(Received for publication, April 1, 1997, and in revised form, May 8, 1997)
From the Transcriptional induction of genes is an
essential part of the cellular response to interferons. We have
established a cDNA library from human lymphoblastoid Daudi cells
treated for 16 h with human The interferons (IFNs)1 are a family
of secreted multifunctional proteins that exert a broad spectrum of
biological activities. First characterized for their potent antiviral
properties, it has now been established that they are involved in a
number of regulatory functions such as control of cell proliferation,
differentiation, and regulation of the immune system (1, 2). Binding of
both type I IFN (IFN- The diverse biological actions of IFNs are thought to be mediated by
the products of specific but usually overlapping sets of cellular genes
induced in the target cells. More recently, some interferon-induced
proteins have been located within discrete nuclear structures termed
nuclear bodies (19-22). Previously defined by electron microscopy as
dense 0.3-0.5-nm diameter spherical particles, the nuclear bodies,
so-called PML (for promyelocytic leukemia protein) nuclear bodies (PML NBs), appear
characteristic of large multiprotein complexes associated with the
nuclear matrix (23-28). These structures are distinct from other well
described subnuclear domains such as the nucleolus, the interchromatin
granules, the perichromatin fibrils, and the coiled bodies (for review, see Ref. 29). Among the PML NB-associated proteins, PML is the best
documented. PML was originally discovered as a fusion protein with
retinoic acid receptor- Although the function of PML NBs is still unknown, some observations
suggest that they may represent preferential targets for viral
infection and thus could play a role in the mechanism of antiviral
action of IFNs. In particular, after adenovirus infection, the viral
E4-ORF3 protein is targeted to PML NBs and causes their reorganization
from spherical to fibrous structures (37, 38). The human T-cell
leukemia virus type 1 Tax oncoprotein induces a diffuse cytoplasmic
redistribution of the Int-6 protein, which normally colocalizes with
PML in the absence of Tax expression (39). This delocalization appears
to be specific for Int-6 because Tax does not alter the global speckled
staining pattern of PML. The herpes simplex virus type 1 immediate-early protein Vmw110 (ICPO), which is implicated in the
control of reactivation of latent herpes simplex virus type 1, transiently colocalizes after viral infection to PML NBs and
subsequently disrupts these structures (40, 41). In the same way, PML
NBs are reorganized after human cytomegalovirus infection (42, 43). The
Epstein-Barr virus-encoded nuclear antigen EBNA-5 (44), the adenovirus
E1A protein, and the SV40 large T antigen (37), other viral members of
the oncoprotein family, are also found in close association with PML
NBs. These observations, coupled with the fact that the PML, SP100, and
NDP52 proteins are all induced by IFN (19-22), have suggested that PML NBs may play a role in the viral infection process. The elucidation of
the actual role of PML NBs in the cell and their implication in the
mechanism of action of IFN will be through the identification of new
PML NB-associated proteins and the characterization of their behavior
under physiological stimuli.
We have therefore established a cDNA library from IFN-treated Daudi
cells and made use of differential screening to search for as yet
unidentified IFN-regulated genes (45). In the course of this study, we
have isolated a human cDNA encoding a novel protein that shares
strong similarity with the nuclear Xenopus protein XPMC2
(46). This new gene will be referred to as ISG20 for
interferon-stimulated gene product of 20 kDa. Using laser confocal
immunofluorescence analysis, we demonstrate that ISG20 protein is
closely associated with PML and SP100 in the newly described nuclear
structures termed PML NBs.
Human lymphoblastoid Daudi
cells were grown in suspension in RPMI 1640 medium supplemented with
10% (v/v) fetal calf serum. Hamster fibroblast (CCL39),
SV40-transformed monkey kidney epithelial (COS-7m6), human
hepatocarcinoma (CCL13), and human HeLa cells were grown in monolayer
cultures in Dulbecco's modified Eagle's medium containing 10% (v/v)
fetal calf serum. For IFN induction, exponentially growing cells were
exposed for 16 h to 500 IU/ml human lymphoblastoid IFN
(HuIFN- For RNA
purification, the cells were pelleted and washed in phosphate-buffer
saline, and total mRNAs were isolated by the guanidine thiocyanate
method as described previously (47). RNAs were fractionated by
electrophoresis on a 10% (v/v) formaldehyde-containing 1.2% (w/v)
agarose gel and transferred to nylon membranes (Hybond-N, Amersham
Corp.). The multiple-tissue Northern blot membrane
(CLONTECH) was a gift of Dr. P. Fort.
Prehybridizations were performed at 42 °C for 12 h in a mixture
containing 50% (v/v) formamide, 0.75 M NaCl, 50 mM sodium phosphate, pH 7, 1 mM EDTA, 0.2%
(w/v) SDS, 5 × Denhardt's solution, 10% (w/v) dextran sulfate,
and 100 mg/ml denatured salmon sperm DNA. An additional 12-h
hybridization was performed in the presence of 106 cpm/ml
32P-labeled random-primed cDNA probe. Stringent
washings were then conducted at 65 °C in 0.1 × SSC (0.15 M NaCl and 0.015 M sodium citrate) before
autoradiography.
For run-on
experiments, the cells were washed twice with phosphate-buffered saline
and lysed with a Dounce homogenizer in 10 volumes of 0.25 M
sucrose containing 10 mM Tris-HCl, pH 8.3, 10 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, and 0.5% (v/v) Nonidet P-40. Nuclei
were purified through a sucrose cushion as described previously
(48).
Poly(A+) RNAs were isolated from total
mRNAs using the Dynabeads biomagnetic separation system (Biosys
S. A.), and the cDNA library was constructed in the Plasmid DNAs of individual clones were prepared, and their
sequences were determined by the Sanger dideoxy sequencing method (T7
sequencing kit, Pharmacia Biotech Inc.). The complete sequence of
ISG20 cDNA was obtained on both strands by overlapping
sequenced fragments of the original clone after subcloning in the
pBluescript II KS vector. Searches for sequence homologies in the EMBL
and GenBankTM Data Banks as well as sequence analyses were performed by
using the BISANCE facilities (49).
CCL13, CCL39,
and COS cells were grown in Dulbecco's modified Eagle's medium
supplemented with glutamine, antibiotics, and 10% fetal calf serum
(Life Technologies, Inc.). Transfections were performed by the calcium
phosphate precipitation method for CCL13 and by the LipofectAMINE
procedure (as outlined by Life Technologies, Inc.) for CCL39 and COS
cells. Cells were grown as monolayers on glass coverslips in 35-mm
Petri dishes containing 2 ml of culture medium and transfected with 2.5 µg of expression vector DNA and 2.5 µg of carrier pSG5 plasmid.
24 h after transfection, the medium was replaced by fresh culture
medium, and the cells were further incubated for 24 h.
Immunofluorescence was performed as previously described (26) with
anti-PML or anti-SP100 polyclonal antibodies (diluted 1:200) and with
the 12CA5 anti-HA monoclonal antibody (15 ng/µl). Slides were viewed
using a Leica confocal microscope. Image files were processed with the
Adobe Photoshop program.
Total RNAs were extracted from
human lymphoblastoid Daudi cells treated for 16 h with 500 IU of
HuIFN- The kinetics of expression of the RNA hybridizing to the
ISG20 cDNA probe was analyzed by probing a Northern blot
of total RNAs isolated from Daudi cells treated for various times with HuIFN-
The specificity of induction of the ISG20 mRNA in
response to treatment with the various types of IFN was then analyzed.
As Daudi cells failed to respond to HuIFN- To determine whether the modulation of ISG20 occurs at the
transcriptional level, nuclear run-on assays were conducted with nuclei
purified from Daudi cells treated or not with HuIFN- To determine the tissue specificity of ISG20 expression, the
694-nucleotide insert was used to probe a set of RNAs isolated from
several tissues (multiple-tissue Northern blot membrane from CLONTECH). As shown in Fig. 2,
ISG20 is strongly expressed, in the absence of exogenous IFN
treatment, in peripheral blood leukocytes, in lymphoid tissues (such as
spleen or thymus), and in colon and lung. Various basal levels were
detected in other tissues. The same blots were stripped and
rehybridized with the control
To investigate the
function of ISG20, the nucleotide sequence of the
694-nucleotide ISG20 cDNA fragment was determined. A single open reading frame (ORF) of 537 nucleotides was identified (Fig.
3). The ISG20 ORF predicts a gene product (gpISG20) of
179 amino acids with a relative molecular mass of 20.4 kDa. Analysis of
the deduced amino acid sequence revealed a very basic protein with 7 lysine and 19 arginine residues (Lys + Arg = 14.5% of total residues) and a pI of 9.2. Using algorithms to predict the presence of
A nucleotide comparison by computer search did not reveal any
significant homology between ISG20 and the sequences
referenced in data bases. However, a search for amino acid sequence
homologies revealed that the complete ISG20 protein shares amino acid
cluster homologies with the Xenopus laevis XPMC2 gene
product (46) and with a theoretical ORF present in the genome of
Saccharomyces cerevisiae (GenBankTM accession number
Z74822). Expression of XPMC2 cDNA has been shown to
rescue in the fission yeast Schizosaccharomyces pombe
several mitotic catastrophe mutants defective in both Wee1 and Mik1
kinases. These redundant kinases negatively regulate Cdc2 kinase by
phosphorylating a conserved tyrosine residue. The XPMC2 gene product
acts as a negative cell cycle regulator by competing with mitotic
substrates for phosphorylation by Cdc2 kinase (46). The role and the
regulation of the S. cerevisiae ORF remain unknown.
On the basis of the amino acid homology between ISG20 and XPMC2, whose
amino acid sequence alignment is presented in Fig. 4, it
is tempting to speculate that ISG20 may be one of the elements that
participate in the negative regulation of cell division, which would be
consistent with the anti-growth properties of IFNs as found in
particular in Daudi cells. We evaluated this hypothesis by analysis of
the cell cycle distribution of cells overexpressing ISG20. To this aim,
COS-7m6 cells were transiently transfected with a vector expressing
ISG20. Transfection was performed by the LipofectAMINE procedure. The
cells were collected 72 h later and analyzed with a flow cytometer
after propidium iodide staining. Under these experimental conditions,
no significant alteration in the cell cycle distribution was observed
(data not shown).
The recent release in the data bases of a human 5 The ISG20 amino
acid sequence does not enclose a canonic bipartite nuclear localization
signal, but the presence of lysine- and arginine-rich domains and the
small size of the protein suggest that gpISG20 might be targeted to the
nucleus. To determine the subcellular localization of ISG20, CCL13,
CCL39, and COS cells were transfected with a tagged ISG20 protein. A
fusion cDNA between the open reading frame of ISG20 and the HA
epitope peptide sequence of the influenza virus was cloned under the
control of the cytomegalovirus promoter in the pJ7
Binding of IFNs to their specific cell-surface receptors triggers
the rapid nuclear translocation of a complex formed by association between the various phosphorylated STAT proteins (see the
Introduction). This mechanism results in the induction of specific sets
of genes that mediate the various biological functions of IFNs. The
proteins encoded by theses genes exhibit cytoplasmic, nuclear, or
cell-surface localization. Cytological analysis has revealed a complex
functional organization within the nucleus. Some nuclear proteins
localize within discrete and functionally distinct classes of nuclear
domains. The two most documented are the nucleolus, in which genes are transcribed and ribosomal RNA is processed (54), and the presumptive splice sites resulting from the association of small ribonucleoprotein particles with specific sites on the nuclear matrix (55-57). Recently, some IFN-induced proteins have been described firmly bound to the
nuclear matrix, forming discrete nuclear structures termed NBs or PML
NBs, distinct from the subnuclear domains previously described
(19-28). In this report, we describe the isolation and characterization of a cDNA encoding a novel PML NB-associated protein designated ISG20, the expression of which is induced after HuIFN treatment.
Comparison of the amino acid sequence of ISG20 with the sequences of
EMBL and GenBankTM Data Banks revealed significant homologies between
ISG20 and the X. laevis XPMC2 gene product (46). Using a
genetic complementation method, XPMC2 has been identified to functionally rescue a fission yeast mitotic catastrophe mutant defective in both Wee1 and Mik1 kinases (46). These homologies raise
the possibility that ISG20 might act as a negative regulator of cell
division induced by IFNs. However, ISG20 did not appear to affect cell
growth in transient transfection experiments. Alignment of the amino
acid sequences of ISG20 and XPMC2 showed that ISG20 is much shorter
than XPMC2 and is homologous to the C-terminal half of XPMC2 protein.
Interestingly, a truncated XPMC2 protein that retains only its
C-terminal half is not able to rescue the mitotic catastrophe
phenotype. According to the presence within ISG20 protein, of a
presumptive structural coiled-coil domain that is supposed to mediate
protein-protein interactions, we can imagine that interactions between
ISG20 and other proteins are required to mimic, in human, the XPMC2
function. However, we have no direct evidence to accredit this
hypothesis at present. In addition, ISG20 shares a strong amino acid
homology with a theoretical ORF present in the genome of S. cerevisiae. The function of this ORF remains unknown. Since
protein conservation during evolution usually affects essential
cellular functions, the comprehension of the regulation and biological
activity of the S. cerevisiae ORF will be important to
determine the actual role of ISG20. The knockout of the yeast gene is
now in progress to allow this study.
We have examined the subcellular localization of ISG20 protein. Ectopic
expression of a fusion protein between ISG20 and the HA epitope peptide
revealed that ISG20 is predominantly nuclear and gives a punctuate
staining pattern. Interestingly, the number and size of the nuclear
dots containing ISG20 were variable from one cell to another. These
data can reflect a modulation of PML NBs throughout the cell cycle.
Using confocal immunofluorescence microscopy, we demonstrated that
ISG20 is closely associated with PML and SP100 within the large
multiprotein complexes termed PML NBs (19-28). To determine whether a
direct interaction between these proteins can occur, a yeast two-hybrid
study was performed. This was conducted using ISG20 fused to the GAL4
DNA-binding domain and PML or SP100 fused to the GAL4-activating
domain. No direct interaction was detected between these proteins when
coexpressed in yeast Y187 cells.
Viruses require the host cell machinery for their multiplication cycle,
and they have developed various strategies to circumvent the antiviral
activities induced in the cells by IFNs. Various viral proteins such as
the adenovirus E4-ORF3 protein (37, 38), the human T-cell leukemia
virus type 1 Tax oncoprotein (39), the herpes simplex virus type 1 Vmw110 protein (40, 41), the cytomegalovirus promoter IE1 protein (43),
and the SV40 large T antigen (37) concentrate in the PML NB speckles,
suggesting that PML NBs play a major role during infection by oncogenic
viruses. The fact that the PML NB-associated proteins PML, SP100,
NDP52, and ISG20 are all inducible by IFNs (Refs. 19-22 and this
report) strongly suggests the implication of a such subnuclear
structure in the mechanism of IFN action. Further studies on the
modulation of Int-6 and PIC-1, a newly described PML NB-associated
protein (58, 59), by IFNs might be worth being performed to confirm this hypothesis. The disruption of PML NBs during viral infection reflects the ability of viruses to circumvent the antiviral activities of IFNs. Eventual modifications of ISG20 subcellular localization after
various viral infections are underway to increase the understanding of
the role of ISG20 in the mechanism of antiviral action induced by IFNs.
The availability of specific antibodies against this protein is
essential to determine its function, and their preparation is now in
progress.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) X89773. We thank J. Nessen, B. Lebleu, and C. Montavon.
Institut de Genetique Moleculaire de
Montpellier-UMR 9942,
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
/
-interferon (IFN) and made use
of differential screening to search for as yet unidentified
IFN-regulated genes. In the course of this study, we have isolated a
human cDNA that codes for a 20-kDa protein sharing striking
homology with the product of the Xenopus laevis XPMC2 gene.
This new gene is induced by both type I and II IFNs in various cell
lines and will be referred to as ISG20 for
interferon-stimulated gene product of 20 kDa. Confocal
immunofluorescence analysis of the subcellular localization of ISG20
protein reveals that it is closely associated with PML and SP100 gene
products within the large nuclear matrix-associated multiprotein
complexes termed the PML nuclear bodies.
/
) and type II IFN (IFN-
) to different
cell-surface receptors (3, 4) activates transduction pathways via
tyrosine phosphorylation of latent cytoplasmic transcription factors
termed STAT factors (for signal transducer and
activator of transcription) (5-11). The STAT
factors are assembled to form the specific transcription complexes
ISGF3 (for interferon-stimulated
gene factor 3) for IFN-
/
and
GAF (for IFN-
activation factor)
for IFN-
. These transcription factors act at different
cis-acting DNA elements termed the IFN-stimulated responsive
element for ISGF3 and the IFN-
activation site for GAF and are
located in the promoter region of IFN-induced genes (12-18).
in malignant hematopoietic cells with a
t(15:17) translocation characteristic for patients with acute
promyelocytic leukemia (reviewed in Refs. 30-32). A tumor cell growth
suppressor function and a role in human oncogenesis were reported for
PML (33-36). In addition to PML, the nuclear bodies include at least
two other IFN-induced proteins, NDP52 (for nuclear
dot protein 52) (19) and the SP100
protein, originally identified as an autoantigen in patients with
primary biliary cirrhosis (20, 24). It is interesting to note that the
number and morphology of PML NBs are variable, particularly throughout the cell cycle and in some pathological contexts. In acute
promyelocytic leukemia, PML NBs are disrupted into a microparticulate
pattern as a consequence of the expression of the PML-retinoic acid
receptor-
oncoprotein. Retinoic acid treatment triggers a
reorganization of the nucleus to generate normal appearing PML NBs,
which in turn is linked to differentiation of acute promyelocytic
leukemia cells (26-28).
Cell Cultures and Antibodies
/
; obtained from Hayashibara Biochemical Laboratories
Inc.) or 500 units/ml IFN-
(a gift of Roussel-UCLAF, Paris, France).
For the generation of anti-PML and anti-SP100 antibodies, a PML
cDNA NcoI-SmaI fragment (positions 80-1421
in the cDNA sequence) and the full-length coding region of SP100
cDNA were inserted into a glutathione S-transferase gene fusion vector to generate GST-PML and GST-SP100 hybrid proteins. Recombinant proteins produced in Escherichia coli were
purified using glutathione-Sepharose 4B columns and were used for
rabbit immunizations.
ZAP
cDNA synthesis system (Stratagene). The library was plated at low
density to obtain individual plaques and transferred to nylon membranes
(Hybond-N). A single round of screening was performed by successive
hybridization of a single filter using 32P-labeled cDNA
probes (2 × 106 cpm/ml) obtained from
poly(A+) RNAs of untreated or IFN-treated Daudi cells.
Prehybridization, hybridization, and washing of the filter were
performed as described for Northern blot analysis. Clones exhibiting a
variation in signal intensity were isolated, and the pBluescript
phagemid vectors containing inserts were excised using the ExAssit-SORL
system (Stratagene). Phagemid DNAs were then extracted and used to
probe Northern blot membranes.
Construction and Screening of cDNA Library from
HuIFN-/
-treated Daudi Cells
/
. An oriented cDNA library was constructed using the
Stratagene
ZAP cDNA synthesis kit. 5000 primary recombinant
clones were screened successively with single-stranded
32P-labeled cDNA derived from exponentially growing
untreated cells and with cDNA from IFN-treated cells. Spots
exhibiting a variation in signal intensity were selected, and
pBluescript phagemid vectors containing inserts were excised using the
Stratagene ExAssit-SORL system. DNAs were prepared and used to probe a
Northern blot containing total RNA extracted from Daudi cells treated
for various times with HuIFN-
/
. Clones exhibiting differential
expression upon Northern blot analysis by comparison with an invariant
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (50) probe
were selected. Complete sequence examination of positive clones
revealed that one that contains a 694-nucleotide insert encoded a new
IFN-induced mRNA, which will be referred to as
ISG20.
/
. As shown in Fig. 1A, the
ISG20 mRNA accumulated rapidly after the onset of IFN
treatment. An 8-fold increase in its steady-state level was reached
after 16 h of exposure to IFN. Hybridization to a GAPDH
probe used as an invariant control confirmed that each lane of the blot
contained an equal amount of total RNA (Fig. 1A).
ISG20 participates in the primary response of IFN action since this induction was not dependent on continuous protein (data not
shown).
Fig. 1.
Northern analysis of IFN-induced
ISG20 mRNA. Total RNAs (20 µg/lane) were
separated on 1.2% formaldehyde-agarose gel, transferred to nylon
membrane, and hybridized to a 32P-labeled ISG20
cDNA probe. The same blot was reprobed with a GAPDH probe to ensure
that equal amounts of RNA were loaded in each lane. A, time
course of ISG20 mRNA induction with HuIFN-/
. Daudi
cells were treated with HuIFN-
/
(500 units/ml) for the indicated
times. B, specificity of ISG20 mRNA induction
by HuIFN-
/
and HuIFN-
. HeLa cells were treated with
HuIFN-
/
(500 units/ml) or HuIFN-
(500 units/ml) for the
indicated times. C, run-on analysis of transcription
elongation of the ISG20 gene. Run-on assays were carried out
with nuclei from Daudi cells treated (IFN) or not (control
(Ct)) with HuIFN-
/
for 16 h. The labeled RNAs
were hybridized with a filter containing the indicated probes.
pSKII, pBluescript II KS vector.
[View Larger Version of this Image (42K GIF file)]
due to the lack of
functional receptors, HeLa cells were treated with 500 units/ml
HuIFN-
/
or HuIFN-
, and total RNAs were extracted and analyzed
as described above. As shown in Fig. 1B, the kinetics of
induction of ISG20 mRNA was found to be similar with the
two types of IFN, although HuIFN-
/
was found to be a stronger
inducer.
/
as
described previously (48). The labeled RNAs were hybridized with
filters containing ISG20, GAPDH, and pBluescript II KS
vector probes. GAPDH and the pBluescript II KS vector were used as
invariant and negative controls, respectively. The data presented Fig.
1C clearly demonstrate that the increase in ISG20
expression by IFN occurs at the transcriptional level, in keeping with
the mechanism of induction of the majority of IFN-induced genes.
-actin provided by the manufacturer as
an invariant control.
Fig. 2.
Tissue specificity of ISG20
expression. A, a multiple-tissue Northern blot membrane
(CLONTECH) was hybridized to a 32P-labeled ISG20 cDNA probe. The tissue
source of the mRNA sample in each lane is indicated at the top. The
same blot was stripped and rehybridized with a -actin cDNA probe
as a control. Kb, kilobase.
[View Larger Version of this Image (31K GIF file)]
-helices (51) and coiled-coil structures (52), region 78-107 of
gpISG20 is strongly predicted to form a coiled-coil domain (Fig. 3).
This structural domain is known to mediate protein-protein interactions
(for review, see Ref. 53), suggesting that ISG20 can act as a component
of a multiprotein complex; this important point will be discussed later
on the basis of the subcellular localization of ISG20 protein. The
coiled-coil region is bordered on both sides by potential
phosphorylation sites. A tyrosine kinase phosphorylation site is
located from amino acids 44 to 52, and a phosphorylation domain is
located from amino acids 106 to 134, which contains several potential
protein kinase C, casein kinase II, and cAMP-dependent
protein kinase phosphorylation sites (Fig. 3).
Fig. 3.
Nucleotide sequence and predicted amino acid
sequence of ISG20 cDNA. The complete nucleotide
sequence of ISG20 cDNA (bottom line) and the
predicted amino acid sequence (top line) are shown. The
nucleotides and amino acids are numbered to the right of the sequence.
The predicted coiled-coil region is boxed. The two
phosphorylation domains are underlined.
[View Larger Version of this Image (34K GIF file)]
Fig. 4.
Alignment of amino acid sequences of ISG20
and XPMC2 proteins. The proteins are indicated on the left. The
positions of the conserved amino acids are indicated by
asterisks, and the conserved hydrophobic residues by
periods.
[View Larger Version of this Image (39K GIF file)]
-EST sequence
(GenBankTM accession number R02224) with a high nucleotide identity to
the XPMC2 cDNA clearly demonstrates that ISG20 is not
the human homolog of XPMC2, but is a member of a new family of
proteins. Surprisingly, human EST-like ISG20 corresponds to a short RNA
and encodes a protein homologous only to the carboxylic half of the
XPMC2 protein.
vector
(pJ7TagHA-ISG20). Cells were transfected by the calcium phosphate
precipitation or LipofectAMINE method and then analyzed by
immunofluorescence using the 12CA5 monoclonal antibody. The ectopically
expressed ISG20 was predominantly nuclear and gave a speckled
distribution pattern in all cell types (Fig. 5A). This localization was not dependent on
the level of tagged ISG20 expression since the same pattern was
observed in COS cells, which express a high level of transfected
cDNA as compared with CCL39 and CCL13 cells. However, in CCL13
cells, ISG20 was diffusely distributed throughout the nucleoplasm in
~30% of the positive cells. This percentage strongly suggests that
the change in the intranuclear distribution of ISG20 might be dependent
on the progression of the cell cycle. The nuclear dots containing ISG20
were dispersed throughout the nucleoplasm and were variable in size and
number per nucleus (Fig. 5A). The speckled nuclear staining
pattern of ISG20 is reminiscent of the labeling observed for other
cellular proteins such as PML and SP100, which colocalize within large multiprotein complexes called PML NBs (23-28). To compare the
intranuclear localization of ISG20 with that of endogenous PML, double
staining immunofluorescence experiments were performed on the
ISG20-transfected CCL13 cells using the 12CA5 monoclonal antibody and
an anti-PML polyclonal antiserum (26). The punctuate pattern of
overexpressed ISG20 is coincident with that of endogenous PML as shown
in Fig. 5B. To examine whether ISG20 and PML exactly
localized within the same nuclear structures, confocal
immunofluorescence microscopic analysis was performed in the CCL13
cells. The localization of ectopically expressed ISG20 was compared
with that of the two major endogenous components of NBs, the PML and
SP100 proteins. As shown in Fig. 6, all of the nuclear
dots containing ISG20 were coincident with the dots containing both PML
and SP100 proteins as revealed by double labeling with the 12CA5
monoclonal antibody and PML (Fig. 6A) or SP100 (Fig.
6B) polyclonal antiserum. The ISG20 labeling pattern was
closely associated rather than perfectly overlapping with the PML/SP100
immunofluorescence pattern. Moreover, there were additional PML or
SP100 dots that did not contain detectable levels of ISG20 protein. It
is not clear at present whether or not the level of ectopically
expressed ISG20 protein is sufficient for occupying all the PML NB
sites or whether this represents a preferential association of ISG20
with a subset of PML NBs.
Fig. 5.
Subcellular localization of ISG20 protein.
A, nuclear localization by immunofluorescence of ectopic
HA-ISG20 in transiently transfected CCL39, COS, and CCL13 cell lines;
B, localization by double immunofluorescence labeling of
ectopic hemagglutinin-ISG20 (right panel) and endogenous
human PML (left panel) in CCL13 cells.
[View Larger Version of this Image (41K GIF file)]
Fig. 6.
Laser confocal immunofluorescence analysis of
colocalization of ISG20 with PML and SP100 in CCL13 cell line.
A, localization by double immunofluorescence labeling of
ectopic HA-ISG20 (left panel) and endogenous human PML
(center panel) in HA-ISG20-transfected CCL13 cells. The
single confocal images were superimposed (right panel).
B, localization by double immunofluorescence labeling of
ectopic HA-ISG20 (left panel) and endogenous human SP100
(center panel) in HA-ISG20-transfected CCL13 cells. The
single confocal images were superimposed (right
panel).
[View Larger Version of this Image (73K GIF file)]
*
This work was supported by grants from the Association pour
la Recherche contre le Cancer, INSERM, CNRS, the Association Nationale pour la Recherche sur le Sida, and the Federation des Centers de Lutte
contre le Cancer.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.
¶
To whom correspondence should be addressed: Inst. de Genetique
Moleculaire de Montpellier-UMR 9942, CNRS, BP 5051, 1919 Route de
Mende, 34033 Montpellier Cedex 1, France. Tel.: 4-67-61-36-61; Fax:
4-67-04-02-45.
1
The abbreviations used are: IFNs, interferons;
HuIFN, human interferon; PML NBs, PML nuclear bodies; HA,
hemagglutinin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ORF,
open reading frame.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.