From the Molecular Medicine Unit, University of Leeds, St. James's University Hospital, Leeds LS9 7TF, United Kingdom
Received for publication, October 18, 2000, and in revised form, February 22, 2001
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ABSTRACT |
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Herpesvirus saimiri (HVS) is the
prototype A number of viruses including herpes, adenovirus, influenza, and
retroviruses replicate in the host cell nucleus. In order to complete
their virus replication cycle, they have devised a number of mechanisms
to transport viral nucleic acids into and out of the nucleus. In
particular, a variety of viruses encode nucleocytoplasmic shuttle
proteins, which specifically mediate the nuclear export of viral RNA.
Such virally encoded proteins include human immunodeficiency virus type
1 (HIV-1)1 Rev, herpes
simplex virus type 1 (HSV-1) ICP27, influenza virus NEP, adenovirus
E4orf6, and the herpesvirus saimiri (HVS) ORF 57 protein (1-6).
Herpesvirus saimiri (HVS) is the prototype ORF 57 is a 52-kDa multifunctional trans-regulatory protein homologous
to genes identified in all classes of herpesviruses. Transactivation of
late viral genes by ORF 57 occurs independently of target gene promoter
sequences and appears to be mediated at a post-transcriptional level
(11). In addition to its transactivation properties, ORF 57 is
responsible for repression of viral gene expression, which correlates
with the presence of introns within the target gene (11-12). ORF 57 also redistributes both U2 and SC-35 splicing factors during an
HVS infection into intense distinct nuclear aggregations (13).
Recent analysis has demonstrated that the ORF 57 protein has the
ability to bind viral RNA and shuttle between the nucleus and
cytoplasm, and is required for efficient cytoplasmic accumulation of
virus mRNA. This suggests that ORF 57 plays a pivotal role in
mediating the nuclear export of viral transcripts (6).
An intriguing question regarding the functioning of virus-encoded
nucleocytoplasmic shuttle proteins is the mechanism they utilize to be
transported through the nuclear pore complex. Macromolecule trafficking
into and out of the nucleus is mediated by soluble transport receptors
(reviewed in Ref. 14). These receptors bind specific proteins or RNA
cargoes and interact with nuclear pore proteins, which subsequently
allow the translocation of the receptor cargo through the nuclear pore
complex. Recently, CRM-1 (for chromosomal region maintenance 1) or
exportin 1, a protein that shares homology with members of the
importin-karyopherin nuclear transport pathway, has been identified as
a nuclear export receptor for proteins, including HIV-1 Rev, carrying a
leucine-rich NES in a process that also requires the GTP-bound
form of Ran (15-16). Furthermore, CRM-1 has been shown to interact
with nuclear pore complex proteins, namely the nucleoporins CAN/Nup214
and Nup88 (16), suggesting that CRM-1 is the bridging protein for the
interactions of NES-containing proteins and the nuclear pore complex.
However, it has recently been shown for the herpesviruses HSV-1 and
Epstein-Barr virus that, although export of some viral RNAs require the
CRM-1 pathway, a proportion of viral RNA export can be mediated by a
CRM-1-independent pathway (17-18). This suggests that at least some
herpesvirus nucleocytoplasmic shuttle proteins may function through a
distinct, as yet unidentified, export mechanism.
In addition, it is tempting to speculate that the virally encoded
nucleocytoplasmic shuttle proteins must interact with cellular nuclear
import pathways. The most widely characterized transport pathway
mediates the nuclear import of proteins that contain a classical
nuclear localization signal (NLS) (reviewed in Ref. 14). These basic,
generally lysine-rich NLS serve as recognition sites for an NLS
receptor termed importin In this report we have investigated the nucleocytoplasmic shuttling
mechanism utilized by the HVS ORF 57 protein. The yeast two-hybrid
system was employed to identify interacting cellular proteins using ORF
57 as bait. Here we show that ORF 57 interacts with importin Yeast Two-hybrid Screen for ORF 57-interacting Proteins--
The
GAL4-based yeast two-hybrid system screening technique (30) was
employed to identify ORF 57-interacting proteins. The "bait"
plasmid was constructed by PCR amplication of the ORF 57 coding region
using forward and reverse primers. These oligonucleotides incorporated
BamHI and PstI restriction sites for the
convenient cloning of the PCR fragment into pGBT9
(CLONTECH), to derive the GAL4 DNA-binding domain
fusion, pDBD57. A human kidney cDNA-GAL4 activation domain fusion
library in the vector pACT2 (CLONTECH) was utilized
to identify ORF 57-interacting proteins. The bait plasmid was
transformed into the Saccharomyces cerevisiae strain HF7c
(CLONTECH). Clones were selected on minimal
synthetic dropout medium in the absence of tryptophan. Yeast clones
harboring the bait plasmids were then sequentially transformed with the
"prey" library. Positive clones, potentially harboring ORF
57-interacting species, were identified both by their ability to grow
on media without tryptophan, leucine, and histidine and by the
detection of Plasmid Constructs--
The yeast two-hybrid importin
The yeast two-hybrid ORF 57 deletion series was produced by a PCR-based
method using a series of forward and reverse primers. The
oligonucleotides incorporated BamHI and PstI
restriction sites for the convenient cloning of the PCR products. Each
fragment was inserted into the yeast two-hybrid expression vector,
pGBT9, in frame with the GAL4-DBD, to derive the deletion series
pDBD57
The importin Viruses, Cell Culture, and Transfections--
HVS (strain A11)
was propagated in owl monkey kidney cells, which were maintained in
Dulbecco's modified Eagle's medium (Life Technologies, Inc.)
supplemented with 10% fetal calf serum. COS-7 cells were also
maintained in Dulbecco's modified Eagle's medium supplemented with
10% fetal calf serum. Plasmids used in the transfections were prepared
using Qiagen plasmid kits according to the manufacturer's directions.
Transfections were performed using LipofectAMINE (Life Technologies,
Inc.) as described by the manufacturer, using 2 µg of the appropriate DNAs.
Co-immunoprecipitation Assays--
COS-7 cells either remained
untransfected, were transfected using 2 µg of the appropriate DNAs,
or were infected with HVS at a multiplicity of infection of 1. After
48 h, cells were harvested and lysed with lysis buffer (0.3 M NaCl, 1% Triton X-100, 50 mM HEPES buffer,
pH 8.0) containing protease inhibitors (leupeptin and
phenylmethylsulfonyl fluoride). For each immunoprecipitation, 10 µl
of the ORF 57 polyclonal (31) or GFP monoclonal antiserum (CLONTECH) were incubated with protein A-Sepharose
beads (Amersham Pharmacia Biotech) for 16 h at 4 °C. The beads
were then pelleted, washed, and incubated with each respective cell
lysate for 16 h at 4 °C. The beads were then pelleted and
washed, and precipitated polypeptides were resolved on a 12%
SDS-polyacrylamide gel and analyzed by immunoblot analysis.
Immunoblot Analysis--
Polypeptides were resolved on a 12%
SDS-polyacrylamide gel, then soaked for 10 min in transfer buffer (25 mM Tris, 192 mM glycine, 20% methanol (v/v)).
The proteins were transferred to nitrocellulose membranes by
electroblotting for 3 h at 250 mA. After transfer, the membranes
were soaked in PBS and blocked by preincubation with 2% (w/v) nonfat
milk powder for 2 h at 37 °C. Membranes were incubated with a
1:400 dilution of the anti-importin GST Pull-down Assays--
The importin Immunofluorescence Analysis--
Cells were fixed with 100%
ice-cold methanol for 10 min. The cells were rinsed in PBS and blocked
by preincubation with 1% (w/v) nonfat milk powder for 1 h at
37 °C. A 1:100 dilution of anti-ORF 57 polyclonal antiserum (31) or
1:50 dilution of B23 monoclonal antibody was layered over the cells and
incubated for 1 h at 37 °C. Texas Red-conjugated immunoglobulin
(Dako; 1:200 dilution) was added for 1 h at 37 °C. After each
incubation step, cells were washed extensively with PBS. The immune
fluorescence slides were examined using a Zeiss Axiovert 135TV inverted
microscope with a Neofluar 40× oil immersion lens.
Northern Blot Analysis--
Northern blot analysis was performed
as previously described (6). Total, nuclear, and cytoplasmic RNA was
isolated from transfected cells and separated by electrophoresis on 1%
denaturing formaldehyde-agarose gel. The RNA was transferred to
Hybond-N membranes and hybridized with 32P-radiolabeled
random-primed probes specific for gB and actin coding sequences.
Importin
Clones that fulfilled all these criteria were sequenced and BLAST
searched against the EMBL/GenBankTM data base. Analysis revealed that
seven ORF 57-interacting clones corresponded to importin In Vitro Co-immunoprecipitation of ORF 57 and Importin Importin
To confirm these results in vitro, GST pull-down assays were
performed. The importin The ORF 57 Amino-terminal Arginine-rich Sequence Is Required for
Importin
To confirm whether the ORF 57 amino-terminal arginine-rich sequence was
required for the interaction with importin The ORF 57 Amino-terminal Arginine-rich Sequence Functions as an
NLS--
To determine if the arginine-rich sequence functions as a
NLS, the subcellular localization of the ORF 57 amino-terminal deletion series was analyzed. These constructs contained a carboxyl-terminal GFP
fusion tag, allowing direct visualization. Transient p57N
To confirm that the arginine-rich sequence functions as a NLS, and
enables nuclear import of a heterologous protein, the NLS was fused
with GFP. COS-7 cell monolayers were transfected with either pEGFP-C1
or p57NLS-GFP, and the subcellular localization of GFP was observed.
The results show that cells transfected with pEGFP-C1 displayed a
fluorescence pattern throughout the cell, in both the nucleus and
cytoplasm. However, the fluorescence pattern observed in
p57NLS-GFP-transfected cells was confined to the nucleus and in
particular the nucleolus (Fig. 5b).
Furthermore, to determine the importance of the arginine and lysine
residues for ORF 57 nuclear localization, a range of site-directed mutations were constructed, p57NLSM1-4, incorporated the alteration of
varying residues within the putative NLS to alanine (Fig.
5c). COS-7 cell monolayers were transfected with either
p57GFP or p57NLSM1-4, and the subcellular localization of GFP was
observed. The results show that cells transfected with p57GFP displayed
a nuclear fluorescence pattern as observed previously. However, the
fluorescence pattern observed in p57NLSM1-4-transfected cells was
confined to the cytoplasm (Fig. 5d). This suggested that the
ORF 57 amino-terminal arginine-rich sequence functions both as a NLS
and possibly as a nucleolar localization signal, and the arginine and
lysine residues are essential for this function.
ORF 57 Redistributes Importin
To ascertain the subcellular localization of each importin
To ascertain if the importin ORF 57 Reduces Nuclear Import of Other NLS-containing
Proteins--
As demonstrated above, ORF 57 redistributes importin
ORF 57-Importin HVS ORF 57 is a member of the virus-encoded nucleocytoplasmic
shuttle proteins involved in mediating the nuclear export of virus
transcripts. (6). Other functionally homologous virus proteins include
HIV-1 Rev, adenovirus E4orf6, HSV-1 ICP27, and influenza virus NEP
(1-5). In this report, we have utilized the yeast two-hybrid system
and co-immunoprecipitation analysis to demonstrate that ORF 57 interacts with importin Interestingly, a number of viral proteins have been shown to interact
with importin Further analysis of the ORF 57 (RRPSRPFRK), EBNA-1, and NP NLSs show
very limited, if any, homology. The EBNA-1 NLS, KRPRSPSS, has been
shown to be required for both importin Insights into the binding of NLS-containing proteins to importin Interestingly, upon nuclear import of the ORF 57-importin In conclusion, our data demonstrate that the multifunctional ORF 57 nucleocytoplasmic shuttle protein interacts with importin -2 herpesvirus. This is an increasing important subfamily
of herpesviruses due to the identification of the first human
-2
herpesvirus, Kaposi's sarcoma-associated herpesvirus. The HVS open
reading frame (ORF) 57 protein is a multifunctional trans-regulatory
protein homologous to genes identified in all classes of herpesviruses.
Recent analysis has demonstrated that ORF 57 has the ability to bind
viral RNA and to shuttle between the nucleus and cytoplasm, and is
required for efficient nuclear export of viral transcripts. Here we
have investigated the nucleocytoplasmic shuttling mechanism utilized by
the ORF 57 protein. The yeast two-hybrid system was employed to
identify interacting cellular proteins using ORF 57 as bait. We
demonstrate that ORF 57 interacts with importin
isoforms 1 and 5. In addition, the binding of ORF 57 to importin
was mediated by the
importin
hydrophobic internal armadillo repeats. An ORF 57 amino-terminal arginine-rich sequence, which functions as a nuclear
localization sequence, was also required for this interaction.
Furthermore, the ORF 57 protein is responsible for the redistribution
of importin
into the nucleoli. These results identify novel
cellular interactions essential for the functioning of this important
herpesvirus regulatory protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-2
herpesvirus, or rhadinovirus (7), an increasingly important family of
viruses due to the recent identification of the first human
-2
herpesvirus, Kaposi's sarcoma-associated herpesvirus (8). Gene
expression during HVS lytic replication is sequentially regulated and
occurs in three main temporal phases: immediate-early, delayed-early,
and late. The two major HVS transcriptional regulating proteins are
encoded by the open reading frames (ORFs) 50 and 57 (9-12).
or karyopherin
(19), which forms a
heterodimeric complex with importin
or karyopherin
. Importin
functions as a transport adapter molecule binding to the nuclear
pore complex via a direct interaction with specific nucleoporins (20,
21). Once in the nucleus, binding of Ran-GTP to importin
causes
dissociation of the import complex (22, 23). Once released from the
cargo, importin subunits are then recycled to the cytoplasm. Importin
is recycled rapidly, whereas the export of importin
is mediated
by the nuclear export factor, CAS, which binds importin
preferentially in the presence of Ran-GTP (24-26). In the cytoplasm
the importin molecules are released by the action of RanBP1 and RanGAP1
(27-29), allowing participation in additional rounds of nuclear import.
isoforms 1 and 5. Confirmation of this interaction was provided by
co-immunoprecipitation experiments from transfected and infected cells.
In addition, the binding of ORF 57 to importin
is mediated via the
hydrophobic, internal, importin
armadillo (arm) repeats. Moreover,
an ORF 57 amino-terminal arginine-rich sequence, which functions as an
NLS, is required for this interaction. Furthermore, the ORF 57 protein
is responsible for the redistribution of importin
into the nucleoli.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity. Plasmids were isolated from
positive yeast clones, selecting for pACT2 cDNA library plasmids by
transformation of the leucine auxotroph into Escherichia
coli strain HB101. To confirm the specificity of the interactions,
pACT-2 library plasmids were transformed into yeast strains harboring
no plasmid, yeast containing pGBT9 vector only, yeast containing pLAM5
(a GAL4 human lamin C fusion), or pDBD57. Only those library plasmids
demonstrating a requirement for the pDBD57 plasmid for induced
expression of HIS3 or lacZ reporter genes were
considered further and selected for DNA sequencing.
1 and
5 deletion series were produced by a PCR-based method using a series
of forward and reverse primers. The oligonucleotides incorporated
BamHI and XhoI restriction sites for the
convenient cloning of the PCR products. Each fragment was inserted into
the yeast two-hybrid expression vector, pACT2, in frame with the
GAL4-AD, to derive the deletion series pAD
1
1-6 and
pAD
5
1-5. The bacterial expression importin
1 and
5
deletion series were produced by cloning the
1 and
5 deletion PCR
fragments into pGEX5T, to derive the GST fusion constructs,
pGST
1
1-4 and pGST
5
1-5.
1-5. The ORF57-GFP and ORF 57 amino-terminal deletion series
were again generated by PCR amplication, again using a series of
forward and reverse primers. These oligonucleotides incorporated
BamHI and PstI restriction sites to facilitate
cloning of the PCR product into the eukaryotic expression vector,
pcDNAGFP (Invitrogen), to yield p57GFP and p57N
1-3GFP. To
produce p57NLS-GFP, oligonucleotides encoding the putative ORF 57 NLS,
nucleotides in bp 78580-78820 of the published sequence (7), were
synthesized. These oligonucleotides incorporated BamHI and
XhoI restriction sites, for convenient cloning. The
oligonucleotides were annealed and ligated with pEGFP-C1 (CLONTECH) to create an in-frame carboxyl-terminal
fusion of the NLS sequence and GFP. To produce p57NLSM1-4, containing
alterations within the putative ORF 57 NLS, oligonucleotides that
incorporated the alteration of arginines and lysine to alanine residues
were synthesized encompassing nucleotides in bp 78677-78774 of the published sequence. These oligonucleotides incorporated
BglII and PflMI restriction sites, for convenient
cloning into pBKRSV57 (11), previously digested with BglII
and PflMI, thereby replacing the wild type coding region
with the site-directed mutated sequence.
1-GFP and
5-GFP constructs were generated by PCR
amplication of the complete
1 and
5 cDNA sequences using forward and reverse primers. These oligonucleotides incorporated BamHI and XhoI restriction sites to facilitate
cloning of the PCR products. Each fragment was inserted into the
eukaryotic expression vector, pcDNAGFP (Invitrogen) to yield
p
1-GFP and p
5-GFP, respectively. These constructs contained a
carboxyl-terminal GFP fusion tag, which allowed direct visualization of
the importin
-GFP proteins using fluorescence microscopy. To produce
pSV40NLS-RFP, oligonucleotides encoding the SV40 NLS, PKKKRKV, were
synthesized. These oligonucleotides incorporated XhoI and
BamHI restriction sites, for convenient cloning. The
oligonucleotides were annealed and ligated with pDsRed1-N1 (CLONTECH) to create an in-frame carboxyl-terminal
fusion of the SV40 NLS and RFP. The sequences of all primers used in
this report can be obtained directly from the authors.
1,
5 polyclonal antisera or a
1:1000 dilution of anti-GFP (CLONTECH), washed with
PBS, and then incubated for 1 h at 37 °C with a 1:1000 dilution
of secondary immunoglobulin conjugated with horseradish peroxidase
(Dako) in blocking buffer. After five washes with PBS, the
nitrocellulose membranes were developed using ECL (Pierce).
-deletion series were
expressed as GST fusion proteins in E. coli DH5
. A fresh
overnight culture of transformed E. coli was diluted 1 in 20 with LB medium. After growth at 37 °C for 2 h, the culture was
induced with 1 mM IPTG and grown at 37 °C for an
additional 4 h. The cells were harvested and resuspended in 0.1 volume of lysis buffer (100 mM Tris-HCl, pH 8.0, 200 mM NaCl, 1% Triton X-100). Cells were sonicated and stored
on ice for 30 min, and cellular debris pelleted. The recombinant
protein was purified from crude lysates by incubation with
glutathione-Sepharose 4B affinity beads. The protein-bound beads were
then incubated with untransfected or appropriate transfected COS-7 cell
lysates previously treated with lysis buffer for 16 h at 4 °C.
The beads were then pelleted and washed, and precipitated polypeptides
resolved on a 12% SDS-polyacrylamide gel. The proteins were then
transferred to nitrocellulose membranes by electroblotting and probed
as described previously.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 and
5 Interact with HVS ORF 57--
To identify
ORF 57-interacting cellular proteins, 1.5 × 106
independent cDNA clones of a human kidney cDNA library fused to the GAL4 activation domain were screened. 24 clones were identified that activated histidine and
-galactosidase reporter gene expression in the presence of the ORF 57-DBD fusion protein. The specificity of
the interaction was confirmed by transforming the putative ORF
57-interacting cellular clones into yeast strains harboring no plasmid,
yeast containing pGBT9 vector only, yeast containing pLAM5 (a GAL4
human lamin C fusion), or pDBD57. Only those library plasmids
demonstrating a requirement of pDBD57 for induced expression of
histidine and
-galactosidase reporter genes were considered further.
1/karyopherin
2/Rch1 and that four clones corresponded to
importin
5/karyopherin
1/hSRP1.
1 and
5--
To confirm whether the observed interaction of ORF 57 with
importin
1 or
5 could also be observed in vitro,
co-immunoprecipitation studies were performed. Control untransfected
COS-7 cells were compared with cells transfected with pRSVORF57, a
eukaryotic expression vector encoding the complete coding region of ORF
57 (11), or HVS-infected cells (multiplicity of infection of 1). After
24 h, the cells were harvested and cell lysates utilized in
co-immunoprecipitation analysis using an anti-ORF 57 polyclonal
antiserum. Polypeptides precipitated from untransfected, transfected,
and infected cellular extracts were then resolved on SDS-PAGE and
transferred to a nitrocellulose membrane. Immunoblot detection was
performed using antiserum specific for importin
1 or
5. The
results demonstrate that, in both ORF 57-transfected and HVS-infected
cells, ORF 57 specifically interacts with both importin
1 and
5
(Fig. 1).
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Fig. 1.
ORF 57 interacts with both importin
1 and
5. To determine
whether ORF 57 interacts with importin
, co-immunoprecipitations
were performed. Control total lysate (lane 1),
untransfected (lane 2), p57-transfected
(lane 3), and HVS-infected (lane
4) cell extracts were immunoprecipitated using ORF 57 antiserum. Bound proteins were resolved on a 12% SDS-PAGE gel and the
presence of importin
1 (a) and
5 (b) were
detected by Western blot analysis.
1 and
5 Interact with ORF 57 via Their Armadillo
Repeats--
To map the domains within importin
1 and
5 required
for their specific interaction with ORF 57, a series of importin
1 and
5 truncations were expressed as fusions with GAL4-AD (Fig. 2a). Competent yeast strain
HF7c was co-transformed with pDBD57 and each importin truncation AD
plasmid, and assessed for their ability to grow on selective medium.
The results indicated that the ORF 57 binding region maps to amino
acids 291-450 of importin
1 and amino acids 253-459 of importin
5, encompassing the central 5-8 arm repeats in both cases. Smaller
deletions within these regions of either importin
abolished the
interaction, suggesting that all the 5-8 armadillo repeats are
required for ORF 57 binding.
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Fig. 2.
ORF 57 interacts with arm 5-8 repeats of
importin . a, a series of
importin
1 and
5 deletions were generated by PCR amplication and
inserted into pACT2. Each deletion plasmid was co-transformed into HF7c
with pDBD57 and assessed for the ability to grow on selective medium.
Results of a positive interaction are indicated by +, whereas no
interaction is indicated by
. b, i, control GST
alone and the GST-importin
1 and
5 deletion series were expressed
in E. coli and purified from crude lysate by incubation with
glutathione-Sepharose 4B affinity beads; ii, protein
extracts of p57GFP-transfected cells were incubated with each
GST-importin
fusion protein. Bound proteins were resolved on a 12%
SDS-PAGE gel, and the presence of ORF 57-GFP was detected by Western
blotting using anti-GFP antiserum.
1 and
5 deletion series were expressed as
GST fusion proteins. Protein extracts prepared from p57GFP-transfected cells were incubated with each GST-importin
fusion protein bound to
glutathione beads. To confirm the expression of ORF57-GFP in each
experiment, fluorescence microscopy was utilized (data not shown).
Bound proteins were then separated by SDS-PAGE, and the presence of
ORF57-GFP was detected by Western blotting using a GFP monoclonal
antibody (Fig. 2b). Results demonstrated that ORF 57 specifically bound to GST-importin
1
3 and GST-importin
5
5, corresponding to the respective arm 5-8 repeats. An ORF57-GFP tagged
expression vector was utilized in this experiment due to the lack of
reactivity of the ORF 57 polyclonal antiserum in Western blot analysis,
as reported previously (31). A GFP alone control was also utilized in
this experiment and was shown not to interact with any of the
GST-importin
fusion proteins (data not shown).
Binding--
To delineate the domains within ORF 57 required for its specific interaction with importin
1 and
5, a
series of ORF 57 deletions were expressed in yeast as fusions with
GAL4-DBD (Fig. 3a). Competent yeast strain HF7c was co-transformed with either importin
1-AD or
importin
5-AD and each of pDBD57
1-6, and assessed for the ability to grow on selective medium. The results indicated that the
amino terminus of ORF 57 is required for its interaction with importin
1 and
5. Further deletions identified a relatively arginine-rich
region, encompassing bp 78580-78820 of the published sequence (7),
required for the interaction with importin
1 and
5.
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Fig. 3.
The ORF 57 amino terminus is required for
importin binding. A series of ORF 57 deletions was generated by PCR amplication and inserted into pGBT9.
Each deletion plasmid was co-transformed into HF7c with pAD
1
3 and
pAD
5
5 and assessed for the ability to grow on selective medium.
Results of a positive interaction are indicated by +, whereas no
interaction is indicated by
.
1 and
5,
co-immunoprecipitation analysis was performed using an ORF 57 amino-terminal deletion series (Fig. 4).
Control untransfected COS-7 cells were compared with cells transfected
with the pORF57-GFP or p57N
1-3. To confirm the expression of ORF
57-GFP and deletions in each experiment, fluorescence microscopy was
utilized (data not shown). After 24 h, the cells were harvested
and cell lysates utilized in co-immunoprecipitation analysis using the
GFP antiserum. Polypeptides precipitated were resolved on SDS-PAGE and
transferred to a nitrocellulose membranes, and immunoblot detection was
performed using antiserum specific for importin
1 or importin
5.
The results demonstrate that the amino-terminal arginine-rich sequence
of ORF 57 is required for its interaction with importin
1 and
5 (Fig. 4).
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Fig. 4.
The ORF 57 amino-terminal arginine-rich
sequence is required for importin binding. a, an ORF 57 amino-terminal deletion
series was generated by PCR amplication and inserted into pcDNAGFP.
b, control or p57N
1-3-transfected COS-7 cell extracts
were immunoprecipitated using GFP-antiserum. Bound proteins were
resolved on a 12% SDS-PAGE gel, and the presence of importins
1 and
5 was detected by Western blot analysis.
1-3 transfections were performed, and the resulting fluorescence pattern was subsequently evaluated. pcDNAGFP was used as a control and displayed, as expected, a fluorescence signal throughout the cell nucleus and cytoplasm. In contrast, p57GFP and p57N
1 resulted in a
distinct nuclear localization reminiscent of that observed previously
with HVS-infected and ORF57-transfected cells (11). However, p57N
2
and p57N
3 resulted in fluorescence restricted to the cytoplasm. This
indicated that the arginine-rich sequence contained within the amino
terminus is required to direct the ORF57 protein to the nucleus (Fig.
5a).
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Fig. 5.
The ORF 57 amino-terminal arginine-rich
sequence functions as a NLS. To determine the subcellular
localization of each ORF 57 amino-terminal deletion mutant, COS-7 cells
remained untransfected (i) or were transfected with pGFP
(ii), p57GFP (iii), p57N 1 (iv),
p57N
2 (v), or p57N
3 (vi). b,
COS-7 cells remained untransfected (i) or were transfected
with pEGFP-C1 (ii) or p57NLS (iii). After 24 h, the subcellular localization of GFP was observed using fluorescence
microscopy. (c) Mutational analysis of the ORF 57 NLS. A range of
site-directed mutations were constructed replacing the arginine or
lysine residues of the ORF 57 NLS. d, COS-7 cells remained
untransfected (i) or were transfected with p57GFP
(ii), p57NLSM1 (iii), p57NLSM2 (iv),
p57NLSM3 (v), p57NLSM4 (vi). After 24 h, the
subcellular localization of GFP was observed using fluorescence
microscopy.
1 and
5 into the
Nucleolus--
To determine the effect of ORF 57 on the subcellular
localization of importin
1 and
5, indirect immunofluorescence was
performed. Initially, the cDNAs of importin
1 and importin
5
were inserted into the eukaryotic expression vector, pcDNAGFP,
allowing direct visualization of the importin
1 and
5 proteins.
To confirm the molecular weight of importin
1-GFP and
5-GFP
fusion proteins, Western blot analysis of transiently transfected cells
was performed. Western blot analysis demonstrated that importin
1-GFP and importin
5-GFP encode the predicted 82- and 86-kDa
proteins, respectively (data not shown).
-GFP
construct, transient transfections were performed and the fluorescence
pattern was evaluated. Importin
1-GFP resulted in a distinct
perinuclear staining pattern, with faint fluorescence throughout the
nucleus and cytoplasm (Fig.
6a). To determine if ORF 57 affected the subcellular localization of importin
1 and
5, dual
immunofluorescence was performed using importin
1-GFP and ORF57
co-transfected (Fig. 6a) or HVS-infected cells (data not
shown). Cells only expressing importin
1 resulted in the distinct
perinuclear staining as previously described. In contrast, cells
expressing ORF 57 showed a drastic redistribution of the importin
1-GFP (Fig. 6a). This analysis showed that ORF 57 expression resulted in a strong nuclear fluorescence of importin
1-GFP. Moreover, the fluorescence was concentrated in nuclear
compartments, varying in number between 2 and 5 per cell, that
resembled nucleoli (Fig. 6a). Essentially identical results
were observed with importin
5-GFP in the presence of the ORF 57 protein (data not shown).
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Fig. 6.
The ORF 57 protein redistributes
importin into the nucleolus.
a, to determine whether ORF 57 affects the relocalization of
importin
-GFP, COS-7 cells were transfected with p
1-GFP
(i and ii) and p
1-GFP or p57 (iii
and iv). ORF 57 was detected using an anti-ORF 57 polyclonal
antiserum and an anti-rabbit Texas Red conjugate. Importin
was
directly visualized using fluorescence microscopy. b, to
determine whether ORF 57/importin
nuclear aggregations are
concentrated in the nucleolus, untransfected (i) or
p
1-GFP- and p57-transfected cells were labeled with a specific B23
monoclonal antibody and detected using an anti-rabbit Texas Red
conjugate (v). Importin
was directly visualized using
fluorescence microscopy (vi).
nuclear aggregations in the presence
of ORF 57 are concentrated in the nucleolus, indirect dual
immunofluorescence was performed. Importin
1-GFP- and ORF 57-co-transfected cells were labeled with a monoclonal antibody, specific for a major nucleolar protein, B23. Results demonstrated that
in the presence of ORF 57, importin
1 was localized into nuclear
aggregations, as described previously. Moreover, these distinct nuclear
aggregations co-localized with B23 (Fig. 6b). This suggests
that ORF 57 redistributes importin
1 and
5 into the nucleolus.
1 and
5 into distinct nucleolar aggregations. To determine
whether this redistribution affected the nuclear import of secondary
NLS-containing proteins via the importin nuclear import pathway,
transient transfections were performed. COS-7 cells were transfected
with pSV40NLS-RFP, a transfer vector encoding a carboxyl-terminal
fusion of the SV40 NLS and RFP, in the absence or presence of ORF57.
Results demonstrate that that cells transfected with pSV40NLS-RFP
displayed a fluorescence pattern confined to the nucleus. However, in
contrast, cells expressing ORF 57 showed a reduced presence of
SV40NLS-RFP in the nucleus and an increased amount in the cytoplasm
(Fig. 7). This analysis suggests that ORF
57 may sequester importin
in the nucleolus, thereby reducing
nuclear import of secondary NLS-containing proteins via the importin
pathway.
View larger version (18K):
[in a new window]
Fig. 7.
ORF 57 inhibits nuclear import of other
NLS-containing proteins. COS-7 cells were transfected with
pSV40NLS-RFP, in the absence or presence of p57GFP. After 24 h,
the subcellular localization of SV40NLS-RFP (i and
iii) and 57GFP (ii and iv) was
observed using fluorescence microscopy.
Interaction Is Required for Viral RNA Nuclear
Export--
We have previously demonstrated that ORF 57 is a
nucleocytoplasmic shuttle protein which mediates the nuclear export of
viral mRNAs (6). To determine whether the interaction of ORF 57 and importin
is required for viral RNA nuclear export, Northern blot
analysis was performed. Total, nuclear, and cytoplasmic RNA were
isolated separately from COS-7 cells transfected with pUCgB, a transfer
vector containing the full-length coding region and promoter of the HVS
late glycoprotein B gene, in the absence and presence of pRSVORF57
or p57NLS
3. The RNA was then separated by electrophoresis,
transferred to Hybond-N membranes, and hybridized with
32P-radiolabeled random-primed probe specific for the HVS
gB and actin coding regions (Fig. 8). The
results demonstrate, as described previously, ORF 57 is required for
the efficient cytoplasmic accumulation of late viral transcripts (6).
However, the deletion of the ORF 57 NLS results in the retention of
late viral transcripts in the nucleus. This suggests that the ORF
57-importin
interaction is required for ORF 57's nucleocytoplasmic
shuttling ability and therefore the efficient nuclear export of late
viral transcripts.
View larger version (55K):
[in a new window]
Fig. 8.
The ORF 57 NLS is required for the efficient
nuclear export of viral mRNA. COS-7 cells transfected with
pUCgB in the absence (lanes 1, 4, and
7) and presence of pRSVORF57 (lanes 2,
5, and 8) or p57NLS 3 (lanes
3, 6, and 9). Total (lanes
1-3), nuclear (lanes 4-6), and
cytoplasmic (lanes 7-9) RNA was then isolated
and separated by electrophoresis on a 1% denaturing
formaldehyde-agarose gel. The RNA was transferred to Hybond-N membranes
and hybridized with a 32P-radiolabeled random-primed probes
specific for the HVS gB and actin coding sequences.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
isoforms 1 and 5. Importin
functions as
an adapter molecule binding NLS-containing molecules and the transport
receptor, importin
(reviewed in Ref. 14). In contrast to importin
, several isoforms of importin
exist in higher eukaryotes. These
can be grouped into three main subfamilies, which differ from one
another in their amino acid comparison by ~50% (32, 33). The
presence of multiple isoforms of importin
suggest that each isoform
may import specific substrates. There have been several experiments to
support this theory. The nuclear import of the transcription factor
Stat1 is mediated by importin
5, but not importin
1 (34).
Moreover, Nadler et al. (35) utilized pull-down assays to
demonstrate that importin
1 and importin
5 share distinct binding
affinities for various NLSs. However, recent analysis has demonstrated
that all importin
isoforms could import most substrates with a
similar efficiency (33). At present, we have not determined whether all
the importin
isoforms have distinct ORF 57 specificities.
and in turn exploit the importin-mediated pathway to
enter the host cell nucleus. The influenza virus nucleoprotein (NP) has
been shown to bind both importin
1 and
5 isoforms (36-38). Furthermore, the Epstein-Barr virus EBNA-1 protein, which is required for the replication and stable maintenance of the viral genome (reviewed in Ref. 39), has also been shown to interact with importin
1 and
5 isoforms (40, 41). Moreover, nuclear import of the human
papillomavirus (strain 11) L1 major capsid has been shown to be
mediated by the importin pathway via importin
5 binding (42). More
recently, HIV-1 Rev has been shown to utilize the importin-mediated
pathway but independently of importin
(43, 44). The HIV-1 Rev
arginine-rich NLS has been demonstrated to interact directly with
importin
which mediates the import of Rev, using in
vitro import assays (43, 44).
1 and
5 binding. Moreover,
additional upstream and downstream sequences are required for importin
1 binding (45). In contrast, the influenza A NP contains two
overlapping non-conventional NLSs (38). Mutational analysis by alanine
scanning identified differing motifs required for importin
1 and
5 binding: SXGTKRSYXXM for importin
5 and TKRSXXXM for importin
1 binding (38). Although data
presented in this report suggest that ORF 57 utilizes the same NLS for
both importin
1 and
5 binding, it cannot be excluded that the NLS contains overlapping motifs that specify importin
isoform binding.
have been revealed by examination of the crystal structure of yeast and
mammalian importin
molecules (46, 47). Sequence analysis has
revealed that importin
is composed of three distinct domains (20,
48): a basic amino-terminal importin
-binding domain; a large
central domain composed of 8-10 arm repeats; and an acidic carboxyl
terminus, which mediates an interaction with the nuclear export factor,
CAS (24-26). Co-crystallization studies of importin
and a
monopartite NLS has identified two possible binding sites (46, 47). The
major site lies at the amino-terminal end of the receptor between the
first and fourth arm repeats, and the minor site is located at the
carboxyl terminus between arm repeats 4 and 8. At both sites, the NLS
binds in an extended antiparallel conformation, via tryptophan and
asparagine residues (46, 47). Moreover, the bipartite nucleoplasmin NLS
simultaneously binds to both major and minor sites (46, 47). From these
studies we infer that ORF 57 contains a monopartite NLS, which
specifically binds to the minor repeats at the carboxyl terminus
between the fourth and eight arm repeats. Similar observations have
demonstrated that this is the minimal region required for the
interaction of importin
1 with EBNA1-NLS (40) and importin
2 with
LEF-1 NLS (49).
complex,
we have demonstrated that this complex is directed to the nucleolus.
Results herein suggest that the ORF 57 amino-terminal arginine-rich
domain mediates both nuclear and nucleolar localization. Similar
nucleolar targeting has been observed with HIV-1 (50-52). However, no
functional role has yet been attributed to this localization and it is
the matter of some debate. It has recently been reported that Rev
induces the redistribution of the nucleoporins Nup98 and Nup214, in
addition to the nuclear export factor CRM-1, into the nucleolus (53).
These findings suggest that assembly of the Rev-nuclear export complex
occurs in the nucleolus. However, data generated utilizing an HIV-1
nucleolar-localized ribozyme suggest that HIV-1 transcripts undergo
nucleolar trafficking (54). This has led to speculation that a
ribonucleoprotein particle containing HIV-1 RNA, regulatory proteins,
and cellular factors, involved in the post-transcriptional modification
or export and translation of HIV-1 transcripts, occurs in the
nucleolus. Whether the nucleolus plays similar roles in ORF 57 functioning is unknown and warrants further investigation.
isoforms
1 and 5. The binding of ORF 57 to importin
is mediated by the
hydrophobic, internal, importin
armadillo repeats. An ORF 57 amino-terminal arginine-rich sequence, which functions as an NLS, was
also required for this interaction. Furthermore, the ORF 57 protein is
responsible for the redistribution of importin
into the nucleoli.
Moreover, preliminary experiments suggest that this redistribution
reduce nuclear import of secondary NLS-containing protein via the
importin-mediated nuclear import pathway. These results suggest that
ORF 57 is a nucleocytoplasmic shuttle protein, which re-enters the
nucleus via the importin
-mediated pathway and this interaction is
necessary for the efficient nuclear export of viral RNA transcripts.
Future studies will be directed to determine which cellular pathway ORF
57 utilizes to exit the nucleus and the functional significance of the
nucleolus in the functioning of this important herpesvirus regulatory protein.
![]() |
ACKNOWLEDGEMENTS |
---|
We are very grateful to Matthias Köhler, Dirk Görlich, Peter Palese, and David Matthews for providing antibody reagents and expression constructs used in this work. We thank Alex Markham for critical reading of this manuscript.
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FOOTNOTES |
---|
* This work was supported in part by grants from the Medical Research Council (MRC) and Yorkshire Cancer Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a MRC studentship.
§ Recipient of a MRC fellowship. To whom correspondence should be addressed. Tel.: 44-113-2066328; Fax: 44-113-2444475; E-mail: a.whitehouse@leeds.ac.uk.
Published, JBC Papers in Press, March 16, 2001, DOI 10.1074/jbc.M009513200
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ABBREVIATIONS |
---|
The abbreviations used are: HIV-1, human immunodeficiency virus type 1; HSV, herpes simplex virus; HVS, H. saimiri; PCR, polymerase chain reaction; bp, base pair(s); ORF, open reading frame; NLS, nuclear localization sequence; GFP, green fluorescent protein; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; DBD, DNA-binding domain; AD, activation domain; NP, nucleoprotein.
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