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J Virol, February 1998, p. 1043-1051, Vol. 72, No. 2
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Resistance to Virus Infection Conferred by the
Interferon-Induced Promyelocytic Leukemia Protein
Mounira K.
Chelbi-Alix,*
Frédérique
Quignon,
Luis
Pelicano,
Marcel H. M.
Koken, and
Hugues
de Thé
CNRS UPR 9051, Centre Hayem, Hôpital
St. Louis, 75475 Paris Cedex 10, France
Received 4 August 1997/Accepted 31 October 1997
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ABSTRACT |
The interferon (IFN)-induced promyelocytic leukemia (PML) protein
is specifically associated with nuclear bodies (NBs) whose functions
are yet unknown. Two of the NB-associated proteins, PML and Sp100, are
induced by IFN. Here we show that overexpression of PML and not Sp100
induces resistance to infections by vesicular stomatitis virus (VSV) (a
rhabdovirus) and influenza A virus (an orthomyxovirus) but not by
encephalomyocarditis virus (a picornavirus). Inhibition of viral
multiplication was dependent on both the level of PML expression and
the multiplicity of infection and reached 100-fold. PML was shown to
interfere with VSV mRNA and protein synthesis. Compared to the IFN
mediator MxA protein, PML had less powerful antiviral activity. While
nuclear body localization of PML did not seem to be required for the
antiviral effect, deletion of the PML coiled-coil domain completely
abolished it. Taken together, these results suggest that PML can
contribute to the antiviral state induced in IFN-treated cells.
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INTRODUCTION |
Interferons (IFNs) are a family of
secreted proteins with antiviral, antiproliferative, and
immunomodulatory activities. The molecular basis of the IFN response,
in particular the antiviral and antiproliferative effects, is not yet
fully understood. More than 100 genes are known to be IFN induced, but
the physiological roles of the majority of their products are not yet
recognized. Only a few IFN-induced proteins, namely, the p68 protein
kinase, the 2',5'-oligoadenylate (2'5'A) synthetase, and certain Mx
family proteins, have been shown to display intrinsic antiviral
activities (reviewed in references 37, 39 and
41). While IFN-treated cells are resistant to a
large variety of virus infections, these three known effectors confer
protection only against some RNA viruses, implying the existence of
complementary pathways.
The PML (promyelocytic leukemia) gene has been identified through its
fusion to the RAR
gene in the t(15;17) translocation found in
patients with acute promyelocytic leukemia (reviewed in reference
44). The PML protein shares a C3HC4 (RING finger) zinc binding motif (14) with a large group of polypeptides
which perform heterogeneous functions ranging from transactivation of viral genes to DNA repair or peroxisome assembly (reviewed in references 4 and 15). PML belongs
to a subfamily of nine proteins defined by the additional presence of
one or two other cysteine-rich motifs, the B boxes, as well as a very
long coiled-coil region (35), which is implicated in PML
homodimerization (21, 32).
PML has a speckled nuclear expression pattern which is the consequence
of the localization of the protein to nuclear bodies (NBs) (10,
12, 23, 45). PML colocalizes on these structures with an
autoantigen of primary biliary cirrhosis, Sp100 (43). The
functions of NBs are unknown, but they seem not to be sites of
replication, transcription, or splicing (42). Analysis of the 5' genomic sequences of PML revealed both a functional
IFN-/
-stimulated response element, ISRE, and an IFN-
activation site, GAS (40), demonstrating that PML is a
primary target gene of IFNs. That the two NB-associated proteins PML
(8, 24, 40) and Sp100 (19) are IFN induced
suggests a role for this nuclear structure in the IFN response. An
important point is to find which, if any, of the biological effects of
IFN could be mediated by PML. Recently, we and others have shown that
overexpression of PML suppresses the growth of some cell lines
(22). At present, the molecular basis of the
antiproliferative effect of PML is not understood. These findings could
allow PML to be included in the pathways responsible for IFN-induced
cell growth suppression.
Here we demonstrate that in the absence of IFN, constitutive
overexpression of PML but not of Sp100 confers resistance to infection
by vesicular stomatitis virus (VSV) and influenza A virus but not by
encephalomyocarditis virus (EMCV), identifying a novel pathway in the
mechanism of IFN antiviral action.
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MATERIALS AND METHODS |
Cell cultures.
Human glioblastoma astrocytoma U373 MG,
Chinese hamster ovary (CHO) cells, mouse GP+E
86 cells
(26), and L929 cells were grown at 37°C in Dulbecco's
modified Eagle's medium. The human histocytic lymphoma cell line U937
was grown in RPMI 1640. All media were supplemented with 10% fetal
calf serum. CHO cells, GP+E86 cells (transfected with the empty or
PML encoding vector), and CHO cells overexpressing Sp100 were kept in
medium supplemented with 0.5 mg of hygromycin (GIBCO) per ml. U373 MG
control cells (transfected with the empty vector) or the same cells
overexpressing PML were kept in medium supplemented with 0.5 mg of G418
per ml. Swiss 3T3 mouse cells transfected by the empty vector or vector expressing MxA (31) were a kind gift from J. Pavlovic and
were grown in the same medium supplemented with 0.5 mg of G418 per ml.
Construction of expression vectors and cell lines.
The PML
cDNA was inserted in different vectors: the
BglII-BamHI fragment (positions 48 to 2084 [11]) was ligated into the retroviral vector
M3P-SVhygro (17), whereas the complete cDNA on
an EcoRI fragment was inserted in the pSG5 vector or the
bicistronic pCIN vector (neomycin resistance) (36). The C
terminal PML mutant, PML Stop 504, was constructed by inserting an
oligonucleotide with an in-frame stop codon at the SacI site
of the PML cDNA (in the pSG5 construct). The coiled-coil PML mutant
PML
(216-333) was created by total digestion of the pSG5 PML vector
with BssHII and religation. The RING finger PML mutant
Q59C60/EL was described previously
(21). This mutation results in a change of the amino acids
glutamine and cysteine at positions 59 and 60 into aspartic acid and
leucine, respectively. The cytoplasmic PML mutant, Stop 381, results
from insertion of three stop codons in the unique Sse8387 I
site (nucleotide 1228 of the PML insert) of the pSG5 PML vector. For
the Sp100 construct, the region containing bp 32 to 1548 was amplified
by PCR from the original construct (43) with the nucleotides
-5' oligo (5'gaagatctgccgccATGGCAGGTGGGGGCGGC3') and -3' oligo
(5'GAGGGTCAGGTAAAGAAGATTAGagatcttc3') and
inserted in the BglII site of the pSG5 vector. This was done
to remove an in-frame upstream stop codon, to optimize the ATG, and
finally to create flanking BglII sites (underlined) for easy
cloning. The amplified Sp100 used was completely verified by sequence
analysis.
Stable transfections of CHO, GP+E86, and U373 MG cells.
Stable CHO or GP+E86 clones were obtained by lipofection (Gibco/BRL)
with pSG5 constructs cotransfected with DSPhygro or M3P-SVhygro-derived constructs and subsequent hygromycin
selection (final concentration, 0.5 mg/ml). Stable PML-expressing U373
MG clones were obtained via transfection with the pCIN-PML construct and subsequent neomycin selection (final concentration, 0.5 mg/ml). Control cells were generated in the same way with the empty vectors. Resistant colonies were examined for PML or Sp100 expression by indirect immunofluorescence, and positive pools were subjected to a
round of subcloning by limiting dilution. As a consequence of the
antiproliferative effect of PML, some of these clones tend to lose
their expression. Therefore, expression of the clones was verified
every six passages by immunofluorescence and Western blot analysis. The
apparent molecular weight of the PML mutant proteins was in agreement
with the molecular weight of the mutations made.
Interferons and anti-interferon antibodies.
Human IFN-
was from Triton Biosciences (Alameda, Calif.), and anti-human IFN-
(G-030-501-553), IFN- (G-028-501-568), and IFN- (G-034-501-565)
antibodies were from the National Institutes, of Health. U373 MG PML
cells were grown for two passages in the presence of 104
neutralizing units of anti-IFN-// antibodies per ml.
Virus stocks and virus yield assay.
Stocks of the WSN strain
of influenza A virus (4 × 108 PFU/ml), VSV (6 × 108 PFU/ml), or EMCV (8 × 108 PFU/ml)
were prepared from supernatants of virus-infected CHO cells. Cells were
seeded in 24-well plates for 5 h at 37°C and then infected with
virus at a multiplicity of infection (MOI) ranging from 0.1 to 3. At
the times indicated in the legends, cultures were frozen and thawed
three times and centrifuged to remove cell debris. The supernatants
were serially diluted, and the virus titers were measured by a plaque
assay on L929 or CHO cells and expressed as plaque-forming units per
milliliter of supernatant.
Determination of IFN titers.
Culture media from mouse
GP+E86 control and GP+E86 PML cells infected or not infected with
VSV or influenza virus at a MOI of 0.1 for 16 h were subjected to
titer determination on L929 cells, and those from U373 MG control and
U373 MG PML cells were subjected to titer determination on HeLa cells.
To inactivate the virus present, the culture supernatants from infected
cells were brought to pH2 for 24 h and neutralized before the
titer determination. All the cells were challenged with VSV. IFN
titers, determined as the amounts of IFN required to produce 50%
inhibition of the cytopathic effect, were expressed in relation to the
human IFN- reference (G-023-902-527) or mouse IFN reference (Ga 02 901 511) of the National Institutes of Health.
Immunofluorescence and Western blot analysis.
Rabbit and
mouse anti-human PML and rabbit anti-human Sp100 antibodies were
produced as described previously (10, 34). Anti-VSV
antibodies were a kind gift from D. Blondel. The cells were fixed in
4% paraformaldehyde for 15 min at 4°C and then 100% methanol for 5 min at 4°C. Immunofluorescence was performed with anti-PML,
anti-Sp100, or anti-VSV antibodies diluted 1/500 to 1/1,000 and
revealed with fluorescein isothiocyanate (FITC)-conjugated secondary
antibodies. For Western blot analysis, the cells were scraped in
phosphate-buffered saline, centrifuged, and lysed in 0.25 ml of 125 mM
Tris (pH 7)-1% sodium dodecyl sulfate-10% glycerol-0.75 mM
phenylmethylsulfonyl fluoride. Each sample was sonicated to reduce the viscosity. Western blot analysis was performed by standard procedures (20). A 40-µg portion of total-cell extracts
was analyzed with rabbit polyclonal anti-PML or anti-VSV antibodies, each diluted 1/2,000 and revealed by enhanced chemiluminescence (Amersham).
Northern blot analysis.
Total RNA was extracted with a
Bioprobe Systems RNA extraction kit standard. After the RNA was blotted
on nitrocellulose membranes (Scheicher & Schuell), the Northern blot
analysis was performed by random priming (Boehringer Mannhein)
radiolabelled VSV N, NS, M, G, or glyceraldehyde-3-phosphate
dehydrogenase probes. VSV N, NS, M, and G probes were generated as
previously described from plasmids (a gift from D. Blondel) pGN2
(13), pGNS1 (13), pKOM1 (5), and pSVG
(18), respectively.
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RESULTS |
IFN- inhibits VSV and influenza virus replication in human
monocytic cell line U937 in the absence of the MxA protein.
The
IFN-induced human MxA protein inhibits the multiplication of VSV and
influenza virus by an unknown mechanism (31, 41). The level
of this known anti-VSV and anti-influenza virus mediator is not
increased in the monocytic cell line U937 cells after IFN- treatment
(38). To assess the capacity of human IFN- to induce PML
expression and to inhibit VSV and influenza virus replication in these
cells, U937 cells were treated with 1,000 U of IFN- per ml for
48 h and infected at an MOI of 0.1 with VSV or influenza virus.
Double immunofluorescence was performed with anti-PML and anti-VSV
antibodies. The results presented in Fig.
1 show that IFN- increases PML levels
and inhibits VSV antigen expression. The virus yields in
IFN--treated U937 cells compared to control infected cells were 750 times lower (6 × 104 and 4.5 × 107
PFU/ml, respectively) for VSV and 125 times lower (8 × 104 and 107 PFU/ml, respectively) for influenza
virus. The absence of MxA induction by IFN in U937 cells
(38) suggests that human IFNs protect cells from VSV and
influenza virus infections by a pathway which is independent of MxA
protein expression.
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FIG. 1.
IFN- induces PML protein synthesis and inhibits VSV
antigen expression in the human monocytic cell line U937. U937 cells
were treated with 1,000 U of human IFN- per ml. After 48 h at
37°C, control (C) and IFN-treated cells were infected with VSV at a
MOI of 0.1. Double immunofluorescence were performed 24 h
postinfection with mouse anti-PML antibodies visualized with Texas red
and rabbit anti-VSV antibodies followed by FITC labelling.
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Overexpression of PML confers resistance to infections by VSV and
influenza virus.
Since the PML gene product was shown to have
growth-suppressing properties (22, 25, 30) and thus could be
a candidate for antiproliferative IFN actions, it may also mediate
other IFN-induced biological effects. To test a possible antiviral
effect of PML, cell lines stably expressing PML were constructed
in hamster CHO and mouse GP+E86 cells. PML expression
was verified by immunofluorescence and Western blot analysis (see Fig.
5B and 8B; data not shown). CHO control cells (transfected with the
empty vector) and those overexpressing PML (CHO PML3) were infected
with a picornavirus (EMCV), an orthomyxovirus (influenza A virus), or a
rhabdovirus (VSV) at an MOI of 0.1. At 16 h later, the cells were
stained with carbol methylene blue or used for the determination of
virus yields. Compared to the effect in CHO control cells, which
underwent nearly 100% cell death, the overexpression of PML in CHO
PML3 conferred resistance to lysis by VSV and influenza virus but not EMCV (Fig. 2). No difference in virus
yield was found between parental CHO cells and CHO cells transfected
with the empty vector (data not shown). Inhibition of the cytopathic
effect was accompanied by a decrease in virus multiplication, as shown
by the viral titers (Fig. 2). The highest inhibitions obtained by CHO
PML3 with VSV and influenza A virus were 125- and 100-fold,
respectively, while no effect on EMCV multiplication was observed.
Similarly, in GP+E86 PML cells, 100- and 80-fold decreases in VSV and
influenza virus growth, respectively, compared to those in infected
cells harboring the empty vector were observed (data not shown).
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FIG. 2.
Overexpression of PML, and not of Sp100, confers
resistance against infections by VSV and influenza virus. CHO control
(transfected with the empty vector), CHO PML3, or CHO Sp100 cells were
infected with EMCV, VSV, or influenza A virus at an MOI of 0.1. After
16 h, viral titers were determined as described in Materials and
Methods.
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Overexpression of another NB-associated protein, Sp100, does not
confer antiviral resistance.
Since Sp100 colocalizes with PML onto
NBs (23) and is also IFN induced (19), our
demonstration that PML has antiviral properties raised the question
whether Sp100 could display similar properties. We generated stable CHO
clones which overexpress Sp100 (see Fig. 8B). The capacity of CHO Sp100
to inhibit virus growth was tested and compared to that of CHO PML.
After 16 h of infection with VSV, influenza virus, or EMCV at an
MOI of 0.1, no protective effect against either of these viruses was
found in CHO Sp100, as revealed by carbol methylene blue staining (data
not shown) or by determination of VSV, influenza virus, or EMCV titers
(Fig. 2). Hence, Sp100 does not inhibit their multiplication, making a
direct role of this protein in the IFN-induced antiviral state against
these three viruses unlikely. Taken together, these results establish
that overexpression of PML specifically inhibits the multiplication of
VSV and influenza A virus.
Inhibition of virus replication in IFN-treated U373 MG cells and
U373 MG PML.
To compare levels of PML expression in transfected
cells to those induced in IFN-treated cells, we have overexpressed PML in human cells. This is because no hamster IFN is available and because
our anti-PML antibodies recognize only human PML on Western blots.
Three PML expression vectors (pSG5, M3P-SVhygro, or pCIN neo [see Materials and Methods]) were tranfected in three different human cell lines (U937, HeLa, and U373 MG). In nearly all cases, we
were unable to isolate clones that expressed PML uniformly, confirming
that overexpression of this protein interferes with cell proliferation
(22), particularly in human cell lines. However, after
several assays, we succeeded in isolating clones of human U373 MG with
the pCIN PML construct.
PML levels in these transfected U373 MG cells were compared to those
induced by IFN in control cells. U373 MG cells were treated
for 48 h with 1,000 U of human IFN-
per ml. Equal amounts of
total protein
extracts from control, IFN-treated cells and U373
MG PML were analyzed
by Western blotting. Figure
3A shows that
no
detectable band of PML was found in untreated U373 MG cells
whereas, as
previously described (
8), different forms of PML,
arising
from alternative splicing of a single gene, were induced
upon IFN
treatment. Molecular imaging analysis (system GS525;
Bio-Rad) revealed
that comparable levels of PML were found in
IFN-treated and transfected
cells.
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FIG. 3.
(A) PML level in IFN-treated U373 MG cells and
U373 MG PML. U373 MG cells were treated for 48 h with 1,000 U of
IFN- per ml. Samples (50 µg) of extracts of control IFN-treated
cells and U373 MG PML were analyzed by Western blotting and revealed
with rabbit anti-PML antibodies. Note that all bands visualized by
anti-PML antibodies are likely to be isoforms derived from alternative
splicing of unique gene. Molecular size markers are indicated on the
left. (B) Inhibition of virus replication in IFN-treated U373 MG cells
and U373 MG PML. One series of U373 MG cells was treated for 48 h
with 10, 100 or 1,000 U of IFN- per ml. The second series of cells,
U373 MG control (transfected with the empty vector), U373 MG PML, and
U373 MG PML* (+ anti-IFN-// antibodies [see Materials and
Methods]) was seeded at 37°C for 5 h. The two series were then
infected with VSV or influenza A virus at an MOI of 0.1. After 16 h, viral titers were determined as described in Materials and Methods.
(C) Expression of PML in U373 MG cells inhibits the expression of VSV
antigens (Top) Expression of VSV antigens in infected U373 MG control
cells (transfected with the empty vector) and U373 MG PML.
Immunofluorescence with rabbit anti-VSV antibodies was performed
13 h after infection with VSV at an MOI of 0.1 and revealed by
FITC labelling. (Bottom) Expression of PML in U373 MG and U373 MG PML
cells revealed by immunofluorescence with mouse anti-PML antibodies
visualized with Texas red.
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Then we tested whether U373 MG cells overexpressing PML, like CHO PML
and GP+E
86 PML, display resistance to VSV and influenza
virus. U373
MG PML cells grow much more slowly than the parental
cell line
transfected with the empty vector (data not shown).
To avoid
interference of growth rates by viral replication, all
the experiments
were done within 24 h. U373 MG and U373 MG PML
cells (2 × 10
5 cells in each case) were seeded for 5 h in
Dulbecco's modified
Eagle's medium containing 10% serum and G418 and
then were infected
at an MOI of 0.1 with VSV or influenza virus for
16 h. At that
time point, there were no observable differences in
growth in
uninfected control and U373 PML cells (data not shown).
However,
a clear difference in VSV and influenza virus replication was
found in infected U373 MG and U373 MG PML cells. Overexpression
of PML
leads to a 90-fold decrease in VSV or influenza virus yield
compared to
control cells (Fig.
3B). VSV antigen expression was
monitored in these
cells by immunofluorescence (Fig.
3C). The
PML-induced antiviral state
was associated with a lower VSV protein
expression.
To compare the degree of inhibition of VSV and influenza virus
replication in U373 MG PML to that obtained in IFN-treated
cells, U373
MG cells were treated for 48 h with 10, 100, or 1,000
U of IFN-
per ml. Then, control cells, IFN-treated cells, and
U373 MG PML were
infected with VSV or influenza virus at an MOI
of 0.1 for 16 h. As
shown in Fig.
3B, inhibition of VSV or influenza
virus replication in
U373 MG PML was comparable to that obtained
in control cells treated
with concentrations of IFN between 10
and 100 U/ml.
Resistance in the PML-expressing cells was not due to the presence of
low IFN levels for the following reasons. (i) When culture
media from
mouse GP+E
86 control and GP+E
86 PML cells infected
or not infected
with VSV or influenza virus at an MOI of 0.1 for
16 h were
subjected to titer determination on L929 cells and those
from U373 MG
control and U373 MG PML were subjected to titer determination
on HeLa
cells, their IFN titers were below the detection limit
(less than 2 U/ml); therefore, PML-expressing cells before and
after viral infection
did not produce sufficient IFN to be protective.
(ii) A mixture of
mouse or human anti-IFN-
/
/
antibodies was
unable to reverse
the resistance of GP+E
86 PML or U373 MG PML
cells to VSV or influenza
virus infections (Fig.
3B, PML* and
data not shown). (iii) 2'5'A
synthetase activity was unaffected
by PML overexpression in CHO,
GP+E
86, and U373 MG (Fig.
4), whereas
it was induced in U373 MG control cells by the addition of 10
U of
IFN-
per ml. These experiments demonstrate that overexpression
of
PML did not lead to the induction and secretion of IFN and
that PML
alone could contribute to IFN-induced inhibition of viral
replication.
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FIG. 4.
2'5' A synthetase activity in IFN-treated cells and
cells overexpressing PML. 2'5' A synthetase activity was determined in
cells extracts from CHO control cells (lane 1). CHO PML3 (lane 2),
GP+E86 control cells (lane 3), GP+E86 PML (lane 4), U373 MG control
cells (lane 5), U373 MG PML (lane 6), and U373 MG cells treated for
48 h with 10 U of IFN- per ml (lane 7). All control cells are
cells transfected with the empty vector. The 2'5' A synthetase activity
was determined by chromatographic analysis of the reaction substrate
(ATP) and the products, 2',5'-oligoadenylates (dimer and trimer), as
previously described (7).
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Resistance to VSV and influenza virus replication is dependent on
both PML expression levels and MOI.
To find whether the viral
resistance observed above is dependent on the level of PML expression,
we selected three CHO PML clones expressing different PML levels, as
shown by Western blot analysis (Fig. 5B).
These three clones and CHO control cells were infected with VSV and
influenza virus at an MOI of 0.1. Figure 5 shows that the PML levels in
the CHO PML clones paralleled the resistance to VSV or influenza virus
infections as assessed by the determination of viral titers (Fig. 5A).
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FIG. 5.
(A) PML levels parallel inhibition of virus
multiplication. CHO control cells (transfected with the empty vector)
and CHO PML 1, 2, and 3 clones were infected with VSV or influenza A
virus at an MOI of 0.1. After 16 h, viral titers were determined
as described in Materials and Methods. (B) PML levels parallel the
inhibition of VSV antigen expression. CHO control cells (transfected
with the empty vector) and CHO PML 1, 2, and 3 clones were infected for
13 h with VSV at an MOI of 0.1. Western blot analysis of the
extracts of these cells was done as described in Materials and Methods.
(Top) Revealed with rabbit anti-PML antibodies; (middle) revealed with
anti-VSV antibodies (VSV antigens are indicated at the right); (bottom)
Coomassie brilliant blue (CBB)-stained proteins.
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The effect of overexpression of PML on the inhibition of VSV antigen
expression was also confirmed by Western blot analysis
with anti-VSV
antibodies (Fig.
5B). The five structural proteins
of VSV are the major
nucleocapside, N (40-kDa), the matrix protein,
M (25 kDa), the
glycoprotein, G (69 kDa), and two minor proteins,
the phosphoprotein,
NS (29 kDa), and the polymerase protein, L
(24 kDa) (
3), but
only the G, N, and M proteins were revealed
with our rabbit anti-VSV
antibodies. As shown in Fig.
5B, the
highest inhibition of the VSV
antigen expression was obtained
with the CHO PML clone expressing the
highest PML level. Thus,
at least in the case of VSV, PML appears to
interfere with the
expression of viral proteins, as has been predicted
from immunofluorescence
analysis (Fig.
3C).
The clone expressing the highest level of PML (CHO PML3) was infected
with VSV and influenza virus at different MOIs for 16
h. As the
MOI increased from 0.1 to 1. the PML-expressing clone
showed decreased
resistance to both viruses (Fig.
6A).
These data
suggest that clones overexpressing PML are less resistant to
VSV
and influenza virus infection at high MOI. This situation is
similar
to the effect of an increasing MOI on the antiviral state
induced
by IFN. The inhibitory effect of PML on VSV multiplication was
also tested by Western blot analysis with anti-VSV antibodies
(Fig.
6B), which again showed that the degree of inhibition is
higher at an
MOI of 0.1 than at an MOI of 1.
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FIG. 6.
Resistance of PML-expressing clones to VSV and influenza
virus is MOI dependent. CHO control cells (transfected with the empty
vector) and CHO PML 3 were infected with VSV or influenza A virus at
different MOIs as indicated in the figure. Swiss 3T3 control and Swiss
3T3 MxA were infected at an MOI of 1 with VSV or influenza virus. (A)
After 16 h, the cells were used for the determination of the viral
titers. Antiviral activities are the means of three independent
experiments. (B) CHO control cells and CHO PML 3 were infected
with VSV for 10 h at different MOIs as indicated in the figure.
The results of Western blot analysis are revealed with anti-VSV
antibodies. (Top) revealed with anti-VSV antibodies (VSV antigens are
indicated at the right); (bottom) Coomassie brilliant blue
(CBB)-stained proteins.
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Overexpression of human MxA confers a high resistance to VSV and
influenza virus infections (
31). To compare the ability
of
PML and MxA to inhibit viral multiplication, CHO control, CHO
PML3,
Swiss 3T3 control, and Swiss 3T3 MxA were infected with
VSV or
influenza virus under the same conditions. The overexpression
of MxA
protein inhibits the replication of these two viruses to
a much higher
extent than does PML. At an MOI of 1, PML had a
small protective effect
whereas MxA strongly protected against
these viruses (Fig.
6A).
Moreover, at higher MOI, MxA still protected
against these viruses,
while PML had no effect (data not shown).
Again, VSV antigen expression
was more strongly inhibited by MxA
than by PML expression (see Fig.
8C), demonstrating that MxA is
a more potent effector than PML.
Viral RNA in PML-expressing cells.
To test if overexpression
of PML interferes with viral mRNA synthesis. CHO control and CHO PML3
were infected with VSV at an MOI of 0.5. After 4 h at 37°C,
total RNA was isolated. The RNA preparations were analyzed for the
presence of VSV mRNA N, NS, M, and G by Northern blot analysis. Figure
7 shows that PML had an inhibitory effect
on viral N mRNA synthesis as well as on NS, M, and G mRNAs. A imager 1200 analysis (Biospace) revealed that the concentrations of VSV
N, NS, M, and G mRNAs were about threefold lower in CHO PML3 and U373
MG PML cells than in control cells (Fig. 7 and data not shown),
demonstrating that PML interferes with VSV mRNA expression. Moreover,
at an MOI of 0.5, the synthesis of viral RNA (Fig. 7) or proteins (Fig.
6B) was less inhibited than was the VSV yield (Fig. 6A) by PML
overexpression.
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FIG. 7.
CHO control cells (transfected with the empty vector)
and CHO PML3 were infected with VSV at an MOI of 0.5 for 4 h.
Total RNA was extracted as described in Materials and Methods. Samples
(20 µg of RNA per lane) were analyzed for the presence of VSV N, NS,
M, and G. GPDH, glyceraldehyde-3-phosphate dehydrogenase.
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Requirement of the coiled-coil domain of PML for its antiviral
activity.
Homodimerization of PML and/or PML-RAR was shown to
occur through a long coiled-coil region (amino acids 229 to 360). To see whether the coiled-coil domain, the RING finger domain, or the
C-terminal region of PML was involved in the antiviral state, four PML
mutants were constructed: the C-terminal PML mutant (PML Stop 504), the
coiled-coil PML mutant (PML216-333), the RING finger PML mutant
(Q59C60/EL), and the C-terminal PML mutant
(Stop 381). The structures of these mutants compared to wild-type PML are shown in Fig. 8A. These mutants were
stably transfected in CHO cells. Their expression was verified by
immunofluorescence (Fig. 3C and data not shown), and their product size
was compared to that of the wild-type protein by Western blot analysis
(Fig. 8B). For both the C-terminal PML Stop 504 and the PML Stop 381 mutants, constructed by the insertion of stop codons, a minor band
corresponding to the size of the wild-type PML is expressed, probably
involving a readthrough mechanism (see Discussion).
View larger version (46K):
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|
FIG. 8.
(A) Description of PML mutants. The structures of PML
(including the C3HC4 zinc finger motif, the two B boxes and the
coiled-coil) and of four PML mutants, the C-terminal PML mutant (PML
Stop 504), the coiled-coil PML mutant [PML(216-333)], the RING
finger PML mutant (Q59C60/EL), and the
cytoplasmic PML mutant (Stop 381), are shown. aa, amino acids; NLS,
nuclear localization signal. (B) Western blot analysis of PML in stably
transfected CHO and GP+E86 cells as well as the four PML mutants;
also shown is the overexpression of Sp100 in CHO cells. (C) Analysis of
PML domains involved in the inhibition of VSV antigens. CHO control
(transfected with the empty vector), CHO PML wt (CHO PML3), and the
four mutants of PML were infected with VSV for 13 h at an MOI of
0.1. Swiss 3T3 neo and 3T3 MxA were infected with VSV under the same
conditions and used as control. Western blot analysis of the extracts
of these cells was done as described in Materials and Methods. (Top)
Revealed with anti-VSV antibodies (VSV antigens are indicated at the
right); (bottom) Coomassie brilliant blue (CBB)-stained proteins. (D)
Description of subcellular distributions in stably transfected CHO
cells and the antiviral potentials of wild-type and mutant forms of
human PML protein. Cells were infected with VSV at an MOI of 0.1. After
16 h, viral titers were determined as described in Materials and
Methods.
|
|
Deletion of the coiled-coil domain led to an altered subnuclear
localization characterized by a fine intranuclear network
without
speckles, whereas absence of the C-terminal region did
not impair the
targetting of PML Stop 504 onto the NBs. The RING
finger PML mutant
(Q
59C
60/EL) is nuclear diffuse, and PML mutant
Stop 381 is mostly cytoplasmic (Fig.
8D). No antiviral state against
VSV or influenza A virus was induced by the PML
(216-333)
coiled-coil
mutant, whereas the PML Stop 504, the RING finger PML
mutant (Q
59C
60/EL),
and the cytoplasmic PML
mutant Stop 381 clones, which all have
the coiled-coil domain, were as
efficient as clone CHO PML3 in
inhibiting the multiplication of VSV and
influenza virus, as assessed
by viral titers (Fig.
8D) and Western blot
analysis (Fig.
8C).
Thus, clearly the coiled-coil region of PML is
required for its
antiviral property.
| |
DISCUSSION |
In this study, we provide evidence that overexpression of PML, but
not of Sp100, confers resistance to two RNA viruses, VSV and influenza
virus, suggesting that PML participates in the antiviral state induced
in IFN-treated cells. PML protein has an inhibitory effect on both VSV
mRNA and protein synthesis. At an MOI of 0.5, the VSV yield appears to
be more highly inhibited than is the synthesis of viral RNA or
proteins, suggesting a possible defect in the production of infectious
virus in cells overexpressing PML. As expected, this antiviral effect
against VSV and influenza virus is dependent both on PML expression and
the MOI of the virus. The degree of inhibition of VSV or influenza
virus replication by PML was comparable to that obtained in control
cells treated with concentrations of IFN between 10 and 100 U/ml. This
may lead to the suspicion that overexpression of PML induces IFN
secretion, which could in turn inhibit viral replication. However, our
results clearly establish that overexpression of PML did not induce IFN secretion even after viral infection.
How PML migh inhibit VSV and influenza virus replication is unknown.
Individual expression of previously identified human IFN-mediators has
shown that overexpression of 2'5' A synthetase (6, 9) or p68
kinase (29) confers resistance to EMCV but not to VSV while
overexpression of human MxA inhibits VSV, influenza A virus, and other
RNA virus multiplication but not that of picornavirus, togavirus, or
herpes simplex virus (16, 31, 38). The two known human Mx
proteins (MxA and MxB) (31), like rat (Mx2 or Mx3)
(27) and mouse (Mx2) (46) Mx proteins, are
cytoplasmic. The Mx1 protein has a speckled nuclear localization in
both mouse and rat cells (2, 27, 47). While rat Mx3 and
human MxB are devoid of antiviral properties, rat Mx1 and human MxA are active against both VSV and influenza virus (28, 31, 41). Mouse Mx1 confers resistance only to influenza virus (47),
and Mx2 (mouse or rat) protects against VSV only (2, 28,
46). Thus, PML closely resembles rat Mx1 in both to its
localization and its antiviral properties.
Compared to MxA protein, PML was found to have a less powerful
antiviral activity against VSV and influenza virus replication. However, it appears that IFNs protect cells against VSV and influenza virus by at least two different pathways, one of which is independent of MxA protein. In human U937 cells, IFN- inhibits VSV and influenza virus replication (see above) without inducing MxA protein
(38). PML could be involved in one of these pathways. The
inhibition, in IFN-treated U937 cells, of the VSV yield was 750 times
and that of influenza virus was 125 times greater than in control cells. These results suggest that in addition to PML, other mediators could be implicated in inhibiting VSV replication in this cell line. In
this sense, it has been shown that the IFN-induced human 9-27 protein
could also participate in the inhibition of VSV but not in that of
influenza virus (1). The resistance to VSV or EMCV
infections conferred by IFN was similar in embryonic fibroblasts derived from PML knockout mice and from wild-type mice (24). This is not surprising for EMCV, since overexpression of PML does not affect the replication of this virus (see above), or for VSV, since
Mx2 (46) appears to play a major role, which may mask PML
contribution.
Two of the previously identified antiviral IFN effectors have
relatively well-defined modes of action: 2'5'A synthetase and p68
kinase (reviewed in references 37 and
39). The molecular targets of the Mx and PML
proteins are unknown, although Mx proteins display GTPase activity,
which may be required for their antiviral properties (2, 33,
41). PML mutation analysis revealed that both the RING finger PML
(Q59C60/EL) and the cytoplasmic PML Stop 381 mutants lost the normal PML localization but still possessed an intact
coiled-coil domain and were effective in inhibiting VSV and influenza
virus. Deletion of the PML coiled-coil domain abolished the antiviral
properties against VSV and influenza virus and altered the punctuate
localization of PML onto NBs. Both C-terminal mutants were created by
insertion of stop codons, which could be suppressed by translational
readthrough, leading to some wild-type PML synthesis. However, the
small amount of wild-type PML synthesized cannot explain the protective
effect observed. Influenza virus, whose replication and transcription
are nuclear, and VSV, whose replication takes place entirely in the
cytoplasm, are both inhibited by nuclear PML and cytoplasmic PML Stop
381 proteins. The PML protein, therefore, could inhibit virus
multiplication indirectly by modifying other cellular proteins, which
may then modulate viral replication in the relevant cellular
compartment.
We have shown here that overexpression of human IFN-induced PML affects
VSV and influenza virus replication and interferes with viral mRNA and
protein synthesis. Thus, PML can contribute to the establishment of the
antiviral state in IFN-treated cells. The significant inhibitory effect
of PML makes it a member of the family of IFN-induced proteins
mediating antiviral properties.
| |
ACKNOWLEDGMENTS |
We acknowledge M. C. Guillemin for anti-Sp100 antibodies and
D. Blondel for anti-VSV sera and plasmids containing VSV N, NS, M, and
G. We thank J. Pavlovic for 3T3 MxA cells, H. Will for the Sp100 cDNA,
and S. Rees (Glaxo/Wellcome, Stevenage, United Kingdom) for the pCIN
vector. We also thank C. Chopin for technical assistance. The help of
B. Boursin with the artwork is greatly appreciated.
This work was supported by grants from Ligue contre le Cancer,
Fondation de France, Fondation St. Louis, and ARC.
M.K.C.-A. and F.Q. contributed equally to this work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: CNRS UPR 9051, Hôpital St. Louis, 1, Ave. Claude Vellefaux, 75475 Paris Cedex
10, France. Phone: 33-1-42-06-31-53. Fax: 33-1-53-72-40-90. E-mail: mchelbi{at}infobiogen.fr.
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Abstract
Introduction
