Journal of General Virology (2004), 85, 2315–2326
DOI 10.1099/vir.0.79795-0
Mx1 GTPase accumulates in distinct nuclear
domains and inhibits influenza A virus in cells
that lack promyelocytic leukaemia protein
nuclear bodies
Othmar G. Engelhardt,13 Hüseyin Sirma,2 Pier-Paolo Pandolfi3
and Otto Haller1
1
Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg,
Hermann-Herder-Strasse 11, D-79104 Freiburg, Germany
Correspondence
Othmar G. Engelhardt
[email protected]
2
Heinrich-Pette-Institut für experimentelle Virologie und Immunologie an der Universität
Hamburg, Martinistrasse 52, D-20251 Hamburg, Germany
3
Molecular Biology Program, Department of Pathology, Memorial Sloan-Kettering Cancer
Center, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10021, USA
Received 11 November 2003
Accepted 20 April 2004
The interferon-induced murine Mx1 GTPase is a nuclear protein. It specifically inhibits influenza
A viruses at the step of primary transcription, a process known to occur in the nucleus of infected
cells. However, the exact mechanism of inhibition is still poorly understood. The Mx1 GTPase
has previously been shown to accumulate in distinct nuclear dots that are spatially associated
with promyelocytic leukaemia protein (PML) nuclear bodies (NBs), but the significance of this
association is not known. Here it is reported that, in cells lacking PML and, as a consequence,
PML NBs, Mx1 still formed nuclear dots. These dots were indistinguishable from the dots
observed in wild-type cells, indicating that intact PML NBs are not required for Mx1 dot formation.
Furthermore, Mx1 retained its antiviral activity against influenza A virus in these PML-deficient
cells, which were fully permissive for influenza A virus. Nuclear Mx proteins from other species
showed a similar subnuclear distribution. This was also the case for the human MxA GTPase
when this otherwise cytoplasmic protein was translocated into the nucleus by virtue of a foreign
nuclear localization signal. Human MxA and mouse Mx1 do not interact or form heterooligomers.
Yet, they co-localized to a large degree when co-expressed in the nucleus. Taken together,
these findings suggest that Mx1 dots represent distinct nuclear domains (‘Mx nuclear domains’)
that are frequently associated with, but functionally independent of, PML NBs.
INTRODUCTION
Interferons (IFNs) induce the synthesis of a large number
of proteins, some of which have antiviral activity (Der et al.,
1998). Among these, the Mx proteins are best known for
their capacity to inhibit the multiplication of many RNA
viruses (for a recent review, see Haller & Kochs, 2002). Mx
proteins are large GTPases with an intrinsic tendency to
homooligomerize (Kochs et al., 2002; Melen et al., 1992;
Nakayama et al., 1993). Different Mx proteins show
different subcellular localizations and antiviral specificities.
Interestingly, the subcellular compartment influences the
antiviral spectrum to a great extent. The human cytoplasmic
3Present address: Sir William Dunn School of Pathology, South Parks
Road, Oxford OX1 3RE, UK.
Images showing the localization of mutant Mx1(C71S) are available as
supplementary material in JGV Online.
0007-9795 G 2004 SGM
MxA protein inhibits members of the Orthomyxoviridae
(such as the influenza viruses and Thogotovirus) and, in
addition, a number of RNA viruses known to replicate in
the cytoplasm. In contrast, the nuclear mouse Mx1 protein
is active exclusively against orthomyxoviruses, known
to have a nuclear replication phase (Haller et al., 1995;
Staeheli et al., 1986). Moreover, when Mx proteins are
translocated into a new compartment, they change their
antiviral properties. For example, targeting of the cytoplasmic MxA protein to the nucleus by virtue of a foreign
nuclear localization signal (NLS) changes the antiviral
activity of MxA such that it still inhibits orthomyxoviruses
but loses activity against all other MxA-sensitive viruses
(Zurcher et al., 1992a). When the NLS of the nuclear Mx1
protein is inactivated by site-directed mutagenesis, the
antiviral activity of the resulting cytoplasmic protein against
influenza A viruses is lost. However, when this mutant
Mx1 protein is equipped with a foreign NLS, it again
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2315
O. G. Engelhardt and others
localizes to the nucleus and regains antiviral activity
(Zurcher et al., 1992b). These results, among others, have
led to the suggestion that the activity of Mx proteins
depends on cofactors that are localized to specific subcellular compartments. We previously identified putative
Mx-interacting proteins and demonstrated that these interaction partners localized to subnuclear structures known
as promyelocytic leukaemia protein (PML) nuclear bodies
(NBs) (Trost et al., 2000). We also found that mouse Mx1
forms characteristic dots in close spatial association with
PML NBs (Engelhardt et al., 2001). These findings raised
the intriguing possibility that PML NBs might contribute to
the antiviral effect of Mx1.
PML NBs, also known as ND10, Kr bodies or PML oncogenic domains, are dynamic subnuclear structures. Their
composition, number and size depend on various stimuli
and vary during the cell cycle. In recent years, an increasing
number of proteins have been found to reside within or to
be partially associated with these structures, which are
organized by PML as the main structural component
(Negorev & Maul, 2001). PML NBs seem to have numerous
functions (Borden, 2002; Piazza et al., 2001; Salomoni &
Pandolfi, 2002; Zhong et al., 2000b). They appear to be
involved in the regulation of transcription, in apoptosis
and in cell-cycle regulation. Furthermore, they have been
proposed to be storage depots where proteins not needed
at a given time are kept inactive or modified until they are
recruited to their sites of action (Negorev & Maul, 2001).
Of particular interest is the role of PML NBs in viral
infections. PML and other PML NB constituents such as
Sp100 or ISG20 are inducible by type I IFNs (Gongora et al.,
1997; Grotzinger et al., 1996; Guldner et al., 1992; Lavau
et al., 1995; Nason-Burchenal et al., 1996; Regad & ChelbiAlix, 2001) and it has been reported that PML itself has
antiviral activity (Chee et al., 2003; Chelbi-Alix et al., 1998;
Djavani et al., 2001; Regad et al., 2001). As a countermeasure, certain viruses seem to alter the composition and
possibly the function of PML NBs. Thus, a number of
DNA viruses associate with PML NBs and change their
morphology (Everett, 2001). Less is known about possible
interactions between RNA viruses and PML NBs. Infection
with lymphocytic choriomeningitis virus leads to the relocation of the PML protein from the nucleus to the cytoplasm
(Borden et al., 1998). Rabies virus expresses two proteins
that interact with the PML protein and alter its distribution
(Blondel et al., 2002). Interestingly, both these RNA viruses
replicate in the cytoplasm but have the capacity to alter the
composition of PML NBs in the nucleus. The exact role of
these virus–host interactions is still unclear.
In the present study, we investigated the role of PML NBs
in the nuclear localization of Mx1 and its antiviral activity
against influenza A virus. We found that cells lacking PML
NBs supported influenza virus growth to the same degree
as wild-type cells, indicating that PML NBs are not involved
in virus multiplication. Moreover, the intranuclear localization of Mx1 was unchanged in these cells. Likewise, nuclear
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Mx proteins of different species co-localized with murine
Mx1 when co-expressed, indicating that Mx proteins
prefer particular subnuclear domains, which may constitute
specific ‘Mx nuclear domains’. Finally, the antiviral activity
of Mx1 against influenza A virus was unchanged in cells
lacking PML NBs.
METHODS
Cells and viruses. Mouse embryo fibroblasts (MEFs) were derived
from PML2/2 animals or control wild-type animals at day 13?5 of
gestation and were kept in Dulbecco’s modified Eagle’s medium
(DMEM) plus 20 % FCS. Both types of cell are Mx1 negative for
genetic reasons (Staeheli et al., 1988). Primary MEFs or MEFs
immortalized by spontaneous transformation were used, as indicated. Mouse 3T3 cells, African green monkey VeroCH cells, human
H1299 lung carcinoma cells, human HeLa and human 293T cells
were maintained in DMEM plus 10 % FCS. Influenza A virus
M-TUR is derived from the avian A/Turkey/England 63 (H7N3)
Langham strain and has been adapted to grow in mouse peritoneal
macrophages (Lindenmann et al., 1978). Virus stock was grown on
PML2/2 MEFs. Influenza A virus FPV-B, a mammalian cell-adapted
variant of influenza A/FPVDobson/34 (H7N7), was grown on Swiss
3T3 cells (Israel, 1979).
Antibodies. Monoclonal anti-MxA antibody M143 recognizes a
conserved N-terminal epitope and shows broad cross-reactivity
to Mx proteins from many species, including mouse Mx1 (Flohr
et al., 1999). Polyclonal rabbit anti-Mx1 antibody is a hyperimmune
serum of rabbits immunized with highly purified recombinant Mx1
protein produced in E. coli. Polyclonal anti-influenza A virus hyperimmune rabbit serum has been described previously (Haller et al.,
1976). Polyclonal rabbit antiserum against Daxx (M-112), polyclonal
rabbit (H-238) and goat (A-20) antisera against PML and polyclonal
rabbit antiserum against the myc epitope (A-14) were obtained from
Santa Cruz Biotechnologies. Anti-FLAG tag (M2) and anti-SC35
monoclonal antibodies were from Sigma-Aldrich. Monoclonal
mouse (5P10-p) and polyclonal rabbit anti-p80 coilin antibodies
were kind gifts from Werner Franke (DKFZ, Heidelberg, Germany)
and Angus Lamond (Wellcome Trust Biocentre, University of
Dundee, Dundee, UK), respectively. Secondary antibodies used were
donkey anti-mouse IgG, donkey anti-rabbit IgG and donkey antigoat IgG conjugated to Cy2, Cy3 or Cy5, as required (Jackson
ImmunoResearch Laboratories).
Plasmids. Plasmids pHMG-Mx1 (Kolb et al., 1992), pHMGMx1(K49A) (Pitossi et al., 1993), pHMG-Mx1(K49M) (Pitossi et al.,
1993) and pHMG-FLAG-MxA (Ponten et al., 1997) have been
described previously. Plasmid pHMG-FlagMx1 was kindly provided
by Georg Kochs (Department of Virology, University of Freiburg,
Freiburg, Germany). Briefly, the FLAG sense oligonucleotide (59CGGTACCGAAGATGGACTACAAGGACGACGATGA-39) flanked
by ClaI adaptors and the complementary oligonucleotide were
inserted into the ClaI restriction site of pSP65-Mx1(ClaI) (Zurcher
et al., 1992b); the resulting FLAG–Mx1 cDNA was transferred to
plasmid pHMG (Gautier et al., 1989). The resulting construct
pHMG-FlagMx1 carried the FLAG sequence as a 59-terminal extension of the Mx1 open reading frame. Plasmid pHMG-rat Mx1 was
produced by inserting the rat Mx1 cDNA (Meier et al., 1988) into
plasmid pHMG (Gautier et al., 1989). pEGFP-Mx1 was kindly provided by Jovan Pavlovic (Institute for Medical Virology, University
of Zürich, Zürich, Switzerland), pEGFP-PML was a kind gift from
Hans Will (Heinrich-Pette-Institut, Hamburg, Germany). Plasmid
pCImycTMxA was generated by replacing the BamHI–NotI fragment
of plasmid pCImycPKM-C1 (Engelhardt et al., 2003) with a PCR
product containing the open reading frame of TMxA (Zurcher et al.,
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Journal of General Virology 85
Mx nuclear domains
1992a). Retroviral vector plasmids pRV-Mx1, pRV-Mx1(K49A) and
pRV-Mx1(K49M) were generated by inserting PCR products containing the open reading frames of the respective proteins into
vector pRRL-sin.hCMV.MCS.pre.DEco via the BamHI and EcoRV
sites. Plasmids pRRL-sin.hCMV.MCS.pre.DEco, pRSV-Rev, pMDLg/
pRRE and pHCMV-VSV-G for the generation of lentiviral vectors
have been described previously (Dull et al., 1998; Zufferey et al.,
1998) (kind gifts from Hans Will).
Transfections and transductions. Cells were seeded into six-well
plates or 24-well plates (with or without glass coverslips) and transfected the following day using Lipofectamine (Life Technologies)
according to the manufacturer’s instructions.
Retroviral vector-containing supernatants were produced in 293T
cells. 293T cells in 90 mm dishes were transfected with plasmids
pRSV-Rev, pMDLg/pRRE, pHCMV-VSV-G and pRV-Mx1, pRVMx1(K49A) or pRV-Mx1(K49M) using Metafectene (Biontex) according to the manufacturer’s instructions. Supernatants were harvested
at different time points between 15 and 52 h post-transfection and
titrated by transduction of wild-type MEFs and immunofluorescence
analysis using anti-Mx antibody M143.
For transduction with retroviral vectors, cells were seeded into 24-well
plates, washed with PBS the next day and incubated with retroviral
vector-containing supernatants for 4–6 h in the presence of 8 mg
polybrene ml21 (Sigma-Aldrich). More virus-containing supernatant
was added and cells were incubated at 37 uC overnight. The inoculum
was removed and replaced with DMEM plus 20 % FCS. Cells were
processed for immunofluorescence staining or infection by influenza
A virus (see below) 3–6 days post-transduction.
Antiviral test. PML2/2 or control wild-type MEFs were seeded
into the wells of a 24-well plate and transduced the next day with
retroviral vectors expressing Mx1, Mx1(K49A) or Mx1(K49M). The
cells were split 3 days later into a 24-well plate containing glass
coverslips, infected the following day with influenza A virus M-TUR
and fixed 6 h post-infection in 3 % paraformaldehyde. Immunofluorescence staining was done as described below with a polyclonal
rabbit anti-influenza A virus serum and monoclonal anti-Mx antibody M143. For quantification, cells expressing Mx1 or mutant Mx1
proteins and influenza A virus antigens and cells expressing only
Mx1 proteins without influenza A virus antigens were counted to
determine the infection rate of Mx-expressing cells. The infection
rate of cells not expressing Mx1 proteins on the same coverslips was
determined in parallel.
Immunofluorescence
staining
and
confocal
using MetaMorph software. Images were smoothed by applying a
median filter, followed, where necessary, by conversion to binary
images, pixel erosion and segmentation. Finally, the coordinates of
the centroids of the individual domains were exported to Microsoft
Excel. For each centroid of PML, SC35 or coilin, the distance to the
nearest Mx1 dot was calculated. At least 30 nuclei for each set of
labellings were used for statistical analysis. Mean minimum distances, calculated as the mean of all minimum distances determined
in one experiment, were compared using Student’s t-test. In the
case of coilin–Mx1 measurements, the data were further split. The
mean of all per-cell means of the minimum PML–Mx1 distances
was used as an arbitrary threshold value to classify the degree of
association between PML and Mx1; cells with a per-cell mean minimum distance below this threshold were termed ‘high PML–Mx1
association’, and all others ‘low PML–Mx1 association’. Means of
minimum coilin–Mx1 distances were determined for each of these
two groups and compared using Student’s t-test. Differences were
considered statistically significant for P<0?01.
RESULTS
Influenza A virus growth in PML-deficient cells
PML has been reported to exhibit antiviral activity against
influenza A virus (Chelbi-Alix et al., 1998), while a number
of DNA viruses appear to replicate and/or transcribe in the
vicinity of PML NBs (Everett, 2001). We therefore tested
whether influenza A virus would grow normally in cells
lacking PML. PML-deficient and wild-type MEFs were
infected with 0?0005 p.f.u. influenza A virus strain FPV per
cell and supernatants were titrated at various times after
infection to determine the amount of progeny virus
produced. Fig. 1 shows the growth curves obtained with
the two different cell types. At each time point analysed, the
virus yields in PML-deficient and wild-type cells were
comparable, indicating that the presence or absence of
PML was irrelevant for virus growth in these MEFs.
microscopy.
Immunofluorescence staining was carried out as described previously (Engelhardt et al., 2001). In some experiments, DNA was
counterstained by incubation in Bisbenzimide (0?33 mg ml21 in
methanol; Sigma-Aldrich) for 10 min or by incubation in TO-PRO3 iodide (diluted 1 : 1000; Molecular Probes) for 30 min. Coverslips
were mounted in Mowiol (Sigma-Aldrich) containing DABCO [1,4diazabicyclo(2.2.2)octane; Sigma-Aldrich].
Analysis was done on a Leica DM-IRBE microscope using a 636
objective and appropriate filter sets (Leica Microsystems). Confocal
microscopy was performed on a Leica TCS SP2 attached to a Leica
DM-IRBE microscope with a 636 objective and on a Radiance 2000
confocal microscope (Bio-Rad) attached to a Nikon Eclipse TE300
microscope with a 606 objective. Image acquisition was done using
the appropriate proprietary confocal software, recording the different
channels separately. Figures were assembled using Adobe Photoshop,
Adobe Illustrator and MetaMorph (Universal Imaging Corporation).
Distance measurements and statistical analysis. Confocal
images of single planes were processed separately for each channel
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Fig. 1. Growth of influenza A virus in cells lacking PML NBs.
Spontaneously transformed MEFs from wild-type (m) and
PML”/” (%) mice were infected with 0?0005 p.f.u. influenza A
virus strain FPV-B per cell. At various times after infection (p.i.),
supernatants were harvested and titrated by plaque assay on
VeroCH cells. Growth curves were determined in triplicate and
mean titres±SD are shown.
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O. G. Engelhardt and others
Mx1 localization in cells lacking PML NBs
PML2/2 mice are derived from the inbred strain 129Sv
known to carry a natural defect at the Mx1 gene locus on
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chromosome 16 (Staeheli et al., 1988). Hence, these animals
have a defective Mx1 gene in addition to the targeted
disruption of the PML gene. We therefore used transient
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Journal of General Virology 85
Mx nuclear domains
transfections with Mx1 cDNA expression plasmids or
transductions with lentiviral vectors to express the Mx1
protein in MEFs derived from PML2/2 mice. Control MEFs
derived from the wild-type 129 mice were treated in the
same way. In genetically Mx1-positive mouse strains, Mx1
is typically found in a dot-like pattern in the nucleus of
IFN-treated cells (Dreiding et al., 1985). When expressed
ectopically, the Mx1 protein shows the same characteristic
appearance. However, its apparent distribution in the
nucleus depends, in part, on expression levels, as illustrated
in Fig. 2(a–c). Wild-type MEFs were transduced with a
lentiviral vector expressing Mx1 and double labelled for
immunofluorescence with antibodies against Mx1 and the
PML NB marker protein Daxx. High-level expression of
Mx1 (as usually achieved by IFN treatment in cells derived
from Mx1-positive mice) led to nuclear dots that were
frequently, but not always, juxtaposed to or partially
overlapping PML NBs (Fig. 2a–c, right cell), as previously
described (Chelbi-Alix et al., 1995; Engelhardt et al., 2001).
Low levels of Mx1 led to a more fine-punctate nuclear
distribution and little association with PML NBs (Fig. 2a–c,
left cell). When PML2/2 MEFs were transduced with the
same lentiviral vector, Mx1 staining again exhibited a dotlike pattern, indistinguishable from the pattern observed
in wild-type cells (Fig. 2d, g). In PML2/2 cells, Daxx protein was found either diffusely in the nucleus (Fig. 2e) or
in patchy, diffuse accumulations (Fig. 2h), many of which
coincided with patches of DNA staining (Ishov et al., 1999,
and data not shown). Examination of double-labelled
PML2/2 cells showed that Mx1 had lost its preference for
localizing close to accumulations of Daxx, suggesting that
Mx1 was not directly associating with Daxx (Fig. 2g–i).
picture was similar. Cells doubly transfected with expression
plasmids for GFP–PML and Mx1 showed preferential
accumulation of Mx1 in association with dots containing
PML and Daxx (Fig. 2n–q). In summary, Mx1 is capable
of forming typical dots in the absence of PML NBs.
Nevertheless, these Mx1 dots are localized close to PML NBs,
once these structures are reformed in PML2/2 cells.
Introduction of PML protein into PML2/2 cells by transfection or fusion with wild-type cells (Ishov et al., 1999;
Zhong et al., 2000a) leads to the reformation of PML NBs.
Concomitantly, PML NB constituent proteins, such as Daxx
or Sp100, relocalize to these domains. We therefore tested
whether the expression of PML protein in PML2/2 cells
would affect the localization of Mx1. For this purpose, we
transfected expression plasmids for Mx1 and GFP–PML
into wild-type or PML2/2 MEFs and analysed the localization of PML, Mx1 and Daxx. GFP–PML and Daxx showed
perfect co-localization in wild-type MEFs (Fig. 2j–m). Mx1
was frequently found juxtaposed to and partially overlapping PML NB marker proteins. In PML2/2 MEFs, the
Nuclear Mx proteins localize to distinct
subnuclear domains
Mx1 protein has antiviral activity in the absence
of PML NBs
We assessed whether PML NBs were required for the antiviral activity of Mx1. Wild-type and PML2/2 cells were
transduced with lentiviral vectors expressing Mx1 and
were subsequently infected with influenza A virus. The
cells were fixed 5–6 h after infection and processed for
immunofluorescence analysis, using specific antibodies
against Mx1 and viral proteins. The infection rate was
quantified as indicated in Methods. As controls, two antivirally inactive mutant forms of Mx1, namely Mx1(K49M)
and Mx1(K49A) (Pitossi et al., 1993), were expressed and
analysed in parallel. Fig. 3(a) shows that Mx1 protected
both wild-type and PML2/2 cells from infection. As
expected, Mx1(K49A) had no antiviral effect in either cell
culture. Fig. 3(b) shows the quantification of a representative experiment. Mx1 inhibited infection of PML-negative
cells to the same degree as wild-type cells, while the mutant
proteins Mx1(K49M) and Mx1(K49A) had no effect.
Clearly, Mx1 inhibited influenza A virus in the absence
of PML, indicating that its antiviral function was not
dependent on PML NBs.
Rat Mx1 protein is found in a dot-like pattern similar
to mouse Mx1 in the nucleus of IFN-treated cells (Meier
et al., 1988). We transfected an expression plasmid for
rat Mx1 into wild-type MEFs and performed doubleimmunofluorescence analysis to check whether or not rat
Mx1 was also found associated with PML NBs. Indeed,
staining with anti-Daxx antibodies revealed that rat Mx1
preferentially accumulated in the immediate vicinity of
PML NBs, exactly like mouse Mx1 (Fig. 4a–c). More
importantly, rat Mx1 retained its ability to form nuclear
dots in PML2/2 cells (Fig. 4d–f).
Fig. 2. Mx1 nuclear dots in the absence of PML NBs. (a)–(i) MEFs from wild-type (a–c) or PML”/” (d–i) mice were
transduced with a lentiviral vector expressing Mx1 protein, fixed 6 days later and immunostained with a polyclonal rabbit
antiserum against Daxx protein (green) and a mouse monoclonal antibody detecting Mx1 protein (red). The red and green
channels were recorded separately by confocal microscopy and superimposed electronically (merge). (a)–(c) Two Mx1expressing wild-type cells. The right cell shows the typical Mx1 dots. The left cell expressed small amounts of Mx1 and shows
little association of Mx1 with Daxx. (d)–(f) An Mx1-expressing cell with fine-punctate and diffuse Daxx on a PML”/”
background. (g)–(i) Cell with patchy accumulations of Daxx. (j)–(q) Wild-type MEFs (j–m) and PML”/” MEFs (n–q) were
transfected with expression plasmids pHMG-Mx1 and pEGFP-PML, fixed 2 days later and stained with primary antibodies as
above. GFP–PML was visualized by its autofluorescence. Red, green and blue channel images were recorded separately and
overlaid electronically (m, q). Insets in (m) and (q) show the overlay of the red and green images of a part of the image [the
lower Mx1-positive cell in (m) and the upper cell in (q)]. Bars, 8 mm.
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O. G. Engelhardt and others
(a)
Mx1(K49A)
__
PML /
wt
Mx1
100
(b)
90
Infection rate (%)
80
70
60
50
40
30
20
10
0
PML+/+
__
PML /
Mx1
__
PML+/+
PML /
Mx1(K49A)
__
PML+/+
PML /
Mx1(K49M)
Fig. 3. Mx1 protein has antiviral activity in the absence of PML NBs. Wild-type (wt) and PML”/” MEFs were transduced with
lentiviral vectors expressing Mx1, Mx1(K49A) or Mx1(K49M) proteins, infected with influenza A virus M-TUR 4 days later, fixed
6 h p.i. and stained for immunofluorescence, as described in Methods. (a) Wild-type and PML”/” cells expressing Mx1 or
mutant Mx1(K49A) (red) were infected with influenza A virus and the accumulation of viral antigens was visualized (green).
Arrows indicate cells expressing Mx1 or Mx1(K49A). (b) Quantification of a representative experiment. Infection rates of
Mx-expressing cells (filled bars) and of non-expressing cells (open bars) are shown.
These findings suggested that specific subnuclear domains
exist in which Mx proteins accumulate when targeted to the
nucleus. To verify this, we used a nuclear form of MxA as
a marker. Normally, the human MxA protein accumulates
in the cytoplasm of cells (Staeheli & Haller, 1985), where it
is partly associated with membranes of the smooth endoplasmic reticulum (Accola et al., 2002). It is known to form
homooligomers but not heterooligomers with other Mx
proteins, such as human MxB (Melen & Julkunen, 1997).
MxA has been shown to translocate from the cytoplasm to
the nucleus when equipped with the NLS of the SV40 large
T antigen (Zurcher et al., 1992a). This nuclear MxA protein,
called TMxA, showed a dot-like accumulation pattern in
the nucleus (Ponten et al., 1997; Zurcher et al., 1992a). To
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analyse its subnuclear localization in more detail, we coexpressed human TMxA with mouse Mx1 and analysed
their respective localization. To avoid complications arising
from antibody cross-reactivity, we used tagged versions
of Mx proteins. Mouse Mx1 was tagged at its N terminus
with a FLAG tag, whereas TMxA was expressed with an
N-terminal myc tag. Both tagged proteins showed nuclear
localization patterns indistinguishable from their untagged
wild-type counterparts (data not shown). When cells were
co-transfected with the two expression plasmids and double
labelled with anti-FLAG and anti-myc tag antibodies, a
high degree of co-localization of the two Mx proteins was
observed (Fig. 5a–f). Frequently, dots containing both
FLAG–Mx1 and myc–TMxA were found close to PML
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Journal of General Virology 85
Mx nuclear domains
Fig. 4. Different nuclear Mx proteins display similar localization patterns. Wild-type (a–c) and PML”/” (d–f) MEFs were
transfected with plasmid pHMG-ratMx1 expressing the rat Mx1 protein, fixed 1 day later and stained for immunofluorescence
with a polyclonal rabbit antiserum against Daxx (green) and a monoclonal anti-Mx antibody (red). Red and green channel
images were recorded separately and overlaid electronically. Bars, 8 mm.
NBs (data not shown), suggesting that they were equivalent
to Mx1 or TMxA domains in cells expressing these proteins
individually.
To demonstrate that this co-localization was not simply
due to heterooligomerization between the human and the
mouse Mx proteins, we used a co-translocation assay that
reveals oligomerization of Mx proteins in living cells
(Kochs et al., 1998; Ponten et al., 1997). When nuclear
TMxA and cytoplasmic MxA are co-expressed, they form
homooligomers and translocate into the nucleus together
(Ponten et al., 1997). We therefore asked whether the
nuclear mouse Mx1 protein would be able to drag the
human MxA into the nucleus. We transfected an expression
plasmid encoding the GFP–Mx1 fusion protein, together
with an expression plasmid encoding FLAG–MxA. In cells
co-expressing these two Mx proteins, GFP–Mx1 accumulated in the characteristic nuclear Mx1 dots, whereas FLAG–
MxA remained in the cytoplasm (Fig. 5g–i). These findings
suggested that the co-localization of FLAG–Mx1 and myc–
TMxA described above (Fig. 5a–f) was most likely not the
result of oligomerization of the two proteins, although we
cannot rule out the remote possibility that the nuclear
environment favours Mx1–TMxA interactions that do not
occur elsewhere in the cell. To prove that GFP–Mx1 was
indeed acting like wild-type Mx1 in this assay, we coexpressed GFP–Mx1 and FLAG–Mx1 in cells. We found
perfect co-localization of the GFP and FLAG signals
(Fig. 5j–l). Moreover, GFP–Mx1 accumulated in nuclear
dots that were frequently associated with PML NBs and
inhibited influenza A virus (data not shown).
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We also analysed the relationship of Mx1 dots with other
prominent nuclear structures, namely SC35 speckles and
Cajal bodies. Vero cells transfected with the plasmid
expressing GFP–Mx1 were labelled with anti-PML and
anti-SC35 or with anti-PML and anti-p80 coilin antibodies
and examined by confocal microscopy. As expected, PML
and GFP–Mx1 were frequently found closely associated
(Fig. 6a–d). It should be noted that Vero cells without IFN
treatment have a fairly small number of PML NBs per
nucleus. SC35 speckles were often separated from Mx1
dots; however, a number of SC35 speckles displayed association with Mx1 dots and/or PML NBs (Fig. 6a and b and
enlargements a1–b2). Similarly, Cajal bodies, as revealed
by labelling p80 coilin, were sometimes in close proximity
to PML NBs as well as to Mx1 dots, whereas in other
instances they occupied distinct areas of the nucleus
(Fig. 6c, d). This varied pattern of localization and association prompted us to analyse the distances between these
nuclear structures statistically, similar to previously reported
approaches (Shiels et al., 2001; Wang et al., 2004). The
distance between each PML, SC35 or coilin domain and
their nearest Mx1 dot was determined as described in
Methods. The mean minimum SC35–Mx1 distance was
larger than the mean minimum PML–Mx1 distance
(Fig. 6e), confirming our qualitative observation that Mx1
dots were associated more closely with PML NBs than with
SC35 speckles. In contrast, the mean minimum distance
between coilin and Mx1 dots did not differ significantly
from the mean PML–Mx1 distance. Since the association of
Mx1 with PML NBs depends, at least in part, on expression
levels of Mx1 and its accumulation in bigger dots (see, for
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O. G. Engelhardt and others
Fig. 5. Mx1 and TMxA accumulate in joint domains in the nucleus. Mouse 3T3 cells were transfected with plasmids pHMGFLAG-Mx1 and pCImycTMxA (a–f), pEGFP-Mx1 and pHMG-FLAG-MxA (g–i) and pEGFP-Mx1 and pHMG-FLAG-Mx1 (j–l),
fixed 2 days later and immunostained with a mouse monoclonal antibody against the FLAG-epitope tag (a, d, h, k) and a rabbit
polyclonal antiserum against the myc tag (b, e) as appropriate. Red and green channel images were recorded separately and
overlaid electronically. Bars, 8 mm.
example, Fig. 2a–c), we divided the distance data for Cajal
bodies into two groups, nuclei with ‘low’ and ‘high’ association between PML and Mx1 (see Methods for details).
Comparison of the mean minimum coilin–Mx1 distances
between these two groups revealed that the association
of Cajal bodies with Mx1 dots reflected the degree of
association between PML NBs and Mx1 dots, in as far as
the mean minimum coilin–Mx1 distance was significantly
lower in cells considered ‘high PML–Mx1 association’.
DISCUSSION
In this study, we addressed the question of whether PML
NBs determine the subcellular localization, antiviral activity,
2322
or both, of the IFN-induced Mx1 GTPase. We previously
identified several proteins including Daxx, Sp100, SUMO-1,
BLM, TOPORS and PKM/HIPK-2 as putative cellular
interaction partners of Mx1 (Engelhardt et al., 2001; Trost
et al., 2000). These proteins are known components of
PML NBs (Negorev & Maul, 2001; Rasheed et al., 2002).
Furthermore, PML, as well as Sp100, is also upregulated
by IFN (Chelbi-Alix et al., 1995; Grotzinger et al., 1996;
Guldner et al., 1992; Lavau et al., 1995; Nason-Burchenal
et al., 1996) and PML itself has been reported to possess
antiviral activity, like Mx1 (Chelbi-Alix et al., 1998; Djavani
et al., 2001; Regad et al., 2001). Therefore, the intriguing
possibility existed that Mx1 might take part in the complex
interplay of PML NB constituents. We have shown here
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Journal of General Virology 85
Mx nuclear domains
Fig. 6. Topological relationship between
Mx1 dots and other nuclear domains. (a)–(d)
Vero cells were transfected with plasmid
pEGFP-Mx1, fixed 1 day later and immunostained with a polyclonal rabbit antiserum
against PML (blue) and a mouse monoclonal
antibody against SC35 (red) (a, b) or a
mouse monoclonal antibody against p80
coilin (red) (c, d). Red, green and blue
channel images were recorded separately
and overlaid electronically. Panels labelled
with letters and numbers (e.g. a1) are enlargements of the areas indicated in the corresponding images (a)–(d). White lines signify
the shortest distance between the centroid
positions of stained domains and numbers
next to the lines indicate the distance in mm.
(e)–(f) Determination of the minimum distances between SC35 and Mx1 (e), coilin
and Mx1 (f) and PML and Mx1 (e, f) was
done as described in Methods. Means±SD
are plotted. Asterisks indicate statistically
significant differences. For details on the
grouping of data into cells with low or high
association between PML and Mx1 (PML–
Mx1 assoc. high, PML–Mx1 assoc. low)
indicated in (f), refer to the main text and
Methods.
that in PML2/2 cells Mx1 displayed a pattern of nuclear dots
that was indistinguishable from the pattern observed in
wild-type cells. Moreover, Mx1 had antiviral activity in
PML2/2 cells, indicating that PML and intact PML NBs
are dispensable for the antiviral action of Mx1. Nevertheless, our findings also show that Mx1 preferentially localized
to the vicinity of PML NBs, provided they were reconstituted in PML2/2 cells.
It should be noted that the association of PML NBs and Mx1
is rarely complete. In most cells, only a subset of Mx1 dots
is juxtaposed to or partially overlapping with PML NBs.
Furthermore, cells expressing low levels of Mx1 often show a
fine-punctate distribution of Mx1 with little or no obvious
association with PML NBs, suggesting that expression levels
http://vir.sgmjournals.org
determine localization. Thus, in IFN-treated cells, Mx1 may
first accumulate in the form of small aggregates that fuse and
grow bigger when protein levels increase. Larger aggregates
may then form at particular sites in the nucleus that are
found frequently in the immediate vicinity of PML NBs.
In cells lacking PML NBs, the sites of Mx1 dot formation
seem to be preserved. It remains to be seen whether Mx1
interacts with other proteins in these areas. Protein–protein
interactions in PML NBs and probably other subnuclear
domains are highly dynamic. Daxx, Sp100, SUMO-1,
TOPORS, FLASH, PIAS-1 and PKM/HIPK-2 are partially
associated with a subset of Mx1 domains and may be
engaged in such dynamic Mx1 interactions (Engelhardt et al.,
2001; Trost et al., 2000; this study and data not shown).
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O. G. Engelhardt and others
An intriguing observation was the finding that nuclear Mx
proteins of three different species exhibited an almost
identical subnuclear distribution. The nuclear Mx1 protein
of the rat behaved like mouse Mx1 and displayed dots that
were frequently associated with PML NBs. Surprisingly, the
human MxA protein, known to reside in the cytoplasm,
adopted an Mx1-like localization upon artificial translocation into the cell nucleus. This nuclear form of MxA
co-localized with mouse Mx1 in PML-associated dots and
tracks. This co-localization was unexpected because the
two proteins do not interact, as shown in an in vivo assay.
Obviously, nuclear Mx proteins accumulate in their own
distinct nuclear domains, which we have tentatively called
‘Mx nuclear domains’. These domains are functionally
independent of PML NBs, albeit often closely associated
with PML NBs in wild-type cells. We propose that the
same protein–protein interactions leading to dot formation of mouse Mx1 in the vicinity of PML NBs also apply
to other nuclear Mx proteins, including the nuclear form
of MxA.
We observed that ‘Mx nuclear domains’ were not exclusively
associated with PML NBs, but could also be found in the
vicinity of Cajal bodies and, to a lesser degree, of SC35
speckles. Association of one nuclear domain with more
than one type of other nuclear structure has been reported
in the literature (Wang et al., 2002) and may be a consequence of the highly dynamic nature of most nuclear
structures or of the multitude of protein–protein interactions of which some proteins are capable. The putative
interaction partners of Mx1 are mostly components of PML
NBs, suggesting a biochemical link of Mx1 to PML NBs.
However, it remains possible that proteins will be found
that link Mx1 to other nuclear structures. It is noteworthy
that, in the natural situation of virus infection, Mx1 is
induced by IFN, which also leads to an increase in the
number of PML NBs but not of SC35 speckles and Cajal
bodies, resulting in the high degree of association seen in
mouse cells upon IFN treatment (Engelhardt et al., 2001).
An interesting question is whether dot formation is essential
for Mx1 activity. It is possible that nuclear Mx proteins
require the dot-forming domains in order to exert their
antiviral activity against influenza viruses and other orthomyxoviruses. The sites of virus transcription and replication in the nucleus are presently not known. It is conceivable
that these viral processes occur at or near the ‘Mx nuclear
domains’ described here. Alternatively, Mx1 dots may serve
as storage depots for excess Mx1 protein. This would imply
that active Mx1 is recruited from these depots to sites of
virus transcription and replication. A similar depot model
has also been proposed for PML NBs (Negorev & Maul,
2001). If the sole purpose of Mx nuclear domains is to
serve as storage depots, these domains are expected to be
dispensable for antiviral activity. This is in line with previous findings showing that Mx1(C71S), a mutant form
of Mx1 with a single amino acid exchange at position
71, is antiviral, although this mutant does not form the
2324
characteristic nuclear dots (Toyoda et al., 1995). However,
using our high-expression vectors, this Mx1 mutant was
able to accumulate in nuclear dots in a majority of cells
(see Supplementary Figure in JGV Online), suggesting that
dot formation most likely depends on protein expression
levels, as discussed above. All other mutants of Mx1 with a
diffuse intranuclear distribution are reported not to have
antiviral activity (Garber et al., 1993). On the other hand,
some inactive variants of Mx1 accumulated in dots in the
nucleus (Pitossi et al., 1993) (see also Fig. 3a). Therefore, it
is presently not possible to decide whether or not Mx1 dots
play a direct role in the antiviral activity of Mx1. Further
mutational studies will be needed to clarify this issue.
Our present study does not support the view that PML
itself has antiviral activity against influenza viruses (ChelbiAlix et al., 1998). In our hands, both wild-type and PML2/2
cells were equally permissive for the influenza A virus strain
used. This also suggests that PML NBs do not play an
essential role in the influenza virus life cycle. In contrast,
Mx1 was clearly antiviral, even in the absence of PML. Mx
GTPases are members of the dynamin superfamily of large
GTPases and may have specialized cellular functions in
addition to their antiviral activity (Haller & Kochs, 2002).
It will be interesting to define further the significance of the
‘Mx nuclear domains’ described here for the cellular and
antiviral functions of this class of nuclear GTPases.
ACKNOWLEDGEMENTS
We thank Hans Will, Thomas Hofmann, Jovan Pavlovic, Angus
Lamond and Werner W. Franke for reagents, Georg Kochs for plasmids and helpful discussions and Karin Engelhardt, Peter Staeheli,
Friedemann Weber and Anneke Funk for critically reading the
manuscript. This work was in part supported by a grant from the
Deutsche Forschungsgemeinschaft to O. H. (HA 1582/3-1) and
the BMBF (H. S.). The Heinrich-Pette-Institut is supported by the
Freie und Hansestadt Hamburg and the Bundesministerium für
Gesundheit und Soziale Sicherheit.
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