From the Abteilung Virologie, Institut für Medizinische Mikrobiologie und Hygiene, Universität Freiburg, D-79008 Freiburg, Germany
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ABSTRACT |
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Human MxA protein is an interferon-induced member
of the dynamin superfamily of large GTPases. MxA inhibits the
multiplication of several RNA viruses, including Thogoto virus, an
influenza virus-like orthomyxovirus transmitted by ticks. Previous
studies have indicated that GTP binding is required for antiviral
activity, but the mechanism of action is still unknown. Here, we have
used an in vitro cosedimentation assay to demonstrate, for
the first time, a GTP-dependent interaction between MxA
GTPase and a viral target structure. The assay is based on highly
active MxA GTPase as effector molecules, Thogoto virus nucleocapsids as
viral targets, and guanosine
5'-O-(3-thiotriphosphate) (GTP Human MxA, a 76-kDa GTPase, belongs to the newly defined dynamin
superfamily of high molecular mass GTPases found in yeast, plant, and
animal cells (1). These large GTPases play key roles in fundamental
cellular processes, such as endocytosis (2); intracellular vesicle
transport (3); cell plate formation in plants (4); and in the case of
Mx proteins, resistance to virus infection (5). MxA is induced
exclusively by type I ( GTP binding seems to be crucial for antiviral activity because
mutations within the N-terminal GTP-binding domain destroy the
antiviral activity (14, 15). In addition, the C-terminal part of MxA
seems to play an important role because the coexpression of an
antivirally inactive C-terminal fragment of MxA interferes with
wild-type MxA activity in a dominant-negative manner (15). Furthermore,
a single amino acid exchange in the C terminus affects the antiviral
specificity of wild-type MxA. This substitution results in loss of
activity against vesicular stomatitis virus (VSV)1 while maintaining
wild-type activity against influenza A virus (FLUAV) and Thogoto virus
(THOV), a tick-borne orthomyxovirus (9, 16).
It has been shown that cytoplasmic MxA inhibits primary transcription
of VSV and measles virus, an early step in the viral life cycle that
occurs in the cytoplasm of infected cells (17, 18). In contrast, FLUAV
transcribes its genome in the cell nucleus and is blocked by MxA at a
later step after primary transcription that is still unknown (8).
However, MxA is able to block primary transcription of FLUAV when
translocated to the nucleus by a foreign nuclear translocation signal
(16). Moreover, recombinant MxA is capable of inhibiting the
transcriptional activity of purified viral nucleocapsids in
vitro (19). Nevertheless and despite much effort, biochemical data
demonstrating a physical interaction with viral components are still
missing, and viral target structures are presently unknown.
Most GTPases act as molecular switches, with the GTP-bound form usually
representing the active state (20). Members of the dynamin superfamily
may be unique among GTPases because they seem to behave as
mechanochemical enzymes rather than as molecular switches (21, 22). It
has been shown that the GTP-bound conformation of dynamin
self-assembles around tubular membrane invaginations (23) and that GTP
hydrolysis leads to constriction and vesiculation of dynamin-coated
tubes (22). Moreover, it has been proposed that the ability to form
helical arrays around tubular templates might be a functional link
between all dynamin-like GTPases (21). In fact, MxA also forms
aggregates of ~30 molecules (24) that adopt a helical structure in
solution,2 and C-shaped and
ring-like structures have been described for mouse Mx1 protein
(25).
Biochemical studies revealed that MxA has an intrinsic GTPase activity
characteristic of large GTPases and that a high percentage of MxA
molecules may be complexed with GTP in vivo (24). We reasoned that it is GTP-bound MxA that represents the antivirally active form and that interacts with viral targets. Therefore, we
performed binding studies in the presence of the non-hydrolyzable GTP
analogue GTP Here, we show for the first time an association of MxA with viral
proteins. In its GTP-bound form, MxA associates with the nucleocapsids
of THOV by binding to the nucleoprotein (NP) component. We show that
this interaction is mediated by domain(s) in the carboxyl-terminal
moiety of MxA and can be prevented by a monoclonal antibody directed
against this region. These results suggest that the binding of GTP to
MxA induces an active conformation of the carboxyl-terminal domain.
Furthermore, we suggest that MxA works by recognizing and wrapping
around incoming nucleocapsids in the cytoplasm of the infected cell,
thereby inactivating their function.
Cells and Viruses--
Parental Swiss mouse 3T3 cells and cells
stably transfected and constitutively expressing wild-type human MxA
protein (clone 4.5.15) were the same as described previously (26).
Cells were grown in Dulbecco's modified Eagle's medium containing
10% fetal calf serum with or without 0.5 mg/ml geneticin (G418).
3T3 cells were infected with THOV strain SiAr126 (27) or Dhori virus
(DHOV) strain DHO/India/1313/61 (28) with an input multiplicity of
infection of 10 and incubated for 16 h. For metabolic labeling of
viral proteins, cells were incubated for the last 4 h in
Dulbecco's modified Eagle's medium minus methionine and 50 µCi/ml
[35S]methionine.
Expression and Purification of Recombinant Proteins--
The NP
of THOV was synthesized in Escherichia coli and purified by
means of an N-terminal His tag. Using polymerase chain reaction, a
SmaI site was added at positions 14-19 of the NP cDNA insert of the plasmid pBK-L3 (29). This restriction site was used to
insert the open reading frame of the NP into the blunted BamHI site of the procaryotic expression vector pQE9
(QIAGEN, Inc., Hilden, Germany), yielding the plasmid pHisNP. All MxA
mutants (see Fig. 1A) used in coimmunoprecipitation
experiments derive from the plasmid pHis-MxA, which contains the
His-tagged MxA cDNA (14, 15, 30).
Histidine-tagged proteins were produced in E. coli M15 after
induction with isopropyl- Coimmunoprecipitation--
Cell extracts were prepared in 50 mM Tris, 0.1% Nonidet P-40, 5 mM
MgCl2, and 0.5 mM dithiothreitol, pH 7.5. After
separation of the cell debris (15 min at 12,000 × g),
the supernatants were used for coimmunoprecipitation. First, lysates of
MxA-expressing cells or the E. coli-expressed MxA mutants
were mixed with a polyclonal rabbit anti-MxA antiserum directed against
E. coli-expressed histidine-tagged MxA protein (15) and with
protein A-Sepharose beads (Pharmacia, Freiburg, Germany). After
incubation for 1 h, the immunocomplex bound to protein A-Sepharose
beads was washed with lysis buffer. The complex was then incubated with
35S-labeled lysates of THOV-infected cells for 2 h at
4 °C in the presence of 150 mM NaCl. Following intensive
washing with lysis buffer containing 150 mM NaCl, the bound
proteins were redissolved in SDS sample buffer, separated on a 10%
SDS-polyacrylamide gel, and blotted on a polyvinylidene difluoride
membrane (Millipore Corp., Bedford, MA). Coprecipitated viral proteins
labeled with [35S]methionine were detected by
autoradiography. The MxA proteins or fragments used for precipitation
were detected on the Western blot with a polyclonal mouse anti-MxA antiserum.
To confirm the identity of the coprecipitated, 35S-labeled
viral protein, the immunocomplex was incubated in radioimmune
precipitation assay buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1% Nonidet P-40, 0.25 sodium deoxycholate, and
0.1% SDS) for 2 min at 70 °C. The labeled protein was then
immunoprecipitated using virus-specific antibodies: polyclonal
guinea pig anti-THOV antiserum, monoclonal anti-NP antibody (mAb2), and
anti-glycoprotein antibody (mAb10) (all kindly provided by P. A.
Nuttall) (31-33). The immunoprecipitated proteins were analyzed by
SDS-polyacrylamide gel electrophoresis and autoradiography.
Isolation of Genomic RNA--
Viral particles of THOV were
isolated from supernatants of THOV-infected BHK-21 cells and purified
by sequential centrifugation steps as described (29). Viral genomic RNA
was extracted from purified viral particles by the acid phenol method
(34) and checked for purity by agarose gel electrophoresis (29).
Synthetic RNP Complexes--
RNA transcripts were synthesized
in vitro from Ksp632I-digested pVN1 (a gift from
Florence Baudin and Rob W. H. Ruigrok, EMBL Grenoble Outstation,
Grenoble, France) or from BamHI-digested pBSK-MxA using T3
or T7 RNA polymerase, respectively, as described (35). In
vitro transcription from pVN1 yields a short model vRNA of ~81
nucleotides with the complementary terminal sequences of segment 5 (NP)
of FLUAV (strain FPV.GI, H7N1) that forms a panhandle structure.
In vitro transcription of pBSK-MxA yields a
2200-nucleotide-long RNA transcript coding for MxA (36). After digestion of the DNA template with DNase I, free nucleotides were removed by gel filtration. The RNA was extracted with phenol/chloroform and precipitated with ethanol.
To prepare artificial RNPs, viral RNA from THOV virions (600 ng) or
synthetic RNA molecules (VN1, 200 ng; and MxA, 560 ng) were mixed with
purified recombinant NP (3 µg) for 10 min at 25 °C in 30 µl of
10 mM Tris, 100 mM NaCl, 5 mM
MgCl2, and 1 mM dithiothreitol, pH 7.5. These
RNP preparations were directly used in the precipitation assays.
Cosedimentation of MxA and RNPs in Glycerol Gradient
Centrifugation--
MxA-expressing cells and cells infected with THOV
were lysed in 20 mM Hepes, pH 7.5, and 0.5 mM
dithiothreitol. The lysate of MxA-expressing cells was then centrifuged
at 100,000 × g for 1 h. The lysate of the
virus-infected cells was cleared by a short centrifugation at
10,000 × g for 10 min. Lysates were mixed and incubated in a volume of 200 µl for 15 min at 37 °C in buffer A
(25 mM Hepes, pH 7.5, 25 mM KCl, 2.5 mM magnesium acetate, 5 mM EDTA, 150 mM potassium glutamate, 2 mM ATP, 16.7 mM creatine phosphate, 3.5 units of creatine phosphokinase,
and 200 µM GTP
To inhibit cosedimentation, the lysates were preincubated for 10 min at
25 °C with monoclonal antibodies directed against the THOV
nucleoprotein (mAb2) or against the MxA protein (2C12) (37). A
monoclonal antibody specific for THOV glycoprotein (mAb10) and a
polyclonal mouse antibody directed against FLUAV (h38III) were used as controls.
MxA Interacts with the NP of THOV--
To search for viral target
proteins of MxA, the following strategy was used. Wild-type and mutant
forms of MxA (Fig. 1A) were immobilized on protein A-Sepharose beads using polyclonal anti-MxA antibodies. These MxA-loaded beads were subsequently used to
coprecipitate proteins from lysates of THOV-infected cells. To allow
detection of putative interaction partners, newly synthesized proteins
were radioactively labeled by incubating the infected cells with
[35S]methionine. Fig. 1B shows that wild-type
MxA synthesized in transfected 3T3 cells, but not a control preparation
without MxA, was able to precipitate a 35S-labeled viral
protein from THOV-infected cells (lanes 1 and 2).
The radiolabeled protein had an estimated molecular mass of 52 kDa and
was identified as the NP of THOV by immunoprecipitation with
monospecific anti-NP antibodies (Fig. 1D). The same viral protein was precipitated by wild-type MxA produced in E. coli (Fig. 1B, lane 3). To determine which
region of MxA was critical for NP binding, we produced various
truncated forms of MxA in E. coli, as schematically depicted
in Fig. 1A. The presence of wild-type (76 kDa) and mutant
forms of MxA in the coprecipitation reactions was confirmed by Western
blot analysis demonstrating bands of the expected sizes in each
reaction sample (Fig. 1C). A truncated form of MxA lacking
the last 90 amino acids ( MxA Binds to Nucleocapsids of THOV--
NP is the most abundant
component of viral nucleocapsids, also referred to as viral
ribonucleoprotein complexes (vRNPs). We therefore investigated whether
MxA would recognize NP in these viral structures. Binding was assessed
in a cosedimentation assay that enabled us to test the capacity of MxA
to cosediment with THOV RNPs during ultracentrifugation in a
discontinuous glycerol gradient. A gradient system was established that
allowed us to distinguish between free MxA and MxA bound to vRNPs.
Lysates were prepared from cells expressing recombinant MxA (26) and
were then mixed with lysates obtained from either THOV-infected or uninfected control cells. The mixture was separated by glycerol gradient centrifugation, and fractions of the gradient were analyzed by
immunoblotting with antibodies to MxA and to the NP of THOV. The
NP-specific bands allowed easy identification of fractions containing
vRNPs. As shown in Fig. 2A,
MxA alone was not able to penetrate into the gradient beyond fractions
5 and 6. Instead, MxA stayed on top of the glycerol layer. The
nucleocapsids present in lysates of THOV-infected cells sedimented as a
broad peak into fractions of high density exhibiting a sedimentation
behavior that was very distinct from that of free MxA (data not shown). When MxA-containing lysates were mixed with lysates of infected cells,
an altered distribution of MxA was observed. MxA now cosedimented with
vRNPs to fractions of higher density, suggesting a tight association of
MxA with these viral structures. In addition, we were able to
demonstrate cosedimentation of MxA with purified vRNPs isolated from
THOV particles (data not shown).
Next, we wanted to know whether this association was virus-specific. We
have previously shown that MxA is an efficient inhibitor of THOV, but
is inactive against DHOV, another tick-borne orthomyxovirus (9).
Therefore, we tested whether MxA would similarly bind to DHOV
nucleocapsids. Fig. 2A shows that there was no
cosedimentation of MxA with vRNPs of DHOV. The nucleocapsids present in
lysates of DHOV-infected cells penetrated deeply into the gradient like THOV nucleocapsids. In contrast, MxA remained on top of the gradient as
in gradients with uninfected control cell lysates, indicating a failure
to interact. These findings suggest that the capacity to interact in
our in vitro test system and the ability to block viral
multiplication in vivo are most likely two facets of the same phenomenon.
Cosedimentation of MxA with vRNPs Depends on GTP Monoclonal Antibody 2C12 Prevents the Association of MxA with
vRNPs--
We used monoclonal antibody 2C12 to abrogate
cosedimentation of MxA with vRNPs. This antibody is known to recognize
a conserved epitope on rodent and human Mx proteins (37, 38) and to
neutralize the antiviral effect of murine Mx1 protein (39). In
addition, this antibody is also able to neutralize the antiviral
activity of human MxA against THOV when microinjected into the
cytoplasm of MxA-expressing cells (data not shown). Therefore, we
tested whether this antibody would also prevent the interaction of MxA with vRNPs. Lysates of MxA-expressing cells were preincubated with
antibody 2C12 and then mixed with lysates of THOV-infected cells and
subsequently subjected to glycerol gradient centrifugation. Under these
conditions, MxA did not cosediment with the nucleocapsids, indicating
that preincubation with antibody 2C12 prevented the interaction of MxA
with the viral target structure (Fig.
3A). In contrast, addition of
a non-Mx-specific antibody (h38III) had no effect (Fig. 3C).
Preincubation of THOV lysates with a monoclonal antibody directed
against the NP of THOV likewise prevented the MxA-vRNP interaction
(Fig. 3B), whereas addition of a monoclonal antibody
directed against the THOV glycoprotein (Fig. 3D) was without
effect. These results support the idea that cosedimentation was
mediated by a specific interaction between MxA and the NP component of
vRNPs.
RNA-bound NP Is the Binding Partner of MxA--
In addition to NP,
vRNPs consist of genomic vRNA and the three viral polymerase subunits.
The results of the coprecipitation assay (Fig. 1) and the antibody
inhibition experiment (Fig. 3B) suggested NP as the binding
partner of MxA. To futher elucidate the role of NP in the
cosedimentation assay, we constructed artificial RNPs consisting only
of genomic RNA and recombinant NP of THOV. NP containing six extra
histidine residues at the N terminus was produced in E. coli, isolated by Ni2+ affinity chromatography, and
further purified by heparin column chromatography (see "Experimental
Procedures"). The pure recombinant NP had an estimated molecular mass
of 52 kDa and exhibited RNA-binding capacity (29). The genomic vRNA
preparation from purified virions consisted of a mixture of all six
genomic RNA segments varying in length from 900 to 2200 nucleotides
(29). Synthetic RNPs were produced by incubating vRNA with the purified
NP in an appropriate binding buffer. Interestingly, these artificial
RNPs had a similar sedimentation behavior in the glycerol gradient as
authentic RNPs extracted from infected cells (Fig.
4B). More important, MxA cosedimented with these structures and reached the bottom of the gradient together with these artificial RNPs (Fig. 4B). In the absence of
artificial RNPs, MxA remained at the top of the gradient, as expected
(Fig. 4A).
These positive results encouraged us to use in vitro
synthesized RNA molecules instead of RNA extracted from virions. First, we generated an 81-nucleotide-long model RNA that contained the complementary terminal sequences of segment 5 of influenza A virus. It
has been shown that this molecule is able to form a double-stranded panhandle structure similar to the genomic influenza viral RNA segments
(35). Indeed, preincubation of this model RNA with recombinant NP of
THOV resulted in the formation of artificial RNPs capable of dragging
MxA into the gradient (Fig. 4C). It was conceivable that
this effect was dependent on the panhandle structure of the viral RNA
molecules. To further investigate this assumption, we synthesized a
linear 2200-nucleotide-long RNA molecule that was predicted not to
contain such a secondary structure. This RNA was then used as backbone
to build up artificial RNPs. Again, MxA coprecipitated with such RNPs,
and GTP Specificity of vRNP Recognition--
Previous attempts to
demonstrate a physical interaction of MxA with target structures of
FLUAV or VSV have invariably failed. Here, we used THOV because this
orthomyxovirus shows an extraordinarily high sensitivity to the
inhibitory effect of MxA. In cell culture, virus titers are on average
1,000,000-fold lower in MxA-expressing cells than in similarly infected
control cells (9). By comparison, FLUAV and VSV titers are reduced at
best 100-1000-fold in the presence of MxA (26). Accordingly, MxA
transgenic mice were found to be completely resistant to infection with
THOV, but showed only partial resistance to infection with FLUAV or VSV
(11). We believe that our ability to identify THOV nucleocapsids as targets for MxA is a direct reflection of the extreme MxA sensitivity of THOV. Consistent with this reasoning is the finding that
nucleocapsids of DHOV were not recognized by MxA (Fig. 2A).
DHOV belongs to the same genus of tick-transmitted orthomyxoviruses as
THOV (40). However, DHOV is insensitive to inhibition by MxA (9).
Sequence comparisons of the NP genes of these two viruses showed that
the degree of amino acid sequence similarity is only ~43% (29). It
may therefore well be that the target domain for MxA is not conserved
among tick-borne orthomyxoviruses.
We also investigated the cosedimentation behavior of MxA with
nucleocapsids of FLUAV and VSV. In both cases, no clear cosedimentation was observed.3 A possible
explanation is that binding of GTP-bound MxA to vRNPs must be of great
strength in order to be detected in our in vitro cosedimentation assay and that weaker interactions are not readily demonstrable, although they may still be sufficient for mediating resistance in vivo. It is, of course, also possible that MxA
recognizes different target structures in THOV, FLUAV, and VSV.
Finally, we cannot exclude a cooperative effect of additional host cell factors since all our experiments were done in the presence of cellular
extracts. In fact, there is circumstantial evidence that cell
type-specific factors may modulate the antiviral specificity of MxA
action (18, 41).
Nature of the Viral Target Structure--
The vRNPs of THOV
contain five components, namely genomic RNA, NP, and the
RNA-dependent RNA polymerase complex consisting of three
subunits PA, PB1, and PB2. NP is the major protein component of the
vRNP structure (42). Previous studies with FLUAV and mouse Mx1 protein
pointed to the viral polymerase as the target of Mx action because high
level expression of recombinant PB2 could abolish the nuclear Mx1 block
in infected cells, resulting in virus growth (43, 44). However, a
similar effect with human MxA was not reported. Moreover, all attempts
to demonstrate a biochemical interaction of mouse Mx1 or human MxA with
PB2 were unsuccessful (44). Here, we have demonstrated that artificial RNPs consisting only of RNA and viral NP are sufficient to interact with MxA (Fig. 4). This clearly indicates that, at least for THOV, the
polymerase subunits are not the primary target of MxA within the RNP
structure. RNA is also excluded because MxA has no RNA-binding capacity.4 The present data
strongly suggest that MxA binds to the RNP structure by recognizing its
major protein component, NP. A monoclonal antibody directed against NP
prevented vRNPs from associating with GTP-bound MxA, whereas a
monoclonal antibody against the envelope glycoprotein had no effect
(Fig. 3). More importantly, MxA was able to coprecipitate NP from
lysates of THOV-infected cells (Fig. 1). A truncated form of MxA
lacking only 90 amino acids at the carboxyl terminus had lost this
capacity, indicating that the observed interaction of NP with wild-type
MxA was significant and not merely due to nonspecific binding. To
obtain functional evidence for the role of NP, we have tried to
neutralize the antiviral effect by expressing recombinant NP in
MxA-containing cells. However, high level expression of NP proved to be
toxic to the cells, and reliable results were not obtained. It remains
to be seen whether MxA interacts with free NP or preferentially with NP
integrated in RNPs. The coprecipitation assay used did not allow us to
differentiate between these possibilities.
Importance of the Carboxyl-terminal Domain for Viral Target
Recognition--
To define the interactive domains of MxA, we took
advantage of the availability of the broadly Mx-reactive monoclonal
antibody 2C12 (37). We demonstrated that antibody 2C12 is capable of preventing the recognition of THOV nucleocapsids by MxA (Fig. 3). We
have mapped the binding site of this antibody to an internal domain
comprising amino acids 363-574 that is located in the C-terminal half
of MxA (Fig. 1A; data not shown). This domain corresponds to
the rat Mx3 protein domain containing the 2C12 epitope recently mapped
by Johannes et al. (38). Direct evidence for the presence of
interactive domain(s) within the C-terminal half of MxA arises from the
present finding that a C-terminal fragment was sufficient to
coprecipitate THOV NP, whereas truncated forms of MxA with deletions in
the C-terminal moiety had lost this function (Fig. 1B).
Taken together, our results support the idea that the interactive structure of MxA is formed by sequences in the internal domain carrying
the 2C12 epitope and sequences in the extreme C-terminal part. Both
domains are seemingly also involved in the formation of a proper
GTP-binding pocket and in oligomerization (30, 45). Our present results
are supported by previous work using quite different approaches.
Experiments with dominant-negative mutant forms of MxA showed that
sequences in the C-terminal moiety were necessary and sufficient for
interference with wild-type function (15). The C-terminal region of MxA
(Fig. 1A) contains two highly conserved leucine zipper
repeats (46). Leucine repeats form amphipathic Crucial Role of GTP Binding for Antiviral Activity--
Previous
studies indicated that an intact GTP-binding domain is required for
antiviral activity of MxA (14, 15). We show here that GTP binding is
necessary and sufficient for the association of MxA with THOV RNPs
(Fig. 2B). Most likely, binding of GTP leads to a
conformational change of the molecule that allows specific recognition
of viral targets, such as RNPs of THOV (Fig.
5). In addition, GTP binding and
association with target structures may favor MxA-MxA interactions and
stabilize the complex. Similar interactions have been observed with
dynamin, another member of the superfamily of large GTPases. Dynamin is
essential for receptor-mediated endocytosis and synaptic vesicle
recycling (2). It is believed that dynamin forms a collar around the
necks of clathrin-coated pits and helps to bud off vesicles from the
plasma membrane (23). Dynamin can self-assemble into rings and stacks
of interconnected rings in solution (51, 52). In the presence of
GTPS) as a stabilizing factor. We show that MxA tightly interacts with viral nucleocapsids by
binding to the nucleoprotein component. This interaction requires the
presence of GTP
S and is mediated by domains in the carboxyl-terminal moiety of MxA. We propose that GTP-bound MxA adopts an antivirally active conformation that allows interaction with viral nucleocapsids, thereby impairing their normal function.
INTRODUCTION
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Abstract
Introduction
References
/
) interferons (6), accumulates in the
cytoplasm of cells (7), and interferes with the multiplication of
distinct RNA viruses. Work with both transfected cells (8-10) and MxA
transgenic mice (11) demonstrated that MxA is a powerful antiviral
agent. In man, synthesis of MxA is induced during acute viral
infections and may thus protect humans from severe disease (12,
13).
S. For this approach, we used THOV because this virus
represents the most MxA-sensitive virus known to date.
EXPERIMENTAL PROCEDURES
-D-thiogalactopyranoside and
were isolated using Ni2+ chelate agarose chromatography as
described (14). NP was further purified using heparin-Sepharose
(Amersham Pharmacia Biotech) in 20 mM Tris, pH 8.0. It
eluted from the column as a sharp peak at 0.7 M NaCl.
S) as described (23). To analyze the
association of MxA with RNPs, the mixture (200 µl) was loaded onto a
discontinuous glycerol gradient (200 µl of 70%, 250 µl of 60%,
and 50 µl of 50% glycerol in buffer A; total volume of the gradient,
700 µl). The gradient was subjected to centrifugation at 40,000 rpm
for 3 h at 12 °C in an SW 50.1 rotor (Beckman Instruments,
München, Germany). Eight fractions (80 µl each) were collected
from the bottom of the gradient and analyzed by Western blotting
using polyclonal rabbit anti-MxA and guinea pig anti-THOV antibodies.
The primary antibodies were detected using alkaline
phosphatase-conjugated secondary antibodies and
5-bromo-4-chloro-3-indolyl phosphate/nitro blue tetrazolium as substrate.
RESULTS
572-662) had lost the capacity to
precipitate NP (Fig. 1B, lane 4).
Likewise, a MxA mutant with a central deletion of 275 amino acids
(
301-576) was unable to precipitate NP (Fig. 1B,
lane 5). Most significantly, however, a truncated
form consisting of only the C-terminal half of MxA (
1-362) was able
to recognize NP, albeit with seemingly lower efficiency as compared
with the wild type (Fig. 1B, lane 6).
Taken together, these experiments suggest that NP is the viral target
recognized by MxA and that the domains responsible for recognition
reside in the C-terminal moiety of MxA.
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Fig. 1.
The carboxyl-terminal moiety of MxA interacts
with the NP of THOV. A, schematic diagram of wild-type
and mutant MxA proteins purified from E. coli and used in
coprecipitation assays. MxA represents the wild-type protein. The
consensus tripartite motif for guanine nucleotide binding at the N
terminus is represented by three open bars. Hatched
boxes near the C terminus indicate two putative leucine zippers.
The region recognized by monoclonal antibody 2C12 is indicated.
Deletion mutant 572-662 lacks the last 90 amino acids of MxA,
deletion mutant
301-576 lacks a central fragment of 275 amino
acids, and deletion mutant
1-362 consists of the C-terminal half of
the protein. B, wild-type and mutant MxA proteins were
immobilized on protein A-Sepharose beads and incubated with
35S-labeled extracts of THOV-infected cells. Coprecipitated
metabolically labeled proteins were analyzed by SDS-polyacrylamide gel
electrophoresis (10%) and autoradiography. Lane
1, No MxA; lane 2, wild-type MxA
produced in 3T3 cells; lane 3, wild-type MxA
produced in E. coli; lane 4, MxA
mutant
572-662; lane 5, MxA mutant
301-576; lane 6, MxA mutant
1-362.
C, Western blot analysis of the immobilized proteins used in
B demonstrated the presence of wild-type or mutant MxA
proteins in the coprecipitation samples. MxA was detected by a
polyclonal mouse antiserum against E. coli-expressed wild-type MxA. This antibody
detected also a contaminating protein of unknown nature
(lane 4, upper band). Molecular mass
markers are indicated. D, the identity of the coprecipitated
35S-labeled NP was confirmed by immunoprecipitation with
anti-NP antibodies. 35S-labeled THOV NP coprecipitated with
beads containing no MxA (lane 1), or wild-type
MxA (lane 2) was detected by autoradiography. The
coprecipitated protein was reisolated from the MxA immunocomplex and
immunoprecipitated with monoclonal antibody mAb2 specific for NP
(lane 4), with monoclonal antibody mAb10 directed
against the viral glycoprotein (GP; lane 5), or
with a polyclonal anti-THOV antiserum (THOV; lane
6). Lane 3 shows the control without
antibody.
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Fig. 2.
Cosedimentation of MxA with vRNPs depends on
GTP S. A, lysates of
MxA-expressing cells were mixed with lysates of uninfected (panel
a), THOV-infected (panel b), or DHOV-infected
(panel c) cells in the presence of GTP
S. The mixtures
were subjected to glycerol gradient centrifugation, and the resulting
fractions were analyzed by Western blotting using polyclonal antisera
from rabbit and guinea pig specific for MxA and NP, respectively. The
positions of the MxA and NP protein bands are indicated. B,
lysates of MxA-expressing cells were mixed with lysates of
THOV-infected cells and incubated without nucleotides (panel
a) or in the presence of GTP
S (panel b) or GDP
S
(panel c) before analysis.
S--
The
initial experiments were performed in the presence of the
non-hydrolyzable GTP analogue GTP
S (Fig. 2A). Previous
studies have shown that GTP binding is critical for the antiviral
action of Mx proteins (14, 15, 19). Therefore, it was conceivable that
GTP was required for proper interaction of MxA with vRNPs. To address
this question, cosedimentation assays of MxA with THOV nucleocapsids
were performed in the presence or absence of GTP
S. Clearly, MxA
interacted with vRNPs only in the presence of GTP
S (Fig.
2B, panel b). No interaction occurred in the
absence of this nucleotide analogue or in the presence of GDP
S (Fig.
2B, panels a and c). In the presence
of GTP
S, the RNPs moved deeper into the gradient than in the absence
of GTP
S, as indicated by the position of the NP-specific bands. This
shift of RNPs to higher density suggests that large and stable
complexes between MxA and nucleocapsids were formed. We also tested
hydrolyzable GTP and GDP nucleotides. In both cases, MxA did not show
an interaction with vRNPs (data not shown), indicating that the
non-hydrolyzable GTP analogue most likely stabilized an interactive
conformation of MxA.
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Fig. 3.
Inhibition of MxA/vRNP cosedimentation by
anti-MxA or anti-NP antibodies. Lysates of MxA-expressing or
THOV-infected cells were preincubated with monoclonal antibody 2C12
recognizing MxA protein (A) or with monoclonal antibody mAb2
recognizing the NP of THOV (B), respectively. As
controls, a polyclonal mouse antibody (h38III) not directed
against MxA (C) and monoclonal antibody mAb10 specific for
the THOV glycoprotein (GP; D) were used. Lysates
were then mixed with the appropriate partner in the presence of GTP S
and subjected to glycerol gradient centrifugation. The fractions
were analyzed by Western blotting as described for Fig. 2.
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Fig. 4.
MxA interacts with artificial RNP
complexes. Lysates of MxA-expressing cells were incubated with
buffer only (A) or with synthetic RNP complexes
(B-E). To generate artificial RNPs, authentic vRNA isolated
from THOV virions (B), a synthetic model panhandle vRNA of
81 nucleotides (C), or an unrelated linear RNA molecule of
2200 nucleotides (D and E) was incubated with
recombinant NP of THOV. The various samples were subjected to gradient
centrifugation in the presence (A-D) or absence
(E) of GTP S. The fractions were analyzed by Western
blotting as described for Fig. 2.
S was necessary for interaction (Fig. 4, D and
E). These findings clearly demonstrated that MxA interacted
with the RNA-bound NP of THOV and that binding was independent of RNA
secondary structure.
DISCUSSION
-helices that are
known to promote protein-protein interactions (47). Targeted mutations
impairing the amphipathic character of these helices destroy the
antiviral activity of mouse Mx1 protein (48, 49). More convincingly, a
single amino acid exchange within the distal leucine zipper motif
changes the antiviral specificity of MxA such that the mutant protein
lost antiviral activity against VSV, but maintained wild-type activity
against FLUAV and THOV (9, 16). The importance of the C-terminal region
for the antiviral activity was also suggested by comparing the amino
acid sequences of the inactive rat Mx3 and active rat Mx2 proteins
(50). Substitutions of single amino acids in the C-terminal region of
rat Mx3 led to gain of antiviral function in the protein (38). In
summary, these findings indicate that the C-terminal half may expose
domains that interact directly with viral target structures, although
recognition of interposed cellular molecules can presently not be
excluded. The cosedimentation assay presented here will be helpful in
the search for such additional factors.
S, long ring-like structures form around tubular membrane
invaginations of ~25 nm in diameter (23). Recently, Sweitzer and
Hinshaw (22) directly demonstrated that purified dynamin binds to lipid
bilayers and forms helical tubes that constrict and generate vesicles
upon GTP addition. These data suggest that dynamin does not behave as a
molecular switch, but as a force-generating molecule (22). Our present
results support the view that the MxA GTPase is most likely also a
mechanochemical enzyme. Like dynamin, MxA self-assembles into polymeric
structures that resemble the C-shaped and ring-shaped polymers
previously observed with mouse Mx1 protein (25).2 The
present data indicate that MxA polymerizes around nucleocapsids and
that this interaction can be stabilized by GTP
S. Interestingly, studies with VSV revealed that MxA requires GTP binding, but not necessarily GTP hydrolysis, for its inhibitory action. Thus, Schwemmle et al. (19) found that, in the presence of GTP
S, purified
MxA inhibited viral RNA synthesis in an in vitro
transcription system and concluded that GTP binding is sufficient for
inhibition, with no need for GTP hydrolysis. It is possible that, in
this in vitro system, GTP-bound MxA associated with the
nucleocapsids of VSV as proposed here for THOV. However, the situation
may be more complex in infected cells. We propose that GTP-bound MxA
associates with viral nucleocapsids, resulting in an impairment of
nucleocapsid function (Fig. 5). In the case of THOV, translocation of
freshly uncoated nucleocapsids into the nucleus may be blocked. Indeed, we recently found that a sizable fraction of microinjected RNPs of THOV
stays in the cytoplasm of MxA-expressing
cells.5 In addition, tight
binding could result in cotranslocation of MxA together with the
nucleocapsids into the nucleus. Once there, MxA could interfere with a
nuclear step of viral replication. As shown previously, MxA has indeed
antiviral activity when transported to the nucleus with the help of a
foreign nuclear translocation signal (9, 16). Such a consecutive
sequence of viral inhibition would be foolproof and could explain the
very high degree of resistance against THOV that is observed in
MxA-expressing cells.
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Fig. 5.
Model for GTP-dependent
interaction of MxA with vRNPs. Our model proposes that MxA exists
in two conformational forms, an inactive GDP-bound form and an active
GTP-bound form. GTP binding induces a conformational change that allows
tight interaction with viral NP in RNP complexes. The active
conformation is stabilized by GTP S, a non-hydrolyzable analogue of
GTP. Subsequent hydrolysis of GTP to GDP leads to the inactive form of
MxA.
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ACKNOWLEDGEMENTS |
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We thank Peter Staeheli, Friedemann Weber, and Michael Frese for helpful discussions; Patricia A. Nuttall for generous gifts of anti-THOV antibodies; Florence Baudin and Rob Ruigrok for providing pVN1; and Simone Gruber for expert technical assistance.
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FOOTNOTES |
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* This work was supported by Grant Ko 1579/1-2 from the Deutsche Forschungsgemeinschaft and by Grant ZKF-B1 from the Zentrum für Klinische Forschung of the University of Freiburg.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. Tel.:
49-761-2036623; Fax: 49-761-2036626; E-mail:
kochs{at}SKL1.UKL.uni-freiburg.de.
The abbreviations used are:
VSV, vesicular
stomatitis virus; FLUAV, influenza A virus; THOV, Thogoto virus; DHOV, Dhori virus; GTPS, guanosine 5'-O-(3-thiotriphosphate); GDP
S, guanosine 5'-O-2-(thiodiphosphate); NP, nucleoprotein; mAb, monoclonal antibody; RNP, ribonucleoprotein
complex; vRNP, viral ribonucleoprotein complex; vRNA, viral RNA.
2 G. Kochs, U. Aebi, and O. Haller, unpublished results.
3 G. Kochs, unpublished results.
4 G. Kochs and M. Schwemmle, unpublished results.
5 G. Kochs and O. Haller, manuscript in preparation.
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REFERENCES |
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