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INTRODUCTION |
The genome of lentiviruses has evolved from simple retroviruses
encoding the classical gag, pol, and
env structural genes toward complexity, mainly by increasing
the number of additional genes. Lentiviruses are limited to a single
transcription unit, and controlled protein expression is mainly
achieved by a very large usage of alternative and facultative splicing
allowing the production of multiple mRNA with different coding
specificities. Consequently, most mRNA are either unspliced or
partially spliced and would normally be retained in the nucleus until
fully spliced. This apparent problem is circumvented in lentivirus such
as HIV-11 by the use of a
particular transacting factor called Rev, which bypasses the cellular
machinery by specifically inducing the nuclear export of these
incompletely spliced viral mRNA.
HIV-1 Rev is a small nuclear/nucleolar protein that binds specifically
to a highly structured 350-nucleotide-long RNA sequence called the Rev
response element (RRE) present in all incompletely spliced viral RNA
(1-3). The Rev/RRE interaction is an ordered process. Rev binds
initially to a single high affinity site located at the apex of the
longest stem of the RRE (4-7). This nucleation event allows additional
Rev molecules to multimerize on the RRE through co-operative
protein/protein and protein/RNA interactions (3, 4, 8, 9). Rev binding
to the RRE is mediated by a highly basic N-terminal domain, which also
specifies nuclear/nucleolar localization. In addition to this RNA
recognition domain, Rev also carries a short C-terminal leucine-rich
motif now called the nuclear export signal (NES) that authorizes Rev to
shuttle between the nucleus and the cytoplasm (10, 11). Structurally similar NES have now been found in many other viral and cellular proteins (for review see Ref. 12). The NES is transferable to heterologous proteins, and mutations in this domain impair both Rev
shuttling and Rev-dependent export of RNA. Furthermore,
when injected into Xenopus oocyte nuclei, a Rev NES peptide
is able to competitively inhibit the Rev-dependent export
of RNA as well as the export of 5 S rRNA and U1 small nuclear RNA (13).
When Rev multimerizes on the RRE, NES motifs are exposed to the outside of the RNA-protein complex and remain accessible for protein contact (14). Altogether, these observations have suggested that nuclear export
of viral mRNA is mediated by a direct interaction between the Rev
NES and cellular co-factors.
The first attempts to isolate Rev co-factors for export were performed
using the two-hybrid interaction trap in yeast. A human nucleoporin-like protein called hRIP/Rab in human and a new yeast nuclear pore-associated protein, Rip-1p, were shown to interact specifically with functional NES, which was consistent with a role of
Rev in nuclear export of RNA (15-17). Subsequent experiments performed
in yeast have demonstrated that many well characterized yeast or
mammalian nucleoporins (Nup) including Nup214/can, yNup159, yNup100,
Nup153, Nup98, Pom121, yNup49p, yNup57p, yNup100p, yNup116p, yNup145p,
rNup98 also contact the Rev NES with a similar specificity (17-19).
However, direct binding using recombinant proteins has been very
difficult to demonstrate, suggesting that these interactions are
indirect and involve a molecular bridge conserved between yeast and mammals.
Recently the identification of CRM-1 as an essential nuclear export
factor has clarified the problem (20-24). CRM-1 was initially described in Schizosaccharomyces pombe as an essential
protein involved in the control of higher order of chromosome structure and gene expression (25). Leptomycin B, an antifungal and antitumor antibiotic with cell cycle arresting activity, was shown to target specifically CRM-1 (26). Recently, the report of Wolff et
al. (27) showing that leptomycin B was a potent inhibitor of HIV-1 Rev-dependent nuclear export has suggested the possible
implication of CRM-1 in this process. Simultaneously, Fornerod et
al. (28) have found CRM-1 associated with Nup214 and Nup88 in a
dynamic subcomplex localized at the nuclear membrane. They have also
shown that CRM-1 shares significant homology within its N-terminal
domain with a family of Ran-binding proteins including importin
, a cellular protein involved in nuclear import. Definite demonstration of
the implication of CRM-1 in nuclear protein export was shown by
different approaches in yeast and human cells. Taking advantage of a
temperature-sensitive CRM-1 mutant strain of Saccharomyces cerevisiae, two groups reported that nuclear export of
NES-containing proteins is blocked at a nonpermissive temperature (21,
23). Furthermore, CRM-1 has been shown to interact with different NES motifs in vitro and in vivo in a complex that
also includes the Ran GTPase in its GTP-bound form (20, 22, 23). In
addition, binding of leptomycin B to CRM-1 inhibits the formation of
the NES·CRM-1·Ran complex (20).
Although the involvement of CRM-1 in nuclear export as a receptor for
NES-bearing proteins is now well documented, the question of the
translocation of the NES·CRM-1·Ran trimolecular complex through the
nuclear pore complex (NPC) has still to be addressed. For example, we
do not know the exact significance of the association of CRM-1 with
FG-rich proteins including those that have been reported to interact
with Rev in the two-hybrid system (29). Furthermore, a significant
fraction of mammalian NPC-associated proteins, which are possibly
involved in nucleo-cytoplasmic transport, have not been characterized yet.
Here we report the identification of a new FG-rich NLP-1
(nucleoporin-like protein
1) interacting with functional NES sequences including the
Rev NES. Similar to the previously identified Rev-interacting factor
hRIP/Rab, this protein is nuclear and associate with yeast and human
CRM-1. The possible involvement of NLP-1 as well as hRIP/Rab in the
first steps of protein nuclear export through the recruitment of the
NES·CRM-1·Ran complex to the NPC is discussed.
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EXPERIMENTAL PROCEDURES |
Oligonucleotides--
The oligonucleotides used are:
1, 5'-CGGATCCGTCGACATGCTGAGCCCGTCCCAC-3'; |
2, 5'-CCGAAGTCTCTCGAGTCCCAGAAGTTCCAC-3'; |
3, 5'-TGAGCCCGTCCCACTTCAGNNNCCTCCCNNNGAGCGTNNNACTNNNGACTGCAACGAGGATTGTGGAACTTCTGGGACGCAGGGTGTTGGAAGCCCTCAAATA-3'; |
4, 5'-CTAGTATTTGAGGGCTTCCAACACCCTGCGTCCCAGAAGTTCCACAATCCTCGTTGCAGTCNNNAGTNNNACGCTCNNNGGGAGGNNNCTGAAGTGGGACGGGC-3'; |
5, 5'-CTAGAGGGGCGGCGACTGGTGAGTACGCCG-3'; |
6, 5'-AATTCGGCGTACTCACCAGTCGCCGCCCCT-3'; |
7, 5'-CCTGGGCCCGCTAGCTTCTGCAACAACTGC-3'; |
8, 5'-CGGAATTCAGATCTGCTTCTTCCTGCC-3'; |
9, 5'-GGGAATTCTAGCTAGGTAGAGATGGGTGCGAGAGCGTCGG-3'; |
10, 5'-GCGCGGCTAGCGATCTAAGTTC-3'; |
11, 5'-GGAAGATCTAGAGCAGTGGGAATAGGAGCTTTGTT-3'; |
and 12, 5'-CCCAAGCTTAGAGCAACCCCAAATCCCC-3'. |
Yeast Interaction Trap--
The yeast two-hybrid interaction
trap was carried out using the LexA system. PLexRev and pLexM10 were
generated by subcloning respectively the Rev coding sequence derived
from pF31 (3) and its mutated counterpart into the BamHI and
SalI sites of the yeast expression plasmid pLex9 (30).
PLexRev was used to transform the recipient strain L40 (31), and the
resulting L40-pLexRev strain was subsequently transformed with a Gal4
transactivation domain-tagged Epstein-Barr virus-transformed human
peripheral lymphocytes cDNA library using standard procedures (32).
Transformants were selected on L/W/H amino acid-depleted DOBA medium
(Bio 101) for a week at 30 °C. Clones positive for histidine
expression were further tested for
-galactosidase activity using
classical colony lift filter assay (32). Plasmids encoding the preys
were finally recovered on leucine-depleted M9 agar plates after
transformation of bacterial strain HB101 with yeast DNA extracted from
the double positive colonies.
PLex
NES was constructed by deletion of the sequence coding for amino
acids 72-116 in pLexRev. Wild type Rev NES and M10 NES (amino acids
70-96) were amplified by PCR with oligonucleotides 1 and 2, cut with
BamHI and XhoI, and inserted into
BamHI-XhoI digested pLex9 to generate plasmid
pLexNES and pLexNESM10. PGADRev and pGADM10 resulted from the insertion
of the Rev and M10 coding sequence into pGAD424
(CLONTECH) digested with BamHI and
SalI. PLex-NLP-1
and pLex-Nup214c were generated by
inserting the BglII-BglII restriction fragment
from pACT NLP-1
and pACTNup214c into BamHI cut pLexA9
plasmid. Because of a basal activation of the histidine reporter gene,
plates were supplemented with 60 mM 3-amino-1,2,4 triazole
when plasmids pLex-NLP-1
and pLex-Nup214c were used in the
transformation experiment.
NLP-1 Expression Analysis--
Multiple tissue Northern blot
(CLONTECH) was hybridized with a uniformly
32P-labeled NLP-1
cDNA probe generated by random
priming (Appligene). O-glycosylation of NLP-1 was addressed
by incubating in vivo or in vitro expressed
proteins with wheat germ agglutinin (WGA)-Sepharose (Sigma) for 30 min
at 4 °C. After washing in 20 mM Tris, pH 7.5, 200 mM NaCl, and 0.1% Nonidet P-40, proteins were separated on acrylamide gels and visualized either by direct autoradiography or by
classical Western blot. NLP-1 subcellular localization was performed by
Western blot with total, nuclear, or cytoplasmic extracts. HeLa cells
were washed and scraped in ice-cold phosphate-buffered saline (PBS).
Total cellular extract was done by cell lysis in RIPA buffer for 30 min
at 4 °C followed by centrifugation at 12,000 × g
for 15 min. For nucleus and cytoplasm fractionation, cells were
resuspended in 500 µl of lysis buffer (10 mM Tris, pH
7.4, 3 mM CaCl2, 2 mM
MgCl2, 0.5% Nonidet P-40, 1 mM Pefabloc, 2 mM leupeptin) and passed 10 times through a 25-gauge
needle. The nuclei were pelleted by centrifugation at 14,000 × g for 20 s at 4 °C, and the cytoplasmic
fraction-containing supernatant was recovered. The nuclear fraction was
obtained by incubating nuclei in 500 µl of high salt buffer (RIPA
buffer, 500 mM NaCl, 1 mM Pefabloc, 2 mM leupeptin) for 40 min at 4 °C followed by a 15-min centrifugation at 12,000 × g.
Indirect Immunofluorescence--
A GST-NLP-1
fusion protein
was expressed in XL-1 bacteria and purified from inclusion bodies
as described in Ref. 33. Rabbit-specific anti-serum was raised against
recombinant GST-NLP-1
(Covalab) and affinity-purified on a
glutathione-agarose GST-NLP-1
column according to Ref. 34. Indirect
immunofluorescence was done on HeLa cells transiently expressing NLP-1
as described before (35). 48 h after transfection, cells grown on
coverglasses were washed three times with PBS and fixed in 4%
paraformaldehyde for 15 min. They were washed with PBS, permeabilized
in PBS with 0.5% Triton X-100 for 5 min, and incubated in PBS with 2%
bovine serum albumin for another 10 min. Incubation with affinity
purified anti-NLP-1
antiserum was performed for 30 min at room
temperature followed by three washes in PBS. Finally, cells were
incubated with a fluorescein isothiocyanate-conjugated goat anti-rabbit
IgG (Sigma), washed, and mounted in
moviol/1,4-diazabicyclo[2.2.]-octan.
Degenerate NES Library Construction and Screening--
A library
of plasmids expressing fusion proteins linking the LexA DNA-binding
domain to NES mutated Rev proteins was constructed as follows.
Oligonucleotides 3 and 4 in which the sequence coding for leucines 75, 78, 81, and 83 of the Rev NES have been randomized were phosphorylated,
hybridized, and cloned into SpeI-BlpI-cut plasmid
pLexM10. XL1 bacterial strain was transformed with the ligated DNA, and
the resulting plasmid library consisting of approximately 2 × 105 independent clones was finally amplified.
L40-pACT-NLP-1
yeast strain was transformed with the library DNA and
plated on L/W/H amino acid-depleted DOBA medium and incubated for a
week at 30 °C. Yeast transformants that have retained the ability to
interact specifically with NLP-1
were selected, and the DNA of the
corresponding plasmids were recovered as described previously.
Rev Reporter Construct and Expression Plasmids--
Rev mutants
interacting with NLP-1
were tested for their ability to substitute
for Rev and to promote export of a RRE-containing unspliced mRNA.
For this purpose, a new Rev reporter plasmid derived from pAAC plasmid
(36) was constructed by assembling PCR-amplified fragments derived from
pNL4.3 HIV-1 provirus (see Fig. 4A). The DNA fragment coding
for the first tat 5' splice site was generated by hybridizing
phosphorylated oligonucleotides 5 and 6. The DNA sequence coding for
the first tat 3' splice sites was amplified with
oligonucleotides 7 and 8. Plasmid pAACtat5' was constructed by cloning
tat 5' splice site sequence into
XbaI-EcoRI-digested pAAC. Sequences coding for
Gag p17 protein and for the 264-nucleotide-long RRE were amplified with
oligonucleotides 9 and 10 and oligonucleotides 11 and 12. The PCR
fragments were then digested with EcoRI and NheI,
NheI and BglII, and BglII and
HindIII, respectively, for the Gag p17, the tat 3' splice,
site, and the RRE and were subsequently ligated into the
EcoRI- and HindIII-cut pAACtat5'
construct to generate pVRS1. DNA sequences coding for wild type, M10,
M4 as well as for selected Rev mutants were subcloned into pSG5Flag expression plasmid (37). 500 ng of CsCl-purified pVRS1 reporter plasmid
and 20 or 100 ng of each Rev expressing plasmid were transfected into
HeLa cells by the calcium phosphate method using 10 µg of pUC19 as
carrier DNA. Cytoplasmic RNA was prepared 48 h after transfection
and was subjected to RNase protection assay as described before (38).
The RNA probe was prepared using HindIII-cut pMTX91 as
template (a gift from Mark Churcher) and T3 RNA polymerase. The probe
hybridize to 93 nucleotides of the HIV-1 Gag p17 coding sequence
(nucleotides 926-1018 of the pNL4.3 sequence, GenBankTM accession
number M19921).
Mammalian Two-hybrid System--
To monitor interaction between
Rev and its cellular partners in mammalian cells, we have set up a
two-hybrid system homologous to what we have used in yeast. Plasmid
pSG5LexA was constructed by cloning the LexA DNA-binding domain coding
sequence into the pSG5 expression vector (Amersham Pharmacia Biotech)
and was used to generate the "bait" plasmids. The "prey"
constructs derived from pFNV plasmid encoding the VP16 transcriptional
activation domain (amino acids 413-490) (37). To monitor the
interaction between the two proteins, we have used the reporter
plasmid, pSEAP LEXA5X constructed by inserting five LexA-binding sites
into the XhoI site of pSEAP promoter plasmid
(CLONTECH). Transfections were done in HeLa cells
plated in 35-mm Petri dishes. 120 ng of pSEAP LEXA5X and various amount
of the LexA or VP16 hybrids expressing plasmids were co-transfected by
the calcium phosphate method with 5 µg of pUC19 as carrier. 48 h
after transfection, secreted alkaline phosphatase was titrated by
chemioluminescence in 10 µl of cells supernatant using the SEAP
Reporter Gene Assay (Roche Molecular Biochemicals).
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RESULTS |
Two-hybrid Screen for Rev NES-interacting Proteins--
In an
attempt to clone Rev co-factors for nucleo-cytoplasmic export, we
performed a two-hybrid interaction screen in yeast using the Rev
protein as a bait. The L40-pLex-Rev yeast strain was transformed with a
human B-cell line cDNA library cloned in pACT and spread on L/W/H
amino acid-depleted medium. More than 2.5 million clones were plated,
leading to the isolation of 200 clones positive for histidine and
-galactosidase expression. These clones were subsequently tested
with pLex-M10, encoding a fusion between the DNA-binding domain of LexA
and M10, a Rev mutant impaired in its capacity of stimulating export of
HIV-1 mRNAs (Leu78-Glu79
Asp-Leu) (11).
Sixteen clones interacting with wild type Rev but not with M10 were
selected. Sequence analysis revealed that two of theses clones
correspond to the C terminus part of nucleoporin Nup214/can (clone
Nup214c), which has already been shown to interact with nuclear export
signal in the yeast two-hybrid system (18, 19). However, a 1.1-kb
cDNA clone corresponding to an unknown gene was also identified by
this extensive screening. The sequence of a gene called CG1
(candidate gene 1) (39),
corresponding exactly to this clone was subsequently deposited in the
GenBankTM data base (accession number U97198). The cDNA
fragment we obtained, which extends from codons 50 to 408 of the
423-amino acid-long CG1 putative open reading frame, was
called NLP-1
. To evaluate the extent of the interaction, we have
assayed
-galactosidase expression in yeast cells co-transformed by
pLex, pLexM10, or pLexRev and plasmids encoding the two identified
preys. As shown in Fig. 1A,
-galactosidase expression was low with the control plasmids or when
the LexM10 hybrid was used as a bait. However, a dramatic increase of
-galactosidase expression (up to 30 times), was obtained upon
co-transfection of pLexRev with pACTNLP-1
or pACTNup214c.

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Fig. 1.
Specific association of Nup214c and
NLP-1 with wild type Rev nuclear export signal
in yeast. A, interaction in yeast L40 cells revealed by
a liquid culture -galactosidase assay with
o-nitrophenyl- -D-galactopyranoside as
substrate. B, interactions monitored by growth on leucine,
tryptophan, and histidine amino acid-depleted agar plates. Hybrid
proteins consisting of the LexA DNA-binding domain fused to wild type
Rev, M10, Rev NES, or to a 26 amino acid wild type Rev NES or M10
mutated NES were tested for association with NLP-1 and Nup214c.
C, two-hybrid interactions using Nup214c or NLP-1 as bait
and Rev or M10 as prey.
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Interaction was further analyzed by testing the preys against the wild
type NES motif or its mutated counterpart fused to LexA. Results are
reported in Fig. 1B. They clearly demonstrated that the NES
domain alone could account for the Rev/NLP-1
interaction, suggesting
that no additional Rev sequences were necessary for this contact.
Furthermore, we challenged the interaction by swapping the bait by the
preys and vice versa. Plasmids expressing Lex-Nup214c and Lex-NLP-1
fusion proteins were co-transfected with plasmids expressing fusion
proteins between Rev or M10 and the transcriptional activation domain
of Gal4 (Gal4AD). As shown in Fig. 1C, the Rev/Nup214c and
the Rev/NLP-1
interactions are preserved when Nup214c and NLP-1
are tethered to Lex and Rev to Gal4AD.
Sequence Analysis of the NLP-1 Protein--
Having shown that the
Rev/NLP-1
interaction is specific of a functional NES, we decided to
analyze in more detail the gene corresponding to the NLP-1
cDNA
and its associated product. The NLP-1
cDNA fragment was
completely sequenced, and a 1.6-kb cDNA fragment encoding the
full-length 1269-base pair open reading frame was cloned by 5' and 3'
rapid amplification of cDNA ends PCR. The 423-amino acid sequence
deduced from the cDNA is reported in Fig.
2A and is similar to what has
been published for the CG1 gene (39). Statistical analysis
of the sequence reveal that the protein is very rich in serine residues
(one-sixth of the polypeptide is serine) and contains 12 FG repeats,
four repeats being of the PAFG type. The FG repeats are concentrated at
the C terminus part of the protein (amino acids 215-375), which is very reminiscent of the FG motifs found in most nucleoporins. At the
N-terminal side of the protein a putative CCCH type zinc finger motif
is also found. This sequence is homologous to zinc fingers motifs found
in other proteins like U2AF35, U2AF1, U2AF23, or yLEE1. In addition, a
putative coiled-coil region predicted by the COILS program at ISREC
(40) is found between amino acids 171 and 197 and separates the FG-rich
domain from the N terminus. Despite no obvious homologies with any
other proteins in the data bases, BLAST search at National Center for
Biotechnology Information with the FG-rich domain reveal partial
homology with various nucleoporins including Nup1, pom1, Nup159,
Nup214, Nup62, Nup42, etc. Furthermore, we have been able to show that
when translated in a rabbit reticulocyte lysate or when expressed in
human cells, the protein binds to WGA-Sepharose, indicating that it is
O-glycosylated in vitro (Fig. 2B) as
well as in vivo (Fig. 2C). In accordance with
previous observations and in agreement with protein expression, we have decided to name this protein nucleoporin-like protein 1 or NLP-1.

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Fig. 2.
Sequence analysis and glycosylation of the
NLP-1 protein. A, amino acid sequence of the NLP-1 open
reading frame. Repeated motifs including the phenylalanine glycine
dipeptide are boxed. Critical amino acids of both the
N-terminal putative zinc finger and the predicted coiled-coil domain
are indicated with asterisks. Serine residues are
underlined. Arrows indicate the N and C termini
of the polypeptide encoded by the clone NLP-1 . B,
in vitro glycosylation of NLP-1. In vitro
translated 35S-labeled Nup214c, NLP-1, and the Epstein-Barr
virus transactivator EB1 were subjected to WGA-Sepharose
chromatography. WGA-bound proteins were separated on a 12%
polyacrylamide gel. C, in vivo glycosylation of
NLP-1. Total protein extract made from HeLa cells (lane 2)
or HeLa cells overexpressing NLP-1 (lanes 1 and
3) were loaded on a WGA-Sepharose column (lanes 1 and 2) or a glutathione-Sepharose column as control
(lane 3). WGA-bound proteins were eluted and subjected to
SDS-polyacrylamide gel electrophoresis and Western blotting using an
affinity purified anti-NLP-1 antibody.
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NLP-1 Expression and Localization--
NLP-1 mRNA expression
was analyzed by Northern blot using different mRNA sources. A major
band corresponding to a 2-kb mRNA, consistent with the expected
size, is found in all cell lines and tissues tested, indicating that
NLP-1 mRNA is ubiquitously expressed (Fig.
3A). A minor band is also
visible at around 4.4 kb, which may represent either an mRNA
isoform or a cross-hybridization with nucleoporin mRNA.

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Fig. 3.
Expression analysis of the NLP-1
protein. A, hybridization of human multiple tissues Northern blot
(CLONTECH) with 32P-labeled NLP-1
cDNA probe. The same blot was stripped off and rehybridyzed with a
human -actin cDNA probe. Size markers, in kb, are indicated on
the right side. B, Western blot of total (T),
nuclear (N), or cytoplasmic (C) protein extracts
obtained from HeLa cells (I) or HeLa cells over expressing
NLP-1 (II). C, indirect immunofluorescence
performed on HeLa cells transfected with pSG5-NLP-1, a NLP-1 expressing
plasmid, using an affinity purified anti-NLP-1 rabbit polyclonal
antibody. Cells were visualized by fluorescence (left) or by
Nomarski (right) using a LSM 510 ZEISS confocal
microscope.
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Subcellular localization of NLP-1 protein was then addressed both by
cell fractionation and by indirect immunofluorescence on HeLa cells
transiently transfected with pSG5-NLP-1 or mock transfected. A
protein of the expected 45-kDa molecular size was detected by Western
blot using an immunopurified polyclonal antibody (Fig. 3B).
NLP-1 is found solely in total and nuclear protein extract in both mock
and pSG5-NLP-1 transfected HeLa cells. Indirect immunofluorescence on
HeLa transfected cells indicates that NLP-1 protein expression is
restricted to the nucleus and excluded from the nucleolus (Fig.
3C).
Specificity of the NES/NLP-1 Interaction--
To further confirm
that interactions revealed by the two-hybrid assay were specific of a
functional nuclear export signal, we have set up a genetic screen in
yeast to select for Rev NES mutants that have maintained the ability to
interact specifically with NLP-1
. We first constructed a small
library of plasmids derived from pLexM10 by randomly mutating the
sequence coding for the four essential leucines located in the Rev
nuclear export signal (Fig.
4A). This library (2 × 105 independent clones) was used to transform the
L40-pACTNLP-1
yeast strain expressing the Gal4 activation domain
fused to the NLP-1
polypeptide. To eliminate self-activating
construct that could result from the generation of a functional
activation domain in yeast, clones positive for both the histidine and
-galactosidase reporter genes were isolated and reintroduced into
the L40-pACT strain expressing the Gal4 activation domain alone.
Fourteen clones that appeared to be positive with pACTNLP-1
and
negative with pACT in co-transformation experiments were finally
sequenced (Fig. 4A). Among these clones, five were identical
to the wild type, indicating that leucines in the Rev NES are probably
the optimal amino acids required for Rev/NLP-1 interaction in yeast.
Three were essentially wild type except for an leucine to isoleucine mutation, which is already known not to interfere dramatically with NES
activity (41). Finally, four clones had two leucines of four changed to
other hydrophobic residues, isoleucine and phenyalanine, or to
tyrosine. It should be noted that very few of the selected clones have
the same DNA sequence, suggesting that most of them if not all are in
fact independent ones (data not shown). Furthermore, as shown in Fig.
4A, all clones were also positive when assayed with
pACTNup214c, indicating that NLP-1
and Nup214 exhibit the same
specificity of interaction with the Rev NES.

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Fig. 4.
Selection of NES mutants that have maintained
the ability to interact with NLP-1 .
A, the sequence coding for the four essential leucines of
the Rev NES were randomized in pLexAM10, and the resulting mutants were
screened for NLP-1 interaction. The name and the amino acid sequence
of the five selected NES including the wild type NES are reported as
well as the number of clones obtained. In each case we have checked the
possibility of an association with Nup214c, indicated with plus
signs. B, schematic representation of pVRS1, the
reporter construct used to evaluate Rev activation in HeLa cells and
the two possible RNA expressed from this plasmid. C and
D, RNase protection assay was done by hybridizing uniformly
32P-labeled MTX91 RNA probe with RNA extracted from HeLa
cells transfected with 500 ng of pVRS1 and 20 or 100 ng of the
indicated plasmid.
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To definitely correlate the ability of these mutated NES to interact
with NLP-1
with their activity as a nuclear export signal, we
decided to challenge the ability of these sequences to substitute for
the wild type NES in a functional assay. For this purpose, a new Rev
synthetic reporter construct was assembled that drives the expression
of a Rev responsive RNA under the control of the CMV enhancer/promoter
region and the SV40 small t polyadenylation signal. The premessenger
RNA expressed from this reporter gene is composed of sequences coding
for the HIV-1 Gag p17 inhibitory sequence region flanked by the first
tat 5' and 3' splice sites and fused to a
264-nucleotide-long version of the HIV-1 RRE (Ref. 41 and Fig.
4B). When Rev is absent, this RNA undergoes splicing at tat
sites. However, Rev expression leads to cytoplasmic accumulation of the
unspliced version of the RNA (data not shown). As shown in Fig.
4C, RNase protection analysis of transcripts expressed from
our reporter construct revealed that very little Gag containing RNA is
found in the cytoplasm of transfected HeLa cells and that most of the
precursor RNA undergoes complete splicing. However, cytoplasmic
accumulation of full-length Gag p17 RNA is dramatically induced upon
expression of Rev but not M10 or M4, a Rev mutant impaired in its
capacity to bind RNA (10). Having shown that expression of our test RNA
was dependent on a functional Rev protein (and also dependent on the
RRE, data not shown), we recloned the four selected Rev proteins into
the mammalian expression vector pSG5Flag to evaluate their activity in
this new system. As shown in Fig. 4D, the four mutants can
substitute for wild type Rev in inducing the nuclear export of the
unspliced Gag RNA (activity reaching 71-95% of wild type). The former
observation indicates that our screen for NLP-1-interacting Rev mutants
has led to the identification of functional Rev proteins harboring
efficient NES and strengthens the idea that the Rev/NLP-1 interaction
identified in yeast is the result of a functional relationship between
the two proteins.
Rev Interacts with NLP-1 in Mammalian Cells--
Interaction
between the NLP-1 protein and functional NES domains has been
identified in the yeast two-hybrid system. To confirm that this
interaction also occurs in mammalian cells, we decided to set up a
two-hybrid interaction test in HeLa cells similar to what we have been
using in yeast. Vectors were constructed to express Rev and M10 (the
Rev mutant lacking a functional NES) proteins fused to the DNA-binding
domain of LexA. An additional vector expressing NLP-1
fused to the
VP16 transcriptional activation domain was also produced. As a reporter
gene, we have cloned the sequence coding for SEAP (the
secreted form of human alkaline phosphatase; CLONTECH) under the
control of a minimal SV40 promoter and five upstream LexA-binding sites
(Fig. 5A). The ability of NLP-1 to associate with Rev but not M10 was evaluated by titrating the
SEAP concentration in the culture medium after transient expression in
HeLa cells. Basal expression of SEAP from the LexA operator-controlled reporter gene was low and not affected by expression of VP16-NLP-1
(Fig. 5C). However, co-transfection with a plasmid
expressing Lex-Rev induced a significant increase of SEAP expression
suggesting that when fused to LexA, Rev is capable of recruiting to the
promoter vicinity one or several components of the transcriptional
machinery. This activation was not observed with Lex-M10, indicating
that this recruitment directly involves the Rev nuclear export domain. More interestingly and in agreement with the yeast two-hybrid assay,
co-expression of VP16-NLP-1
in the mammalian system produces a
further activation that is directly dependent upon the amount of
transfected plasmid DNA. In addition, M10 failed to interact with NLP-1
as revealed by the absence of SEAP induction upon expression of the
Lex-M10 hybrid protein (Fig. 5C). These findings indicate that Rev and NLP-1 are indeed able to interact in mammalian cells and
that the Rev NES is crucial for this interaction.

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Fig. 5.
The Rev-NLP-1 interaction is reproduced in
mammalian cells. A, schematic view of the reporter
construct used for two-hybrid experiments in HeLa cells. OP
stands for LexA operator binding sites, and SEAP stands for
secreted alkaline phosphatase.
PSV40e and SV40 PA correspond, respectively, to
promoter/enhancer and polyadenylation sequences of SV40. B,
schematic representation of the Lex and VP16 fusion proteins expressed
from the indicated plasmids used in panel C and Fig.
6B. The VP16 hybrids proteins are tagged with the SV40 large
T nuclear localization signal (nls) and the Flag peptide
(Eastman Kodak). C, two-hybrid experiments performed in HeLa
cells. pSEAP LEX5X reporter plasmid was co-transfected with constructs
expressing the Lex or VP16 hybrid proteins as indicated. SEAP activity
in cell culture medium was assayed by chemiluminescence.
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NLP-1 Associates with CRM-1 in Yeast and Mammals--
It has been
reported recently that both HIV-1 Rev and HTLV-1 Rex interact in the
yeast two-hybrid system with a nucleoporin-like human protein called
hRIP/Rab and several yeast and mammalian FG-rich nucleoporins including
yRip1p, hNup214, and hNup153 (15-19). Furthermore, it has been
demonstrated that the yRip1p/Rev interaction in yeast required wild
type CRM-1 and that CRM-1 interacts with various nucleoporins in yeast
(29). Because we have been unable to show any direct and physical
contact between Rev and NLP-1 using biochemical approaches, we reasoned
that the interaction we have detected was likely to be mediated by
CRM-1. Two-hybrid experiments were performed both in yeast and
mammalian cells to address this possibility (Fig.
6). Indeed, using the yeast interaction test, we show that S. cerevisiae CRM-1 associates with
NLP-1
and Nup214c (Fig. 6A). Similarly, in human cells,
human CRM-1 also interacts with NLP-1 as revealed by a 3-4-fold
activation upon co-expression of VP16/CRM-1 and LexNLP-1 in our system
(Fig. 6B).

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Fig. 6.
. NLP-1 interacts with CRM-1 in yeast and HeLa
cells. A, yeast two-hybrid interaction assay. L40 cells
were co-transformed with the indicated plasmids and spread on leucine,
tryptophan, and histidine amino acids-depleted agar plates. Growth on
selective plates is indicated by a plus sign. ND,
not done. B, two-hybrid experiments in HeLa cells using
reporter plasmid pSEAP LEX5X and the LexNLP-1 and VP16 or VP16CRM1
proteins expressing constructs. Reporter gene expression was evaluated
by titrating SEAP activity in cell culture medium by chemiluminescence.
The values are given as multiples of the number of relative light units
obtained for cells transfected with pLexNLP-1 alone.
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DISCUSSION |
In this study we report the characterization of a cellular
protein, encoded by a previously identified gene of unknown function called CG1 (39) that interacts specifically with the Rev
nuclear export signal in the yeast two-hybrid system. Because it has
strong homologies with nucleoporins including multiple FG repeats and a
high serine content and because it is glycosylated in vitro and in vivo, this protein was named NLP-1. In addition, a
putative CCCH type zinc finger and a coiled-coil region are suggested
by a thorough analysis of the NLP-1 primary sequence. Thus, NLP-1 closely resembles the previously identified Rev-interacting protein hRIP/Rab, which also harbors many FG repeats, a high serine content, and a zinc finger of the CCCC type (15, 16). In both cases, the Rev NES
interaction domain is located in the FG-rich, C-terminal half of these
proteins, and immunolocalization experiments have revealed that most
endogenous hRIP/Rab and transiently expressed NLP-1 are nucleoplasmic
with no particular staining at the nuclear envelope. Yet, when
immunolocalization of NLP-1 is performed on HeLa cells, a nuclear
membrane staining is observed, suggesting that endogenous NLP-1 is
membrane-bound (data not shown). However, this apparent discrepancy may
be due to the fact that the NLP-1 antiserum we have used possibly
recognizes additional proteins that we believe may include nucleoporins
(data not shown). Thus, we cannot rule out the possibility that most
NLP-1 is bound to the NPC when expressed at physiological levels. The
use of a monospecific antibody would be necessary to unambiguously
address the NLP-1 intracellular localization.
We have shown that NLP-1 and Nup214 interact specifically with a
functional Rev NES in yeast. To confirm the functional significance of
this contact, we asked whether motifs able to bind to NLP-1 can specify
nuclear export. To do so, we randomized the four essential leucines in
Rev NES and selected for NLP-1-interacting mutants. A similar approach
based on the HTLV-1 Rex/hRIP/Rab interaction was reported previously
and led to the same conclusion, i.e. the ability to bind
FG-rich domains of hRIP/Rab or NLP-1 is predictive of NES function
(41). Taken altogether, the selected sequences also indicate that
although leucines 1, 2, and 4 can be replaced by isoleucine or other
hydrophobic amino acids, the leucine in position 3 is critical (Fig.
4). However, we still have no clear idea about the molecular
interactions that sustain binding of the NES to its receptor. The
consensus sequence for a functional NES proposed by Bogerd et
al. (41) suggests that similar to nuclear localization signals,
these targeting signals are not highly structured. In contrast, the
nuclear export receptor CRM-1 interacts with NES in a
RanGTP-dependent manner, and binding of RanGTP and the NES
to CRM-1 is highly co-operative (20). This suggests that a
RanGTP-dependent conformational change of CRM-1 is probably
required to stabilize NES binding.
It has been shown previously that the FG-rich domain found in many
nucleoporins of the GLFG and the XXFG but not the
FXFG type are capable of interacting with the Rev NES in
both yeast and higher eucaryotic cells (17-19). More recently, CRM-1,
which is highly conserved from yeast to mammals, was shown to be a
major export receptor for leucine-rich NES (20-24). Because CRM-1 was found associated with Nup88 and Nup214 in a dynamic complex (28), it
was conceivable to postulate that CRM-1 was the necessary bridge allowing interaction of NES to nucleoporins in yeast. This hypothesis was indeed clearly demonstrated for yRip1p (29). In the present work,
we show that NLP-1 also interacts with CRM-1, and we believe that the
NES-NLP-1 interaction observed in yeast and HeLa cells is mediated by
CRM-1 as well. However, we cannot rule out the possibility that Rev
binds directly to NLP-1 as well as CRM-1. Considering the size of the
NES motif, if these contacts exist, they might be mutually exclusive.
CRM-1 can bind FG-rich domains both in yeast and human cells (Refs. 28
and 29 and this work). However, we have no information about the
molecular status of CRM-1 within this complex. In particular, we do not
know whether RanGTP and the NES-containing proteins participate to this
complex and whether they have to associate with CRM-1 before it can
bind to nucleoporins. CRM-1 belongs to a family of import/export
receptors that bind to the small GTPase Ran, an essential constituent
of the bidirectional nucleocytoplasmic transport of proteins and RNA
(42). Binding of GTP-bound Ran to import receptors induced dissociation
of the cargo/transporter complex (43, 44). Conversely, binding of
RanGTP to export receptors like Cas, Exportin t, or CRM-1 is believed
to be necessary both for complex assembly with the export substrate and
for the translocation process (20, 45-47). In the case of Cas and
Exportin t, the complex is disassembled in the cytoplasm by the
combined action of RanBP1 and RanGAP1, resulting in GTP hydrolysis (45, 48). For CRM-1-mediated export, NES binding requires RanGTP (20).
Therefore, it is conceivable that binding to the FG domain of
NPC-associated proteins only occurs when CRM-1 is complexed to both
RanGTP and to the NES-containing cargo. Hydrolysis of GTP by RanGAP1
and RanBP1 at the cytoplasmic face of the NPC would completely
dissociate the complex in a way similar to what happens following
RanGTP binding for the import process.
The NPC is thought to be composed of 50-100 different nucleoporins,
but only a fraction of these have been identified so far (for review
see Refs. 49-51). Nucleoporins can be separated into two classes:
integral membrane proteins, which make up the core of the NPC, and the
dipeptide FG-rich nucleoporins, which are mostly located at the NPC
periphery. It is noteworthy that although not necessary for proper
nucleoporin localization, the FG-rich domain is conserved among
eucaryotes and has probably been maintained for a critical function.
NLP-1 and hRIP/Rab are both ubiquitously expressed in humans and are
probably conserved among species. Homologues of hRIP/Rab are likely to
exist in mouse, quail, frog, fly, and even in yeast (15, 16). In the
case of NLP-1, mouse cDNA sequences can be found in the expressed
sequence tag data base that encodes a putative polypeptide with high
sequence homologies to NLP-1. In addition, a S. pombe open
reading frame (GenBankTM accession number AL031349) with minor sequence
homology with NLP-1 is found to be very similar in structure. This
475-amino acid-long open reading frame harbors a CCCH type zinc finger
at its very N terminus with strong homologies with the one found in
NLP-1, a putative coiled-coil in its center, and a C-terminal FG-rich
domain. Consequently, NLP-1 and hRIP/Rab are likely to belong to a
family of proteins well conserved through evolution, which could be
involved in nuclear export. The complex CRM-1/RanGTP/NES cargo is
likely to assemble in the nucleoplasm where it is targeted to the
nuclear envelope for proper translocation. NLP-1 and hRIP/Rab may serve
as transporters helping to direct this complex to the NPC. On the other
hand, zinc fingers, coiled-coil domains, and RNA-binding domains are
found in many nucleoporins, and some of them have RNA or DNA binding
activities (for review see Ref. 50). NLP-1 and hRIP/Rab may also be
involved in other kinds of transport processes or nuclear functions
involving CRM-1, similar to what has been shown for Rip1p (52).