was obtained
from B. Burke. To construct the expression plasmid for hCRM1, its CDS
was PCR amplified using hCRM1 cDNA (15) and inserted into
p37R vector as described above for HA-tagged plasmids. The GFP-NES
plasmid encodes the GFP protein fused to the Rev NES and was a gift
from E. Afonina and G. N. Pavlakis. The authenticity of the
constructs was verified by double-strand sequencing.
Cell culture and transfections.
HLtat is a HeLa-derived cell
line that constitutively produces HIV-1 Tat protein (39).
Human 293 is an embryonic kidney cell line. Cells were maintained in
Dulbecco modified Eagle medium (DMEM) containing 10% fetal calf serum
plus penicillin and streptomycin (53). For microscopy of
living cells, 60-mm-diameter plastic transfection plates were seeded in
DMEM without phenol red. Cells were transfected by the calcium
phosphate coprecipitation technique, using DNA purified on QIAGEN
columns, and total protein was extracted (53). L3luc
luciferase expression plasmid was included in the transfection
mixtures, and the luciferase activity was measured (41). GFP
fluorescence was measured in cell lysates with a CytoFluor fluorimeter
(43). HIV-1 p24gag antigen was
measured by using commercial antigen capture assay kits (Cellular
Products) (53). LMB was a generous gift from B. Wolff
(51). Stock solutions of LMB were made at 0.5 mM in 1:1
dimethyl sulfoxide-isopropanol and stored at 70°C. Working solutions of 0.5 µM LMB in DMEM were kept at 20°C. For 293 and HLtat cells, LMB treatment was performed with 10 nM LMB for 4 h at
37°C.
Antibodies, immunofluorescence, and microscopy.
The rabbit
polyclonal antibodies for Rev (10) and GFP (44)
have been described elsewhere. The monoclonal antibody for the
influenza virus HA epitope was obtained from BAbCo (Berkeley, Calif.).
Rabbit antisera for hCRM1 were obtained from J. van Deursen and G. Grosveld. Affinity-purified rabbit antibodies for Xenopus Nup98 have been described elsewhere (36) and were a generous gift from M. Powers and D. Forbes. For indirect immunofluorescence, cells grown on plastic plates were fixed with 3.7% formaldehyde in
phosphate-buffered saline (PBS) for 10 min at room temperature, made
permeable by treatment with 0.1% Nonidet P-40 or 0.1% Triton X-100 in
PBS for 10 min, and labeled with the appropriate antibodies as
described previously (43). Integrity of the nuclei was
confirmed by using goat polyclonal anti-lamin A antibodies (Santa Cruz
Biotechnology) or, in living cells, a DNA-specific live fluorescent dye
(Hoechst 33342). Cells were observed under appropriate illumination
with a Zeiss Axiovert 135TV microscope. Digital images were acquired with a SenSys charge-coupled device camera (Photometrics) and processed
with IPLab Spectrum software. Digital deconvolution was performed using
the HazeBuster extension of IPLab Spectrum software. Measurements of
GFP fusion proteins in cell lysates were performed as described
previously (52).
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RESULTS |
Nup98 can translocate to the cytoplasm.
We studied the
localization of Nup98, Nup153, and Nup214 in the human HeLa-derived
HLtat cell line by using indirect immunofluorescence. We used an
affinity-purified antibody raised against Xenopus Nup98 which has been shown to react with human Nup98 (36) to
visualize endogenous Nup98 (Fig. 1A), and
we used an anti-HA mouse monoclonal antibody to visualize the HA
epitope-tagged transfected Nup98 (Fig. 1B). Both endogenous Nup98 and
HA-tagged Nup98 show similar subcellular localization. They are found
predominantly in the nucleoplasm, at the nuclear rim, and as dots in
the cytoplasm and over the nucleus but are excluded from the nucleoli
(Fig. 1A, left panel). The nuclear rim association is more prominent upon digital deconvolution microscopy (data not shown). Similar localization of endogenous Nup98 was found in cultured
Xenopus cell line A6 with the same antibody, in agreement
with published data (36).
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FIG. 1.
Nup98, but not Nup153 and Nup214, is able to translocate
to the cytoplasm. Endogenous Nup98 was visualized by using
affinity-purified anti-Xenopus Nup98 serum (A), whereas
HA-tagged Nup98, Nup214, and Nup153 proteins were detected with anti-HA
antibody (B to D). HLtat cells were transiently transfected with 2 µg
of the indicated HA-tagged nucleoporin expression plasmids (B to D) or
not transfected (A). Some cells were treated with 2 µg of actinomycin
D (ActD) per ml for 2 h. The indirect immunofluorescence and
fluorescence microscopy were performed as described in Materials and
Methods, and the images were filtered with a linear filter (3 × 3 kernel), using IPLab Spectrum software. Bar, 20 µm.
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To examine the possible ability of Nup98 to shuttle between the nuclear
and cytoplasmic compartments, we asked whether the
presence of an
inhibitor of transcription affects the localization
of Nup98, as has
been reported for several other shuttle proteins
such as Rev
(
29). Interestingly, we found that the endogenous
Nup98
(Fig.
1A) and HA-Nup98 (Fig.
1B) completely translocate
from the
nucleus to the cytoplasm of the HeLa cells upon treatment
with
actinomycin D in the absence or presence of the translation
inhibitor
cycloheximide. This translocalization was most prominent
in confluent
monolayers of HLtat cells, whereas we did not find
this effect in A6
cells (not shown). These data demonstrate that
Nup98 can be exported
from the nucleus of HeLa
cells.
Upon transient transfection of HLtat cells, HA-Nup214
predominantly localizes to the nuclear rim and can also be detected
in
the cytoplasm (Fig.
1B, left panel), in agreement with published
data
(
25). The HA-tagged Nup153 (Fig.
1C, left panel) localizes
to the nuclear rim and the nucleoplasm, as previously described
(
2). In contrast to Nup98, treatment with actinomycin D did
not affect the localization of either Nup214 or Nup153 (Fig.
1B
and C,
right panels). These findings indicate that Nup98, in contrast
to
Nup153 and Nup214, is a dynamic factor rather than a stationary
component of the
NPC.
Rev recruits Nup98 and Nup214 to the nucleoli.
Next, we
asked whether Rev can associate with intact nucleoporins Nup98, Nup153,
and Nup214 in mammalian cells and thereby affect their subcellular
localization. We expressed these HA-tagged nucleoporins in HLtat cells
in the presence of GFP-tagged Rev or, as a control, the Rev mutant that
lacks the NES [GFP-NES()Rev]. As expected from the previous
studies, both Rev and NES()Rev localized predominantly to the
nucleoli (Fig. 2, bottom panels). As
shown in Fig. 2A, the presence of Rev resulted in the redistribution of
Nup98 to the nucleoli. In contrast, the presence of NES()Rev had no
effect on the localization of Nup98, which remained excluded from the
nucleoli (Fig. 2A). Similarly, we found that Rev, but not NES()Rev,
can recruit Nup214 to the nucleoli (Fig. 2B). In contrast to Nup98 and
Nup214, Rev did not affect the localization of Nup153 (Fig. 2C).
Western immunoblots and fluorescence measurements of GFP-tagged
proteins confirmed the presence of comparable amounts of Rev and
NES()Rev proteins, and similar results were obtained with untagged
Rev proteins, which were visualized by indirect immunofluorescence
(data not shown).
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FIG. 2.
Rev recruits Nup98 and Nup214 to the nucleoli. HLtat
cells were transfected with 0.5 µg of the HA-tagged nucleoporin
expression plasmids in the presence of 1 µg of GFP-Rev or 1 µg of
GFP-NES()Rev expression plasmid as indicated at the bottom of the
figure. The GFP fluorescence and HA indirect immunofluorescence were
detected in the same cells, as indicated to the left. Fluorescence
microscopy was performed as described in the legend to Fig. 1. Bars, 20 µm.
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We further found that in the presence of Rev, the isolated NP domains
of Nup98 and Nup153 are also recruited to the nucleoli
and that this
localization is mediated by the NES of Rev and hCRM1,
since it is
interrupted by LMB (data not shown). The recruitment
of NP domain of
Nup153 is in contrast to our finding of the lack
of intact Nup153 to
codistribute with Rev (Fig.
2) and is most
likely due to the generic
ability of various NP domains to interact
with hCRM1/Rev, as suggested
by Neville et al. (
31).
Taken together, these experiments demonstrate that Rev can recruit
intact Nup98 and Nup214, but not Nup153, to the nucleoli.
This finding
demonstrates that both Nup98 and Nup214 are dynamic
factors and,
importantly, are able to associate with Rev outside
the NPC. These
results further suggest a role of Nup98 and Nup214
in Rev
function.
Rev recruits hCRM1 to the nucleoli.
To examine the association
of hCRM1 and HIV-1 Rev in mammalian cells, we studied whether the
presence of Rev affects the localization of hCRM1. We transfected human
293 cells with the BFP-tagged Rev protein (44) or, as a
control, the BFP-tagged NES()Rev protein. Endogenous hCRM1 was mostly
found at the nuclear rim as well as within the nucleus (Fig.
3A), in agreement with published data (15), while both Rev and NES()Rev localized predominantly
to the nucleoli (Fig. 3A, right panels). Importantly, in the cells that
expressed Rev, but not NES()Rev, a significant fraction of hCRM1 was
found in the Rev-containing nucleoli (left top panel). Such
colocalization was observed in all Rev-expressing cells. Upon treatment
with LMB, the effect of Rev on hCRM1 localization was completely
abolished, whereas no effect of LMB on hCRM1 distribution was observed
in cells that did not produce detectable amounts of Rev or that
expressed the NES()Rev. Similar results were obtained with untagged
Rev and NES()Rev, which were detected by indirect immunofluorescence
as well as in studies using another cell line such as HLtat (data not
shown). Taken together, these findings suggest that Rev associates with
hCRM1 (Fig. 3A), which further allows the recruitment of Nup98 and
Nup214 (Fig. 2) to the nucleoli.
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FIG. 3.
hCRM1 codistributes with Rev and Nup98. (A) Human 293 cells were transfected with 1 µg of BFP-Rev (wild type [wt]) or
BFP-NES()Rev [NES()] expression plasmid as indicated to the
right, and treated with 6 nM LMB for 4 h. The endogenous hCRM1 was
visualized by indirect immunofluorescence with rabbit anti-hCRM1 serum
(15) and rhodamine red-conjugated goat anti-rabbit secondary
antibodies as previously described (43). BFP fluorescence is
shown pseudocolored in green. (B) HLtat cells were transfected with 0.5 µg of HA-Nup98 and subjected to double indirect immunofluorescence
for HA-Nup98 and the endogenous hCRM1, as indicated. Before
immunodetection, cells were extracted in situ with 0.004% digitonin in
0.3× PBS for 10 min at 4°C. Fluorescence microscopy was performed as
described in the legend to Fig. 1.
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We next addressed the question whether HA-Nup98 and hCRM1 codistribute
in the absence of Rev. We found that these proteins
colocalize in
nucleoplasmic foci in transfected HLtat cells (Fig.
3B). Since both
proteins showed diffuse nucleoplasmic localization
(Fig.
1B and
3A), we
performed in situ extraction with digitonin
prior to immunostaining in
order to remove the fraction of proteins
that is loosely bound to
nuclear structures. This treatment revealed
an extensive dot-like
pattern in which HA-Nup98 and hCRM1 colocalized,
as shown in Fig.
3B.
This dot-like pattern of hCRM1 was not observed
in the absence of
HA-Nup98 (Fig.
3B, indicated by an arrow). A
similar but less-extensive
colocalization pattern was obtained
without digitonin treatment (data
not shown). This result shows
that Nup98 is able to colocalize with
hCRM1 independent of Rev
outside the
NPC.
Competitive inhibition of Rev function by the isolated nucleoporin
repeat domains of Nup98 and Nup214.
We then explored the effects
of the isolated NP domains of Nup98, Nup214, and Nup153 on Rev
function. We reasoned that exogenously expressed, isolated NP domains
may be able to titrate soluble factors that interact with the
respective endogenous nucleoporins and that this depletion might affect
Rev-mediated HIV-1 expression, resulting in competitive inhibition.
Therefore, a Rev-deficient molecular clone was cotransfected into human
293 cells in the presence of sufficient amounts of Rev necessary to
achieve maximal HIV expression together with increasing amounts of
plasmids expressing the NP domains of Nup98, Nup214, or Nup153. One day
later, the cells were harvested and cell extracts were analyzed for Gag
production as a measure of Rev-mediated HIV-1 expression. Rev function
was potently inhibited by the NP domains of Nup98 (Fig.
4A) and Nup214 (Fig. 4B). The inhibition
by these NP domains occurred to similar degrees and in a dose-dependent
manner. In contrast, Nup153-NP inhibited Rev function ~100-fold less
(Fig. 4C). In another experiment, we obtained a similar ~100-fold
difference (Fig. 4D) in the inhibition of HIV-1 by Nup98-NP and
Nup153-NP and we also confirmed that similar amounts of GFP-tagged
proteins were present (Fig. 4E). Using the yeast two-hybrid system,
Fritz and Green (16) showed strong interaction of all three
NP domains with Rev. Interestingly, we found a differential effect of
these three NP domains on Rev function reflecting intrinsic properties
of these properties in mammalian cells. Despite the differences in size
and the number of nucleoporin repeats (19 FG repeats within the 227-aa
Nup214-NP versus 39 repeats within the 492-aa Nup98-NP), the extent of
Rev inhibition was very similar for these two NP domains. The NP domain of Nup153 contains 25 FG repeats, which is comparable to those of Nup98
and Nup214. The different numbers of FG repeats probably do not account
for the observed differential effects on Rev function. We conclude that
the observed inhibition by Nup153-NP may reflect the ability of generic
NP repeats to interfere with Rev (45), while the strong
differential effect of the NP domains of Nup98 and Nup214 likely
indicates the direct participation of these nucleoporins in the Rev
pathway.
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FIG. 4.
NP domains of Nup98 and Nup214 specifically affect the
function of Rev. (A to C) Human 293 cells were transfected with 1 µg
of Rev()HIV in the presence of 25 ng of the pBsRev expression plasmid
(Rev/RRE) or 1 µg of the Rev()RRE().CTE Rev-independent HIV-1
(CTE). All transfections included 0.1 µg of L3luc, a luciferase
expression vector. Increasing amounts of expression plasmid Nup98-NP
(A), Nup214-NP (B), or Nup153-NP (C) were cotransfected (plotted on the
x axis in micrograms). Gag production was measured in the
lysates at one day posttransfection (p24gag
measured in nanograms per milliliter) and is plotted on the
y axis. Results from a representative experiment are shown.
Similar results were obtained in three independent transfection
experiments. (D and E) 293 cells were cotransfected with 1 µg of
Rev()HIV in the presence of 25 ng of pBsRev expression plasmid and in
the absence or presence of the indicated GFP-tagged NP expression
plasmids. HIV expression is determined by measuring Gag production and
expressed as a percentage of the value obtained in the absence of the
NP domains (D). Western immunoblot analysis (E) of the extracts shown
in panel D. GFP-tagged proteins were detected by using rabbit anti-GFP
serum for the experiment shown in panel D. The positions of protein
markers (in kilodaltons) are shown to the left. The expected molecular
masses of GFP fusion proteins were 27 kDa (GFP), 77 kDa (GFP-Nup98-NP),
and 86 kDa (GFP-Nup153-NP). n/t, nontransfected cells.
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As a control for the specificity of the inhibition, we used an
otherwise isogenic Rev-RRE-deficient molecular clone of HIV-1
that
contains the type D retrovirus posttranscriptional control
element
(CTE) necessary for expression (
53). CTE has been shown
to
utilize an export pathway distinct from that of Rev-RRE (
34,
38,
52) and involves the cellular TAP protein (
22). We
also
used a luciferase mRNA transcribed from the HIV-1 long terminal
repeat promoter, representing a transcript that does not depend
on
posttranscriptional regulation. The NP domains of all three
nucleoporins tested affected the CTE-mediated expression of HIV-1
(Fig.
4A to C) and the luciferase expression (not shown) similarly
but
to a much lesser extent than the Rev-mediated HIV expression.
Based on
this comparison, we concluded that the observed dose-dependent
inhibition of Rev-mediated expression by Nup98-NP and Nup214-NP
was due
to a preferential inhibition of Rev
function.
The inhibition of Rev function induced by the NP domain of Nup98
can be counteracted by excess Rev.
Since hCRM1 is the likely
molecular link between Rev and the nucleoporins, we asked whether hCRM1
can outcompete the inhibition mediated by the NP domain of Nup98. We
found that cotransfection of more than 1 µg of the hCRM1-producing
plasmid preferentially inhibited the Rev-mediated HIV expression
compared to expression of isogenic HIV-1 mRNAs that are controlled by
the CTE (Fig. 5A). It is plausible that CRM1 is present at the optimal
amounts required for nucleocytoplasmic export and that excess CRM1
disturbs this balance and negatively interferes with processes
involving CRM1-mediated export. In addition, we found that the presence
of excess hCRM1 did not counteract the Nup98-NP-induced inhibition of
Rev-mediated expression (Fig. 5A). One
explanation for the observed lack of complementation is that the amount
of exogenous hCRM1 required for the titration of Nup98-NP interferes
with cellular CRM1-mediated export and thereby with HIV-1 expression
per se. Alternatively, Nup98-NP may bind more strongly to the hCRM1-Rev
complex than to free hCRM1. In this model, hCRM1-Rev complexes, but not
free hCRM1, will preferentially compete for Nup98-NP domain, and the amount of Rev, but not the amount of CRM1, will determine the outcome
of the experiment. To test this model, we cotransfected constant
amounts of the Rev()HIV-1 clone and of the Nup98-NP expression
plasmid necessary to achieve ~100-fold inhibition. The transfection
mixtures also contained increasing amounts of a Rev expression vector.
The amount of Rev used has been shown to allow maximal HIV expression
in the absence of inhibitor (data not shown). As shown in Fig. 5B, the
increasing amounts of Rev led to a dose-dependent rescue of HIV-1
expression. Similar results were obtained when a wide range of Rev and
Nup98-NP concentrations was used (data not shown). The increase in HIV
expression is specific, since increasing amounts of Rev did not affect
the CTE-mediated expression of HIV-1 (data not shown) or luciferase
expression (Fig. 5B). Therefore, our data favor the model in which
Nup98-NP interacts better with Rev-CRM1 than with CRM1 alone.
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FIG. 5.
Rev counteracts the inhibitory effect of the NP of
Nup98. (A) 293 cells were transfected with 1 µg of Rev()HIV and
0.25 µg of pBsRev in the absence or presence of 0.25 µg of
Nup98-NP. In parallel, transfections were done with 1 µg of
Rev()RRE().CTE in the absence of Nup98-NP (CTE). Increasing amounts
of hCRM1 expression plasmid were cotransfected (plotted on the
x axis in micrograms). p24gag
expression was measured and is plotted on the y axis as raw
values (in nanograms per milliliter of lysate). Similar results were
obtained in three independent transfection experiments. Panels A to C
show results from representative experiments. (B) Rev counteracts the
effect of Nup98-NP on HIV-1 expression. Human 293 cells were
transfected with 1 µg of Rev()HIV (Rev/RRE) in the presence of 0.2 µg of Nup98-NP. Transfections included the luciferase plasmid.
Increasing amounts of pBsRev plasmid, starting at 25 ng, were
cotransfected (plotted on the x axis in micrograms).
p24gag expression (in nanograms per milliliter
of lysate) is plotted on the left y axis as raw values.
Luciferase values are plotted on the right y axis as
multiples of 103 firefly units (FFU). (C) 293 cells were
cotransfected with 0.25 or 2 µg of GFP-Rev expression plasmid in the
presence or absence of 0.2 µg of Nup98-NP plasmid, as indicated. At
day 1 posttransfection, some cells were treated with actinomycin D (+)
in the presence of cycloheximide as indicated at the top, and GFP
fluorescence was visualized as described previously (43).
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We further demonstrated that Nup98's NP domain directly affects the
nuclear export of Rev by studying the subcellular localization
of a
fluorescent GFP-tagged Rev protein in living cells. A constant
amount
of the Nup98-NP expression vector necessary to inhibit
HIV expression
was cotransfected with either small or large amounts
of a GFP-tagged
Rev expression plasmid. One day later, the cells
were treated with
actinomycin D and the nuclear export of GFP-tagged
Rev protein was
visualized directly. Figure
5C shows that when
Rev is present at small
amounts, its export is abolished by Nup98-NP.
In contrast, when present
at large amounts, Rev counteracts this
inhibition and regains its
ability to translocate to the cytoplasm.
The same results were obtained
by using untagged Rev proteins
and indirect immunofluorescence (not
shown). To further support
the specificity of the effect of Nup98-NP,
we also studied the
export of a GFP hybrid containing the NES of Rev as
well as the
export of RanBP1, a protein we previously showed to contain
a
Rev-like NES which determines its cytoplasmic accumulation
(
52).
Nup98-NP inhibited the export of both NES-GFP and
RanBP1, demonstrating
that this inhibition reflects the interference of
the NP domain
with CRM1 function in general, and this interference is
independent
of the nature of its
ligand.
Under the conditions leading to the inhibition of Rev's nuclear export
by Nup98-NP, we did not observe reduced nuclear accumulation
of Rev or
NES(
)Rev or inhibition of the nuclear import of human
glucocorticoid
receptor and human hnRNP A1 protein (not shown).
Therefore, the effect
of the isolated Nup98-NP domain is clearly
on export but not import, in
agreement with published data (
2,
35,
36). Taken together,
the data provided in Fig.
5 show
that competitive inhibition of Rev's
function by Nup98-NP is due
to the competitive inhibition of Rev's
nuclear
export.
| |
DISCUSSION |
In this study, we show that in HeLa cells Nup98 can translocate to
the cytoplasm upon actinomycin D treatment and that Rev is able to
recruit both Nup98 and Nup214, but not Nup153, to the nucleolus. We
further provided evidence that the isolated NP domains of Nup98 and
Nup214 are able to competitively inhibit Rev-mediated expression by
interfering with Rev's nuclear export. Taken together, these data
suggest that Nup98 and Nup214, but not Nup153, are dedicated downstream
partners of hCRM1 in human cells. The fact that the interaction of Rev
and these nucleoporins is mediated via hCRM1 and our finding that hCRM1
associates with Rev via its nuclear export signal in the nucleoli
further point to the nucleolus as a integral part necessary for the Rev
function. hCRM1 has previously been shown to be a dynamic factor, able
to relocalize from the nuclear envelope to the nucleoplasm in response
to expression of Nup214-NP (15). The nucleolus has been
implicated as part of normal hCRM1 route, based on the presence of hCRM
in the nucleoli of Nup214-depleted embryos and based on its nucleolar
relocalization upon treatment with actinomycin D in HtTA-1 cells
(15). Since we did not observe actinomycin D-induced
translocation of hCRM1 in 293 and HLtat cell lines (data not shown),
the presence of the NES of Rev thus appears to be necessary and
sufficient for hCRM1's nucleolar recruitment. This observation
indicates that hCRM1 associates with Rev on the normal route of both proteins.
The above findings led us to propose a model of participation of Nup98
and Nup214 in the hCRM1-mediated nuclear export of Rev. We speculate
that transport intermediates assemble in the nucleoli or the
nucleoplasm and that these intermediates include Rev, hCRM1, and Nup98
or Nup214. In this model, Nup98 and Nup214 act as soluble factors by
targeting the preassembled Rev-hCRM1-nucleoporin complexes to the NPC.
Nup98 and Nup214 are proto-oncogenes that have been implicated in human
myeloid leukemias. Chromosomal translocations leading to acute myeloid
leukemia (AML) produced fusions of Nup98's NP domain with HOXA9, a
transcription factor potentially involved in myeloid differentiation
(6, 30), or with a putative RNA helicase, DDX10
(1). The Nup98-HOXA9 fusion was predicted to act through its
HOXA9 moiety by inhibition of HOXA9-mediated differentiation. Based on
the predicted properties of DDX10, the Nup98-DDX10 protein was not
expected to act on the level of transcription. Therefore, the presence
of a NP moiety of Nup98 on both fusions indicates that it may
contribute to AML in a transcription-independent manner. Nup214 is also
activated by fusions of its NP domain to different genes unrelated to
transcription initiation factors (49), leading to myeloid
leukemias. This led us to speculate that Nup98 and Nup214 oncogenic
fusions may contribute to these malignancies by the same underlying
mechanism, mediated through their NP domains.
The NES-containing proteins I
B and inhibitor of protein kinase A
(PKI) regulate the activity and the nucleocytoplasmic transport of NF-B and protein kinase A, respectively. IB and PKI
have been suggested to negatively regulate signal transduction by
clearing their respective substrates from the nucleus to ensure timely signal termination (11, 16, 50). Mitogen-activated protein kinase kinase (MAPKK) has been shown to control the nuclear
accumulation of mitogen-activated protein kinase (MAPK) by a similar
mechanism (20, 24). The nuclear export of MAPKK is mediated
by a Rev-type NES that has been recently shown to interact with hCRM1
(18). Disruption of the NES within constitutively
active MAPKK has been shown to increase its ability to induce
transformation and led to a dramatic increase of activated MAPK in
the nucleus (19). Therefore, the inhibition of NES-mediated
export may be predicted to result in enhanced or constitutive signals
through the above transducers. In our experimental model, the
nucleoporin repeat domains of Nup98 and Nup214 alone or as a fusion to
a neutral moiety, GFP, resulted in extremely strong and specific
inhibition of NES-mediated export, by hCRM1-bridged titration of a
low-abundance NES substrate. The configuration of the Nup98-NP within
GFP hybrid protein used in this report directly mimics the
above-described Nup98 oncogenic fusion proteins. Therefore, the
phenotypes of NP proteins described here may reflect the contribution
of the NP moiety to the phenotypes of Nup98 and Nup214
oncogenic fusion proteins. If there is an analogy between our
experimental system and primary leukemia cells, these oncoproteins may
cause unregulated signal transduction through NF-B, protein
kinase A, or MAPK, contributing to AML. In support of this model,
constitutive activation and MAPK and MAPKK is sufficient for cell
transformation (7, 9, 27, 28) and has been implicated in AML
(47).
| |
ACKNOWLEDGMENTS |
We thank J. Hauber, E. Afonina, G. N. Pavlakis, D. Forbes,
M. Powers, G. Grosveld, and J. van Deursen for reagents and J. Bear for
technical assistance. We are grateful to A. Gragerov, G. N. Pavlakis, and E. Izaurralde for critically reading the manuscript.
This research was sponsored by the National Cancer Institute, DHHS,
under contract with ABL.
| |
ADDENDUM IN PROOF |
We showed by a cell fusion assay that both Nup98 and Nup153
were able to shuttle between nuclei. Nevertheless, Nup153 could not be recruited to the nucleoi by Rev (Fig. 2). Recently, Bogerd et
al. (H.P. Bogerd, A. Echarria, T. M. Ross, and B. R. Cullen, J. Virol. 72:8627-8635, 1998) also found that
NP-Nup214 has a differential effect on Rev/RRE- versus CTE-controlled expression.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: ABL-Basic
Research Program, Bldg. 535, Rm. 110, NCI-FCRDC, Frederick, MD
21702-1201. Phone: (301) 846-5159. Fax: (301) 846-7146. E-mail:
felber{at}mail.ncifcrf.gov.
| |
REFERENCES |
| 1.
|
Arai, Y.,
F. Hosoda,
H. Kobayashi,
K. Arai,
Y. Hayashi,
N. Kamada,
Y. Kaneko, and M. Ohki.
1997.
The inv(11)(p15q22) chromosome translocation of de novo and therapy-related myeloid malignancies results in fusion of the nucleoporin gene, NUP98, with the putative RNA helicase gene, DDX10.
Blood
89:3936-3944[Abstract/Free Full Text].
|
| 2.
|
Bastos, R.,
A. Lin,
M. Enarson, and B. Burke.
1996.
Targeting and function in mRNA export of nuclear pore complex protein Nup153.
J. Cell Biol.
134:1141-1156[Abstract/Free Full Text].
|
| 3.
|
Boer, J.,
J. Bonten-Surtel, and G. Grosveld.
1998.
Overexpression of the nucleoporin CAN/NUP214 induces growth arrest, nucleocytoplasmic transport defects, and apoptosis.
Mol. Cell. Biol.
18:1236-1247[Abstract/Free Full Text].
|
| 4.
|
Boer, J. M.,
J. M. van Deursen,
H. J. Croes,
J. A. Fransen, and G. C. Grosveld.
1997.
The nucleoporin CAN/Nup214 binds to both the cytoplasmic and the nucleoplasmic sides of the nuclear pore complex in overexpressing cells.
Exp. Cell Res.
232:182-185[Medline].
|
| 5.
|
Bogerd, H. P.,
R. A. Fridell,
S. Madore, and B. R. Cullen.
1995.
A novel cellular co-factor for HIV-1 Rev.
Cell
82:485-494[Medline].
|
| 6.
|
Borrow, J.,
A. M. Shearman,
V. P. Stanton, Jr.,
R. Becher,
T. Collins,
A. J. Williams,
I. Dube,
F. Katz,
Y. L. Kwong,
C. Morris,
K. Ohyashiki,
K. Toyama,
J. Rowley, and D. E. Housman.
1996.
The t(7;11)(p15;p15) translocation in acute myeloid leukaemia fuses the genes for nucleoporin NUP98 and class I homeoprotein HOXA9.
Nat. Genet.
12:159-167[Medline].
|
| 7.
|
Brunet, A.,
G. Pages, and J. Pouyssegur.
1994.
Constitutively active mutants of MAP kinase kinase (MEK1) induce growth factor-relaxation and oncogenicity when expressed in fibroblasts.
Oncogene
9:3379-3387[Medline].
|
| 8.
|
Cordes, V. C.,
S. Reidenbach,
A. Kohler,
N. Stuurman,
R. van Driel, and W. W. Franke.
1993.
Intranuclear filaments containing a nuclear pore complex protein.
J. Cell Biol.
123:1333-1344[Abstract/Free Full Text].
|
| 9.
|
Cowley, S.,
H. Paterson,
P. Kemp, and C. J. Marshall.
1994.
Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and for transformation of NIH 3T3 cells.
Cell
77:841-852[Medline].
|
| 10.
|
D'Agostino, D. M.,
V. Ciminale,
G. P. Pavlakis, and L. Chieco-Bianchi.
1995.
Intracellular trafficking of the human immunodeficiency virus type 1 Rev protein: involvement of continued rRNA synthesis in nuclear retention.
AIDS Res. Hum. Retroviruses
11:1063-1072[Medline].
|
| 11.
|
Fantozzi, D. A.,
A. T. Harootunian,
W. Wen,
S. S. Taylor,
J. R. Feramisco,
R. Y. Tsien, and J. L. Meinkoth.
1994.
Thermostable inhibitor of cAMP-dependent protein kinase enhances the rate of export of the kinase catalytic subunit from the nucleus.
J. Biol. Chem.
269:2676-2686[Abstract/Free Full Text].
|
| 12.
|
Feinberg, M. B.,
R. F. Jarrett,
A. Aldovini,
R. C. Gallo, and F. Wong-Staal.
1986.
HTLV-III expression and production involve complex regulation at the levels of splicing and translation of viral RNA.
Cell
46:807-817[Medline].
|
| 13.
|
Fischer, U.,
J. Huber,
W. C. Boelens,
I. W. Mattaj, and R. Luhrmann.
1995.
The HIV-1 Rev activation domain is a nuclear export signal that accesses an export pathway used by specific cellular RNAs.
Cell
82:475-483[Medline].
|
| 14.
|
Fornerod, M.,
M. Ohno,
M. Yoshida, and I. W. Mattaj.
1997.
CRM1 is an export receptor for leucine-rich nuclear export signals.
Cell
90:1051-1060[Medline].
|
| 15.
|
Fornerod, M.,
J. van Deursen,
S. van Baal,
A. Reynolds,
D. Davis,
K. G. Murti,
J. Fransen, and G. Grosveld.
1997.
The human homologue of yeast CRM1 is in a dynamic subcomplex with CAN/Nup214 and a novel nuclear pore component Nup88.
EMBO J.
16:807-816[Medline].
|
| 16.
|
Fritz, C. C., and M. R. Green.
1996.
HIV Rev uses a conserved cellular protein export pathway for the nucleocytoplasmic transport of viral RNAs.
Curr. Biol.
6:848-854[Medline].
|
| 17.
|
Fritz, C. C.,
M. L. Zapp, and M. R. Green.
1995.
A human nucleoporin-like protein that specifically interacts with HIV Rev.
Nature
376:530-533[Medline].
|
| 18.
|
Fukuda, M.,
S. Asano,
T. Nakamura,
M. Adachi,
M. Yoshida,
M. Yanagida, and E. Nishida.
1997.
CRM1 is responsible for intracellular transport mediated by the nuclear export signal.
Nature
390:308-311[Medline].
|
| 19.
|
Fukuda, M.,
I. Gotoh,
M. Adachi,
Y. Gotoh, and E. Nishida.
1997.
A novel regulatory mechanism in the mitogen-activated protein (MAP) kinase cascade. Role of nuclear export signal of map kinase kinase.
J. Biol. Chem.
272:32642-32648[Abstract/Free Full Text].
|
| 20.
|
Fukuda, M.,
Y. Gotoh, and E. Nishida.
1997.
Interaction of MAP kinase with MAP kinase kinase: its possible role in the control of nucleocytoplasmic transport of MAP kinase.
EMBO J.
16:1901-1908[Medline].
|
| 21.
|
Gorlich, D.,
M. Dabrowski,
F. R. Bischoff,
U. Kutay,
P. Bork,
E. Hartmann,
S. Prehn, and E. Izaurralde.
1997.
A novel class of RanGTP binding proteins.
J. Cell Biol.
138:65-80[Abstract/Free Full Text].
|
| 22.
|
Grüter, P.,
C. Tabernero,
C. von Kobbe,
C. Schmitt,
C. Saavedra,
A. Bachi,
M. Wilm,
B. Felber, and E. Izaurralde.
1998.
TAP, the human homolog of Mex67p, mediates CTE-dependent RNA export from the nucleus.
Mol. Cell
1:649-659[Medline].
|
| 23.
|
Izaurralde, E., and S. Adam.
1998.
Transport of macromolecules between the nucleus and the cytoplasm.
RNA
4:351-364[Abstract].
|
| 24.
|
Jaaro, H.,
H. Rubinfeld,
T. Hanoch, and R. Seger.
1997.
Nuclear translocation of mitogen-activated protein kinase kinase (MEK1) in response to mitogenic stimulation.
Proc. Natl. Acad. Sci. USA
94:3742-3747[Abstract/Free Full Text].
|
| 25.
|
Kraemer, D.,
R. W. Wozniak,
G. Blobel, and A. Radu.
1994.
The human CAN protein, a putative oncogene product associated with myeloid leukemogenesis, is a nuclear pore complex protein that faces the cytoplasm.
Proc. Natl. Acad. Sci. USA
91:1519-1523[Abstract/Free Full Text].
|
| 26.
|
Kutay, U.,
F. R. Bischoff,
S. Kostka,
R. Kraft, and D. Gorlich.
1997.
Export of importin alpha from the nucleus is mediated by a specific nuclear transport factor.
Cell
90:1061-1071[Medline].
|
| 27.
|
Mansour, S. J.,
J. M. Candia,
K. K. Gloor, and N. G. Ahn.
1996.
Constitutively active mitogen-activated protein kinase kinase 1 (MAPKK1) and MAPKK2 mediate similar transcriptional and morphological responses.
Cell Growth Differ.
7:243-250[Abstract].
|
| 28.
|
Mansour, S. J.,
W. T. Matten,
A. S. Hermann,
J. M. Candia,
S. Rong,
K. Fukasawa,
G. F. Vande Woude, and N. G. Ahn.
1994.
Transformation of mammalian cells by constitutively active MAP kinase kinase.
Science
265:966-970[Abstract/Free Full Text].
|
| 29.
|
Meyer, B. E.,
J. L. Meinkoth, and M. H. Malim.
1996.
Nuclear transport of human immunodeficiency virus type 1, visna virus, and equine infectious anemia virus Rev proteins: identification of a family of transferable nuclear export signals.
J. Virol.
70:2350-2359[Abstract].
|
| 30.
|
Nakamura, T.,
D. A. Largaespada,
M. P. Lee,
L. A. Johnson,
K. Ohyashiki,
K. Toyama,
S. J. Chen,
C. L. Willman,
I. M. Chen,
A. P. Feinberg,
N. A. Jenkins,
N. G. Copeland, and J. D. Shaughnessy, Jr.
1996.
Fusion of the nucleoporin gene NUP98 to HOXA9 by the chromosome translocation t(7;11)(p15;p15) in human myeloid leukaemia.
Nat. Genet.
12:154-158[Medline].
|
| 31.
|
Neville, M.,
F. Stutz,
L. Lee,
L. I. Davis, and M. Rosbash.
1997.
The importin-beta family member Crm1p bridges the interaction between Rev and the nuclear pore complex during nuclear export.
Curr. Biol.
7:767-775[Medline].
|
| 32.
|
Nigg, E. A.
1997.
Nucleocytoplasmic transport: signals, mechanisms and regulation.
Nature
386:779-787[Medline].
|
| 33.
|
Pante, N., and U. Aebi.
1996.
Molecular dissection of the nuclear pore complex.
Crit. Rev. Biochem. Mol. Biol.
31:153-199[Medline].
|
| 34.
|
Pasquinelli, A. E.,
R. K. Ernst,
E. Lund,
C. Grimm,
M. L. Zapp,
D. Rekosh,
M.-L. Hammarskjöld, and J. E. Dahlberg.
1997.
The constitutive transport element (CTE) of Mason-Pfizer monkey virus (MPMV) accesses a cellular mRNA export pathway.
EMBO J.
16:7500-7510[Medline].
|
| 35.
|
Powers, M. A.,
D. J. Forbes,
J. E. Dahlberg, and E. Lund.
1997.
The vertebrate GLFG nucleoporin, Nup98, is an essential component of multiple RNA export pathways.
J. Cell Biol.
136:241-250[Abstract/Free Full Text].
|
| 36.
|
Powers, M. A.,
C. Macaulay,
F. R. Masiarz, and D. J. Forbes.
1995.
Reconstituted nuclei depleted of a vertebrate GLFG nuclear pore protein, p97, import but are defective in nuclear growth and replication.
J. Cell Biol.
128:721-736[Abstract/Free Full Text].
|
| 37.
|
Radu, A.,
M. S. Moore, and G. Blobel.
1995.
The peptide repeat domain of nucleoporin Nup98 functions as a docking site in transport across the nuclear pore complex.
Cell
81:215-222[Medline].
|
| 38.
|
Saavedra, C.,
B. Felber, and E. Izaurralde.
1997.
The simian retrovirus-1 constitutive transport element, unlike the HIV-1 RRE, uses factors required for cellular mRNA export.
Curr. Biol.
7:619-628[Medline].
|
| 39.
|
Schwartz, S.,
B. K. Felber,
D. M. Benko,
E. M. Fenyö, and G. N. Pavlakis.
1990.
Cloning and functional analysis of multiply spliced mRNA species of human immunodeficiency virus type 1.
J. Virol.
64:2519-2529[Abstract/Free Full Text].
|
| 40.
|
Sodroski, J.,
W. C. Goh,
C. Rosen,
A. Dayton,
E. Terwilliger, and W. Haseltine.
1986.
A second post-transcriptional trans-activator gene required for HTLV-III replication.
Nature
321:412-417[Medline].
|
| 41.
|
Solomin, L.,
B. K. Felber, and G. N. Pavlakis.
1990.
Different sites of interaction for Rev, Tev, and Rex proteins within the Rev-responsive element of human immunodeficiency virus type 1.
J. Virol.
64:6010-6017[Abstract/Free Full Text].
|
| 42.
|
Stade, K.,
C. S. Ford,
C. Guthrie, and K. Weis.
1997.
Exportin 1 (Crm1p) is an essential nuclear export factor.
Cell
90:1041-1050[Medline].
|
| 43.
|
Stauber, R.,
G. A. Gaitanaris, and G. N. Pavlakis.
1995.
Analysis of trafficking of Rev and transdominant Rev proteins in living cells using green fluorescent protein fusions: transdominant Rev blocks the export of Rev from the nucleus to the cytoplasm.
Virology
213:439-454[Medline].
|
| 44.
|
Stauber, R. H.,
P. Carney,
K. Horie,
E. A. Hudson,
N. I. Tarasova,
G. A. Gaitanaris, and G. N. Pavlakis.
1998.
Development and applications of enhanced autofluorescent protein mutants.
BioTechniques
24:462-471[Medline].
|
| 45.
|
Stutz, F.,
E. Izaurralde,
I. W. Mattaj, and M. Rosbash.
1996.
A role for nucleoporin FG repeat domains in export of human immunodeficiency virus type 1 Rev protein and RNA from the nucleus.
Mol. Cell. Biol.
16:7144-7150[Abstract].
|
| 46.
|
Tabernero, C.,
A. S. Zolotukhin,
A. Valentin,
G. N. Pavlakis, and B. K. Felber.
1996.
The posttranscriptional control element of the simian retrovirus type 1 forms an extensive RNA secondary structure necessary for its function.
J. Virol.
70:5998-6011[Abstract].
|
| 47.
|
Towatari, M.,
H. Iida,
M. Tanimoto,
H. Iwata,
M. Hamaguchi, and H. Saito.
1997.
Constitutive activation of mitogen-activated protein kinase pathway in acute leukemia cells.
Leukemia
11:479-484[Medline].
|
| 48.
|
van Deursen, J.,
J. Boer,
L. Kasper, and G. Grosveld.
1996.
G2 arrest and impaired nucleocytoplasmic transport in mouse embryos lacking the proto-oncogene CAN/Nup214.
EMBO J.
15:5574-5583[Medline].
|
| 49.
|
von Lindern, M.,
S. van Baal,
J. Wiegant,
A. Raap,
A. Hagemeijer, and G. Grosveld.
1992.
Can, a putative oncogene associated with myeloid leukemogenesis, may be activated by fusion of its 3' half to different genes: characterization of the set gene.
Mol. Cell. Biol.
12:3346-3355[Abstract/Free Full Text].
|
| 50.
|
Wen, W.,
A. T. Harootunian,
S. R. Adams,
J. Feramisco,
R. Y. Tsien,
J. L. Meinkoth, and S. S. Taylor.
1994.
Heat-stable inhibitors of cAMP-dependent protein kinase carry a nuclear export signal.
J. Biol. Chem.
269:32214-32220[Abstract/Free Full Text].
|
| 51.
|
Wolff, B.,
J. J. Sanglier, and Y. Wang.
1997.
Leptomycin B is an inhibitor of nuclear export: inhibition of nucleocytoplasmic translocation of the human immunodeficiency virus type 1 (HIV-1) Rev protein and Rev-dependent mRNA.
Chem. Biol.
4:139-147[Medline].
|
| 52.
|
Zolotukhin, A. S., and B. K. Felber.
1997.
Mutations in the nuclear export signal of human ran-binding protein RanBP1 block the Rev-mediated posttranscriptional regulation of human immunodeficiency virus type 1.
J. Biol. Chem.
272:11356-11360[Abstract/Free Full Text].
|
| 53.
|
Zolotukhin, A. S.,
A. Valentin,
G. N. Pavlakis, and B. K. Felber.
1994.
Continuous propagation of RRE() and Rev()RRE() human immunodeficiency virus type 1 molecular clones containing a cis-acting element of simian retrovirus type 1 in human peripheral blood lymphocytes.
J. Virol.
68:7944-7952[Abstract/Free Full Text].
|
Journal of Virology, January 1999, p. 120-127, Vol. 73, No. 1
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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-
Shulga, N., Goldfarb, D. S.
(2003). Binding Dynamics of Structural Nucleoporins Govern Nuclear Pore Complex Permeability and May Mediate Channel Gating. Mol. Cell. Biol.
23: 534-542
[Abstract]
[Full Text]
-
Raval, A., Weissman, J. D., Howcroft, T. K., Singer, D. S.
(2003). The GTP-Binding Domain of Class II Transactivator Regulates Its Nuclear Export. J. Immunol.
170: 922-930
[Abstract]
[Full Text]
-
Daelemans, D., Afonina, E., Nilsson, J., Werner, G., Kjems, J., De Clercq, E., Pavlakis, G. N., Vandamme, A.-M.
(2002). A synthetic HIV-1 Rev inhibitor interfering with the CRM1-mediated nuclear export. Proc. Natl. Acad. Sci. USA
99: 14440-14445
[Abstract]
[Full Text]
-
Li, J., Liu, Y., Kim, B. O., He, J. J.
(2002). Direct Participation of Sam68, the 68-Kilodalton Src-Associated Protein in Mitosis, in the CRM1-Mediated Rev Nuclear Export Pathway. J. Virol.
76: 8374-8382
[Abstract]
[Full Text]
-
Sirri, V., Hernandez-Verdun, D., Roussel, P.
(2002). Cyclin-dependent kinases govern formation and maintenance of the nucleolus. JCB
156: 969-981
[Abstract]
[Full Text]
-
Catez, F., Erard, M., Schaerer-Uthurralt, N., Kindbeiter, K., Madjar, J.-J., Diaz, J.-J.
(2002). Unique Motif for Nucleolar Retention and Nuclear Export Regulated by Phosphorylation. Mol. Cell. Biol.
22: 1126-1139
[Abstract]
[Full Text]
-
Zolotukhin, A. S., Tan, W., Bear, J., Smulevitch, S., Felber, B. K.
(2002). U2AF Participates in the Binding of TAP (NXF1) to mRNA. J. Biol. Chem.
277: 3935-3942
[Abstract]
[Full Text]
-
Wiegand, H. L., Coburn, G. A., Zeng, Y., Kang, Y., Bogerd, H. P., Cullen, B. R.
(2002). Formation of Tap/NXT1 Heterodimers Activates Tap-Dependent Nuclear mRNA Export by Enhancing Recruitment to Nuclear Pore Complexes. Mol. Cell. Biol.
22: 245-256
[Abstract]
[Full Text]
-
Lohrum, M. A. E., Woods, D. B., Ludwig, R. L., Balint, E., Vousden, K. H.
(2001). C-Terminal Ubiquitination of p53 Contributes to Nuclear Export. Mol. Cell. Biol.
21: 8521-8532
[Abstract]
[Full Text]
-
Vasu, S., Shah, S., Orjalo, A., Park, M., Fischer, W. H., Forbes, D. J.
(2001). Novel vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export. JCB
155: 339-354
[Abstract]
[Full Text]
-
Savino, T. M., Gebrane-Younes, J., De Mey, J., Sibarita, J.-B., Hernandez-Verdun, D.
(2001). Nucleolar Assembly of the Rrna Processing Machinery in Living Cells. JCB
153: 1097-1110
[Abstract]
[Full Text]
-
Guzik, B. W., Levesque, L., Prasad, S., Bor, Y.-C., Black, B. E., Paschal, B. M., Rekosh, D., Hammarskjöld, M.-L.
(2001). NXT1 (p15) Is a Crucial Cellular Cofactor in TAP-Dependent Export of Intron-Containing RNA in Mammalian Cells. Mol. Cell. Biol.
21: 2545-2554
[Abstract]
[Full Text]
-
Hofmann, W., Reichart, B., Ewald, A., Muller, E., Schmitt, I., Stauber, R. H., Lottspeich, F., Jockusch, B. M., Scheer, U., Hauber, J., Dabauvalle, M.-C.
(2001). Cofactor Requirements for Nuclear Export of Rev Response Element (Rre)-And Constitutive Transport Element (Cte)-Containing Retroviral Rnas: An Unexpected Role for Actin. JCB
152: 895-910
[Abstract]
[Full Text]
-
Strasser, K., Bassler, J., Hurt, E.
(2000). Binding of the Mex67p/Mtr2p Heterodimer to Fxfg, Glfg, and Fg Repeat Nucleoporins Is Essential for Nuclear mRNA Export. JCB
150: 695-706
[Abstract]
[Full Text]
-
Ho, A. K., Shen, T. X., Ryan, K. J., Kiseleva, E., Levy, M. A., Allen, T. D., Wente, S. R.
(2000). Assembly and Preferential Localization of Nup116p on the Cytoplasmic Face of the Nuclear Pore Complex by Interaction with Nup82p. Mol. Cell. Biol.
20: 5736-5748
[Abstract]
[Full Text]
-
Michienzi, A., Cagnon, L., Bahner, I., Rossi, J. J.
(2000). Ribozyme-mediated inhibition of HIV 1 suggests nucleolar trafficking of HIV-1 RNA. Proc. Natl. Acad. Sci. USA
97: 8955-8960
[Abstract]
[Full Text]
-
Kang, Y., Bogerd, H. P., Cullen, B. R.
(2000). Analysis of Cellular Factors That Mediate Nuclear Export of RNAs Bearing the Mason-Pfizer Monkey Virus Constitutive Transport Element. J. Virol.
74: 5863-5871
[Abstract]
[Full Text]
-
Yang, J., Bogerd, H. P., Peng, S., Wiegand, H., Truant, R., Cullen, B. R.
(1999). An ancient family of human endogenous retroviruses encodes a functional homolog of the HIV-1 Rev protein. Proc. Natl. Acad. Sci. USA
96: 13404-13408
[Abstract]
[Full Text]
-
Hussey, D. J., Nicola, M., Moore, S., Peters, G. B., Dobrovic, A.
(1999). The (4;11)(q21;p15) Translocation Fuses the NUP98 and RAP1GDS1 Genes and Is Recurrent in T-Cell Acute Lymphocytic Leukemia. Blood
94: 2072-2079
[Abstract]
[Full Text]
-
Bear, J., Tan, W., Zolotukhin, A. S., Tabernero, C., Hudson, E. A., Felber, B. K.
(1999). Identification of Novel Import and Export Signals of Human TAP, the Protein That Binds to the Constitutive Transport Element of the Type D Retrovirus mRNAs. Mol. Cell. Biol.
19: 6306-6317
[Abstract]
[Full Text]
-
Boyle, S. M., Ruvolo, V., Gupta, A. K., Swaminathan, S.
(1999). Association with the Cellular Export Receptor CRM 1 Mediates Function and Intracellular Localization of Epstein-Barr Virus SM Protein, a Regulator of Gene Expression. J. Virol.
73: 6872-6881
[Abstract]
[Full Text]
-
Elfgang, C., Rosorius, O., Hofer, L., Jaksche, H., Hauber, J., Bevec, D.
(1999). Evidence for specific nucleocytoplasmic transport pathways used by leucine-rich nuclear export signals. Proc. Natl. Acad. Sci. USA
96: 6229-6234
[Abstract]
[Full Text]
-
Kang, Y., Cullen, B. R.
(1999). The human Tap protein is a nuclear mRNA export factor that contains novel RNA-binding and nucleocytoplasmic transport sequences. Genes Dev.
13: 1126-1139
[Abstract]
[Full Text]
-
Bailer, S. M., Balduf, C., Katahira, J., Podtelejnikov, A., Rollenhagen, C., Mann, M., Pante, N., Hurt, E.
(2000). Nup116p Associates with the Nup82p-Nsp1p-Nup159p Nucleoporin Complex. J. Biol. Chem.
275: 23540-23548
[Abstract]
[Full Text]
-
Goodwin, D. J., Whitehouse, A.
(2001). A gamma -2 Herpesvirus Nucleocytoplasmic Shuttle Protein Interacts with Importin alpha 1 and alpha 5. J. Biol. Chem.
276: 19905-19912
[Abstract]
[Full Text]
-
Griffis, E. R., Altan, N., Lippincott-Schwartz, J., Powers, M. A.
(2002). Nup98 Is a Mobile Nucleoporin with Transcription-dependent Dynamics. Mol. Biol. Cell
13: 1282-1297
[Abstract]
[Full Text]
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Abstract
Introduction
