Feb 6, 1990 - Resistance to Influenza Virus and Vesicular Stomatitis Virus. Conferred by Expression of Human MxA Protein. JOVAN PAVLOVIC, THOMAS ...
Vol. 64, No. 7
JOURNAL OF VIROLOGY, July 1990, p. 3370-3375
0022-538X/90/073370-06$02.00/0 Copyright © 1990, American Society for Microbiology
Resistance to Influenza Virus and Vesicular Stomatitis Virus Conferred by Expression of Human MxA Protein JOVAN PAVLOVIC, THOMAS ZURCHER, OTTO HALLER,t AND PETER STAEHELI* Institut fur Immunologie und Virologie, Universitat Zurich, Gloriastrasse 30, CH-8028 Zurich, Switzerland Received 6 February 1990/Accepted 28 March 1990
MxA and MxB are interferon-induced proteins of human cells and are related to the murine protein Mxl, which confers selective resistance to influenza virus. In contrast to the nuclear murine protein Mxl, MxA and MxB are located in the cytoplasm, and their role in the interferon-induced antiviral state was unknown. In this report we show that transfected cell lines expressing MxA acquired a high degree of resistance to influenza A virus. Surprisingly, MxA also conferred resistance to vesicular stomatitis virus. Expression of MxA in transfected 3T3 cells had no effect on the multiplication of two picornaviruses, a togavirus, or herpes simplex virus type 1. Treatment of MxA-expressing cells with antibodies to mouse alpha-beta interferon did not abolish the resistance phenotype. The conclusion that resistance to influenza virus and vesicular stomatitis virus was due to the specific action of MxA is further supported by the observation that transfected 3T3 cell lines expressing the related MxB failed to acquire virus resistance.
nuclear murine protein Mxl, human MxA (1, 29) and MxB (1) both accumulated in the cytoplasm of IFN-treated cells. To investigate the antiviral potentials of the human Mx proteins, we have prepared stable lines of transfected mouse 3T3 cells that constitutively express either human MxA or MxB. In this report, we show that MxA confers a high degree of resistance to influenza A virus and to VSV, but does not interfere with the multiplication of two picornaviruses, a togavirus, or herpes simplex virus type 1 (HSV-1). Transfected 3T3 cells expressing MxB remained susceptible to all viruses that we tested.
Resistance to a large number of RNA and DNA viruses can be induced by treating vertebrate cells with interferons (IFNs). This transient antiviral state is thought to result from the concerted action of a large number of IFN-induced proteins (24, 26). Defining the relative contributions of individual IFN-induced proteins is a formidable task. To date, the physiological roles of only a few IFN-induced proteins have been resolved (22, 26). Genetic and molecular evidence indicated that the IFNinduced murine protein Mxl has intrinsic antiviral activity with selectivity for influenza viruses: constitutive expression in transfected mouse 3T3 cells of a cDNA encoding murine Mxl led to inhibition of influenza virus, but had no effect on vesicular stomatitis virus (VSV) (31). Similarly, constitutive expression in transfected CHO cells of a cDNA encoding IFN-induced human 2-5A synthetase conferred resistance to picornaviruses, but did not make the cells resistant to VSV or herpes simplex virus (HSV) (5). Circumstantial evidence suggested that the IFN-induced protein kinase P1 is responsible for the translation at reduced rates in IFN-treated cells of mRNAs from many different viruses (14, 24, 26). Inhibition of VSV in IFN-treated cells is likely to be caused by more than one IFN-induced protein: viral mRNA translation (18) but also primary transcription of VSV (3) was reduced in IFN-treated cells. The IFN-induced proteins principally responsible for these observed inhibitory effects have not yet been identified. In the murine system, IFN-mediated inhibition of influenza virus is efficient only in cells from animals carrying a functional Mxl gene (30). The mechanism by which Mxl inhibits the multiplication of influenza virus remains controversial (15, 20). The induced proteins responsible for inhibition of influenza virus in IFN-treated human cells were not identified to date. We have previously described two IFN-induced proteins of human cells, called MxA and MxB, that showed high sequence similarities to mouse Mxl (1). In contrast to the *
MATERIALS AND METHODS IFNs and IFN treatments. Recombinant human IFN-aX2 (108 U/mg) was a gift from Biogen SA, Geneva, Switzerland. Mouse IFN-a/x (1.3 x 107 U/mg) was purchased from Lee Biomolecular Research Inc., San Diego, Calif. Confluent cell monolayers were treated with 1,000 U of IFN per ml in culture medium for 18 h prior to protein extraction. Antibodies to IFN. Sheep anti-mouse IFN-a/3 antibodies were a gift from Ion Gresser, Institut de la Recherche Scientifique sur le Cancer, Villejuif, France. Cell cultures were grown for 7 days (two passages) in the presence of 1.6 x 104 neutralizing units of anti-mouse IFN-ac/, antibodies per ml (10). Cells. Swiss mouse 3T3 cells, the human glioblastoma cell line T98G, and BALB.A2G-Mx mouse embryo fibroblasts were grown in Dulbecco modified minimal essential medium containing 10% fetal calf serum. Viruses. Stocks of the FPV-B strain (13) of influenza A virus (3 x 108 PFU/ml), VSV serotype Indiana (2 x 108 PFU/ml), VSV serotype New Jersey (7 x 106 PFU/ml), encephalomyocarditis virus (EMC virus) (1.5 x 109 PFU/ ml), mengovirus (2 x 109 PFU/ml), Semliki Forest virus (SFV; 1.5 x 108 PFU/ml), and HSV-1 (3 x 106 PFU/ml) were prepared from supernatants of virus-infected Swiss 3T3
cells. Virus yield reduction assay. Confluent-cell monolayers were infected for 30 min at 25°C at a multiplicity of infection of 1 PFU per cell. The virus inoculum was removed by two washings with phosphate-buffered saline, and the cultures were incubated at 37°C in Dulbecco modified minimal essen-
Corresponding author.
t Present address: Abteilung Virologie, Institut fur Medizinische Mikrobiologie und Hygiene, Klinikum der Albert-Ludwigs-Universitat, D-7800 Freiburg, Federal Republic of Germany. 3370
VOL. 64, 1990
tial medium containing 10% fetal calf serum. Samples of the culture supernatants were removed 12, 24, and 36 h postinfection, and the virus titers were determined on 3T3 cells by the 50% tissue culture infective dose method. Virus plaque assay. Confluent-cell monolayers were infected at 37°C for 1 h, the virus inoculum was removed, medium containing 2% fetal calf serum and 0.4% Noble agar was added, and the cultures were incubated at 37°C for 36 h. The agar overlay was removed, and the cells were stained with 1% crystal violet in 20% ethanol. Construction of pHMG-Mx expression vectors. The following Mx cDNA fragments were isolated and blunted with T4 polymerase: for murine Mxl, the 2.2-kilobase BamHIBamHI fragment (positions 147 to 2314 [31]) of plasmid pHG327Mx (21); for human MxA, the 2.2-kilobase EcoRISmaI fragment (positions 1 to 2253 [1]); and for human MxB, the 2.9-kilobase BanI-BamHI fragment (position 55 to 3' end [1]). The Mx cDNA fragments were cloned in the proper orientation into the unique EcoRV site of plasmid pCL642 (9), which contains the promoter region, the first noncoding exon, the complete first intron, and the splice acceptor site of the second exon of the murine 3-hydroxy-3-methylglutaryl coenzyme A reductase gene. This plasmid was kindly provided by R. Lathe, Institut de Chimie Biologique, University of Strasbourg, Strasbourg, France. Transfection. Swiss mouse 3T3 cells were transfected with pSV2neo (25) and the Mx cDNA constructs by the calcium phosphate coprecipitation method as previously described (31). Transfected clones were selected in medium containing 500 ,ug of G418 per ml. Resistant clones were examined for Mx expression by indirect immunofluorescence (8), and positive clones were subjected to a second round of subcloning by limiting dilution. Immunofluorescence analysis. Cells were prepared for immunofluorescence analysis as previously described (8). The antisera were diluted 1:50 in phosphate-buffered saline containing 5% normal goat serum. Rhodamine-conjugated goat anti-rabbit immunoglobulin G and goat anti-mouse immunoglobulin G (Nordic; diluted 1:50 in phosphate-buffered saline containing 5% normal goat serum) were used as secondary antibodies. Western blot analysis. Protein extracts were prepared by lysing cells in sample buffer (16). Protein was loaded (200 ,ug per lane) onto 10% polyacrylamide-sodium dodecyl sulfate gels (16). Western immunoblot analysis was performed as previously described (1). RESULTS Permanently transfected cell lines that constitutively express Mx proteins. cDNAs encoding mouse Mxl (31), human MxA (1), or human MxB (1) were cloned downstream of the promoter of the murine 3-hydroxy-3-methylglutaryl coenzyme A reductase gene (9). The resulting DNA constructs were cotransfected with plasmid pSV2neo (25) into Swiss 3T3 mouse cells. These cells were used because they are derived from an influenza virus-susceptible Mx- mouse strain lacking functional Mx genes and therefore are unable to synthesize endogenous Mx proteins (27, 28). Stably transfected cell clones were produced by culturing the cells in medium containing the drug G418. Individual clones were tested for Mx protein expression by indirect immunofluorescence analysis with specific antibodies (1, 27). Clones expressing Mx proteins were obtained at high frequency (up to 50% of the initial G418-resistant cell population). The primary cell clones were subcloned by limiting dilution, and
ANTIVIRAL ACTIVITY OF MxA
3371
FIG. 1. Localization of Mx proteins in transfected Swiss 3T3 cells by indirect immunofluorescence. Control 3T3-SV2neo cells (a) or clonal isolates of transfected 3T3 cells expressing human MxA (b), human MxB (c), or murine Mxl (d) were immunostained with mouse antiserum to a ,-galactosidase-MxA fusion protein (1) (panels a and b), mouse antiserum to a ,-galactosidase-MxB fusion protein (1) (panel c), or rabbit antiserum to a synthetic peptide consisting of the 16 C-terminal amino acids of mouse Mxl (AP5) (19)
(panel d).
stable lines expressing mouse Mxl (3T3-mMxl), human MxA (3T3-hMxA), or human MxB (3T3-hMxB) at a uniform level in close to 100% of the cell population were selected (Fig. 1). Mouse Mxl accumulating in the nucleus of the transfected 3T3 cells (Fig. ld) gave a punctate staining pattern that is typical for this protein (8). Human MxA and MxB both accumulated in the cytoplasm of transfected 3T3 cells (Fig. lb and c). MxA gave a granular staining pattern, whereas MxB stained more uniformly. Similar MxA and MxB staining patterns were observed in human cells treated with IFN-a2 (1, 11, 29). No distinctive immunostaining was observed when 3T3 cells transfected with pSV2neo alone were treated with antibodies to MxA (Fig. la), Mxl, or MxB (results not shown). Constitutive expression of Mx proteins did not significantly affect the growth rates of the transfected 3T3 cell lines. To estimate the relative concentrations of Mx proteins produced by the transfected cells, we prepared Western blots from whole-cell extracts of representative cell clones (Fig. 2). The three independent 3T3-mMxl cell lines contained about as much Mxl as did Mx+ mouse embryo fibroblasts (31) that were treated with IFN-aIp (Fig. 2a). The concentrations of MxA protein in the three independent 3T3-hMxA cell lines (Fig. 2b) and of MxB protein in the three independent 3T3-hMxB cell lines (Fig. 2c) were comparable to that in human T98G cells treated with IFN-a2. The additional bands in Fig. 2 are due to proteolytic degradation of MxA (Fig. 2b) or to cross-reacting antibodies in the polyclonal antisera to Mxl and MxB (Fig. 2a and c). Northern (RNA) blotting experiments showed that the Mx mRNAs of 3T3-mMxl, 3T3-hMxA, and 3T3-hMxB had the predicted sizes, indicating that the transcripts originating from the
J. VIROL.
PAVLOVIC ET AL.
3372
z
z
LL
IL
x
x c )C) iD cq 3T3-mMxl
Z
z N 3T3-hMxA >
._
kDa -. 20m 200
Co
N
m
c,
tL)
c)
c 3T3-hMx3
U-
a)
E-N
co
co
m'-
C
' CO
10C'
0 7) c
c
kDa
c
kD
c
C'
_~
200 -_
-*
-*
1 16 -_ 97 -_.
97 _. W.-.4allow
oovw
- ..
-0*-
MXJ
1__r-. w
66
66 -_-
42
z
(50F
.) cx
LL) o
c
Z
-
(D (3 C; co co
CM
200 116
0
0
ai)
6
A-
a
a
MxA
66 -_
42 -_
-_
4m
116 -_ 97 -_.
42 -_
b
c
FIG. 2. Western blot analysis of Mx proteins in transfected Swiss 3T3 cells. Western blots of 10% polyacrylamide-sodium dodecyl sulfate gels with cell extracts (200 ,ug of protein per lane) of the indicated 3T3-mMxl, 3T3-hMxA, and 3T3-hMxB cell clones were immunostained with (a) antiserum AP5, (b) antiserum to 0-galactosidase-MxA fusion protein or (c) antiserum to P-galactosidase-MxB fusion protein. Extracts of 3T3-SV2neo cells, BALB.A2G-Mx mouse embryo fibroblasts, or T98G cells treated with IFN (+) or left untreated (-) were included as controls. The positions of protein size markers (in kilodaltons [kDa]) are indicated.
transfected Mx cDNA constructs were spliced and processed correctly (results not shown). VSV and influenza virus replication is inhibited in cells expressing human MxA. To assess the antiviral state of the various cell lines, we infected confluent-cell monolayers with viruses representing five different families, namely, Orthomyxoviridae (influenza A virus FPV-B), Rhabdoviridae (VSV serotype Indiana), Picornaviridae (mengovirus and EMC virus), Togaviridae (SFV), and Herpesviridae (HSV-1). All viruses that we used were cytopathic and, except for SFV, lysed susceptible 3T3-SV2neo cells completely within 24 h after infection at 1 PFU per cell. Samples of individual culture supernatants were removed at 12, 24, and 36 h postinfection and assayed for infectivity. For influenza virus, the titers were maximal at 12 and 24 h postinfection and decreased thereafter. The virus yields of VSV, EMC virus, mengovirus, and SFV reached a plateau between 12 and 24 h. HSV-1 titers were maximal 24 h postinfection and had decreased significantly by 36 h. Importantly, the differences in viral titers among individual cell lines remained fairly constant over the whole observation period. Titers of the samples taken at 24 h postinfection are shown in Fig. 3. 3T3-mMxl cell lines expressing the nuclear Mxl showed a high degree of resistance to influenza virus: the viral titers at 24 h postinfection were 300- to 500-fold lower than the titers of the control 3T3-SV2neo cultures (Fig. 3). All our 3T3mMxl cell lines were fully susceptible to the other viruses that were tested: similar titers of VSV, HSV-1, EMC virus, mengovirus, and SFV were measured in the culture supernatants of infected 3T3-mMxl cells and of control 3T3SV2neo cells (Fig. 3). These results confirmed the conclusion of earlier experiments, using less stable cell lines, that mouse Mxl confers selective resistance to influenza viruses (31).
All 3T3-hMxA cell lines expressing the cytoplasmic huMxA showed a high degree of resistance to influenza virus: the viral titers in the 24-h supernatants of the three 3T3-hMxA cell lines studied here were between 100- and 200-fold lower than those of the control 3T3-SV2neo cultures (Fig. 3). Unexpectedly, these cell lines also exhibited a high degree of resistance to infection with VSV. MxA-expressing 3T3-hMxA cells infected with VSV yielded several hundredfold less virus than control 3T3-SV2neo cells (Fig. 3). A similar result was obtained when VSV serotype New Jersey (32) was used as the challenge virus: the MxA-expressing cell line 3T3-hMxA-4.5.15 produced 150-fold less virus than did control 3T3-SV2neo cells (results not shown). Double immunofluorescence analysis of 3T3-hMxA cells infected with VSV or influenza virus revealed that a very small percentage of MxA-expressing cells (fewer than 1%) produced viral proteins at 6 h postinfection, whereas under the same conditions more than 90% of the control 3T3-SV2neo cells produced VSV or influenza virus proteins (results not shown). Thus, the majority of the MxA-expressing cells seemed to resist VSV and influenza virus infection completely. In a few cells of the culture, the virus appeared to escape the control by MxA protein and replicated unhindered. The multiplication of mengovirus, EMC virus, HSV1, and SFV was not affected by human MxA (Fig. 3). In contrast to the cells expressing human MxA, those expressing the cytoplasmic human MxB were fully susceptible to all viruses tested, including influenza virus and VSV (Fig. 3). All of our transfected cell lines remained responsive to mouse IFN-oW/, (results not shown). To rule out the possibility that MxA induces low levels of endogenous IFN which, in turn, would render the cells resistant to VSV, we grew 3T3-SV2neo, 3T3-hMxA, and 3T3-hMxB cells for 7 days in the presence of polyclonal antibodies to murine man
ANTIVIRAL ACTIVITY OF MxA
VOL. 64, 1990
108
.......................................................................................
~~~HSV-1
1
l.., 106
106
3373
..
...
.,.,.,. ..,
_ ,,-... , , ..... ., :. , .- ,.... .:.
.. .... ..;...
.
....
....
....
...
.... ..
....
...
104
E
a
_
_ _ _.1 _---V1-' _.
_
_
_
a_
Is _
0
LO~
1-.
H
8
1 cn
1':':'
...::
....
..1':::
l
l '.1.... 1l'.-. 1Z. '. '.l1'. . 1l :"':"Z
.,)
cn
C') .V 1 0 1 c0
Mengovirus
109 108
........................................................................................
.~~~~~~~..
... .. ..
io6 I
1o5
....
....
10 7
...
... ..
....
I 1
0
104 3T3-SV2neo 3T3-mMxl 3T3-hMxA 3T3-hMxB
3T3-SV2neo 3T3-mMxl
3T3-hMxA 3T3-hMxB
FIG. 3. Inhibition of virus multiplication by Mx proteins. Two pools of pSV2neo-transfected 3T3 cells and independent clonal lines of 3T3-mMxl (3.5.2, 1.7.6, and 1.23.3), 3T3-hMxA (4.5.15, 3.5.5, and 3.12.11) or 3T3-hMxB (3.5.9, 3.46.7, and 3.38.3) cells were infected with 1 PFU of an influenza A virus (FPV-B), a rhabdovirus (VSV serotype Indiana), two picornaviruses (EMC virus [ECMV] and mengovirus), a togavirus (SFV), or a herpesvirus (HSV-1) per cell. The viral titers in the culture supernatants at 24 h postinfection are plotted. TCID50, 50% tissue culture infective dose.
IFN-ac/ (10) prior to infection with VSV. 3T3-hMxA cells subjected to this treatment remained persistent to VSV (Fig. 4). The concentration of the antibody was sufficient to neutralize at least 100 U of exogenous IFN-ot/ per ml, since the addition of 100 U of mouse IFN-o/J3 per ml 18 h prior to infection with VSV did not reduce the virus yields (Fig. 4). Furthermore, 2'-5'-oligoadenylate synthetase activity in 3T3-hMxA cells did not exceed background levels as expected for nonstimulated cells, whereas treatment of 3T3hMxA or of control 3T3-SV2neo cells with IFN-ox/ led to an approximately 50-fold increase in 2'-5'-oligoadenylate synthetase activity (results not shown). These data confirmed that human MxA protein possesses intrinsic anti-VSV and anti-influenza virus activity. To further demonstrate the antiviral potentials of murine Mxl and human MxA, monolayer cultures of 3T3 cells expressing the different Mx proteins were infected with about 50 PFU of influenza A virus, VSV, or SFV per 60-mm culture dish and the viruses were allowed to form plaques under soft agar. As expected from the titer determination data presented above, influenza virus failed to form plaques on 3T3-mMxl and on 3T3-hMxA cultures, but grew on
E
or80
10 5 3T3-SV2neo
3T3-hMxA
3T3-hMxB
FIG. 4. Anti-IFN-a3/, antibodies have no effect on the resistance of 3T3-hMxA cells against VSV. Cultures of control 3T3-SV2neo cells and of a clonal line of 3T3-hMxA (4.5.15) and 3T3-hMxB (3.46.7) cells were grown in either the presence (-) or absence (0) of 1.6 x 104 neutralizing units of sheep anti-mouse IFN-oa/I antibodies (10) per ml for 7 days prior to infection with VSV at 1 PFU per cell. At day 7, 100 U of mouse IFN-oa/1 per ml was added to one set of the antibodytreated cultures (El). At 18 h later these cultures were infected with VSV. Viral titers in the cell supematants at 24 h postinfection were determined. TCID50, 50% tissue culture infective dose.
3374
J. VIROL.
PAVLOVIC ET AL.
E - - - - -y-s - - -.
O.
.
vs
/
...
...
.
.
.
.
Monolayer cultures of control 3T3-SV2neo cells, 3T3-mMxl-7.23.3 cells expressing murine Mxl, 3T3-hMxA-4.5.15 cells expressing human MxA, or 3T3-hMxB-3.46.7 cells expressing human MxB were
infected with about 50 PFU of influenza virus, VSV,
and the viruses
were
or
SFV,
allowed to form plaques under soft agar.
3T3-hMxB and control 3T3-SV2neo cultures (Fig. 5). VSV formed
plaques
Mxl
human MxB
on
cultures of 3T3 cells
cultures of 3T3
protein, but failed cells expressing human
formed
on
or
plaques
all cell lines that
we
expressing murine plaques on MxA protein. SFV to form
studied.
DISCUSSION We have demonstrated that
expression of the cytoplasmic a high degree
human MxA in transfected 3T3 cells conferred
of resistance to influenza A virus and to the rhabdovirus
VSV, whereas the picornaviruses EMC virus and mengovithe togavirus SFV, and HSV-1 were not affected by
rus,
The cytoplasmic human MxB did not interfere with replication of any of the viruses that we tested. In contrast, the related mouse Mxl protein efficiently blocked of influenza A virus but had no detectable the effect on VSV or the other viruses listed above. MxA.
the
replication inhibitory
Selective inhibition of influenza virus also documented in
it has
previous
not been clear whether
interferon-induced antiviral
by mouse
Mxl
was
studies (2, 21, 31), but to date MxA
protein plays
a
role in the
Several indirect
experiments argued the notion that MxA has an intrinsic antiviral activity. of purified MxA failed to protect cells against influenza virus and VSV (33); however, it was not possible to prove that the purified protein was still active. Horisberger et al. (12) compared the decay kinetics of the antiviral state with that of MxA in IFN-treated human state.
against Microinjection
fibroblasts. Since the half-life
apparently longer and influenza virus, the authors postulated that MxA might not play a role in the IFN-induced antiviral state. In contrast to these
than
that of
the antiviral
of MxA
was
state against VSV
indirect
experimental approaches, our experiments with cells expressing MxA permitted us to define its antiviral potential. An interesting aspect of our is that human MxA
transfected
findings
and
Mxl both
strongly interfered with the replication of influenza A although these proteins accumulated in different cell compartments, raising the question of whether these proteins have distinct modes of action. Indeed, micromouse
virus,
injection
of
a
monoclonal antibody
that
recognizes
a
com-
mon epitope of human MxA and mouse and rat Mx proteins did not affect the resistance phenotype of IFN-treated human cells (32), whereas the same antibody neutralized the IFN-induced antiviral state against influenza virus in mouse or rat cells (2). The fact that MxA but not Mxl inhibited the multiplication of VSV would further argue in favor of basically different mechanisms. It is also conceivable that the two Mx proteins act similarly and that VSV escapes the action of the nuclear Mxl only because its entire replication cycle takes place in the cytoplasm (32). Alternatively, MxA might block a distinct virus multiplication step that takes place in the cytoplasm, whereas mouse Mxl might block a different step of the influenza virus replication cycle that takes place in the nucleus. If the two Mx proteins indeed had identical modes of action, we would expect that cytoplasmic forms of mouse Mxl would inhibit both influenza virus and VSV. In support of this notion, indirect immunofluorescence analysis indicated that the subpopulation of IFN-treated mouse cells containing Mxl predominantly in the cytoplasm was resistant to infection with influenza A virus (21). However, a series of Mxl variants deprived of their nuclear localization signal (and therefore accumulating in the cytoplasm) had no antiviral activity against either influenza A virus or VSV (21; T. Zurcher, unpublished results). Since it is unknown whether the amino acids that constitute the nuclear transport signal of Mxl are part of a domain necessary for antiviral function, there is no definite proof that mouse Mxl variants can act in the cytoplasm. We also cannot exclude the formal possibility that small but significant amounts of MxA accumulated in the cell nucleus and that the observed inhibitory effect on influenza virus was due to the action of MxA in the nucleus. In the case of influenza virus infection of cells expressing Mxl and in the cases of influenza virus or VSV infections of cells expressing MxA, viral proteins were not found in significant quantities at 6 h postinfection, suggesting inhibition of some early virus multiplication step(s). There is no agreement on whether Mxl from IFN-treated mouse cells blocks influenza virus at a step before or at primary transcription (15) or prevents influenza virus mRNA translation (20). By using our transfected 3T3 cell lines, it should now be possible to unambiguously identify the viral replication steps susceptible to Mxl and MxA action. The N-terminal moieties of murine, human, and fish Mx proteins show a high degree of sequence conservation (1), suggesting that these regions constitute a functional Mx protein domain. Recently, we and others (12, 26) observed that the conserved regions contain sequence motifs which are found in most GTP-binding proteins (4, 6, 7, 17, 23). In all Mx proteins characterized to date, these motifs occurred in the expected order and with typical spacing, suggesting that Mx proteins possess GTP-binding activity. The nature of the putative guanine nucleotide-dependent biochemical activity of Mx proteins and its functional importance remain to be established. In contrast to the N-terminal moieties, the amino acid sequences in the C-terminal moieties of Mx proteins differ considerably (1). The data presented here demonstrate that different Mx proteins can indeed have distinct biological activities, suggesting that the highly variable regions of Mx proteins constitute the domains which determine specificity. Human MxA and mouse Mxl have intrinsic antiviral activity, indicating that their physiological role is related to virus defense. In contrast, our virus inhibition tests did not yield any suggestions about the physiological function of human
ANTIVIRAL ACTIVITY OF MxA
VOL. 64, 1990
MxB. The derived view that Mx proteins might serve not
only antiviral but also cellular functions is supported by the recent observation that the VPSJ gene of the yeast Saccharomyces cerevisiae codes for a protein that is homologous to vertebrate Mx proteins (J. H. Rothman, C. K. Raymond, T. Gilbert, P. G. O'Hara, and T. H. Stevens, Cell, in press). ACKNOWLEDGMENTS We thank R. Lathe for providing plasmid pCL642, M. Schubert for providing VSV serotype New Jersey, M. Michel for providing SFV, G. Merlin for performing 2'-5'-oligoadenylate synthetase assays, T. Stevens for sharing unpublished sequence data, and E. Frohli and D. Bucher for excellent technical assistance. This work was supported by grants from the Swiss National Science Foundation and by the Canton of Zurich. LITERATURE CITED 1. Aebi, M., J. Fah, N. Hurt, C. E. Samuel, D. Thomis, L. Bazzigher, J. Pavlovic, 0. Haller, and P. Staeheli. 1989. cDNA structures and regulation of two interferon-induced human Mx proteins. Mol. Cell. Biol. 9:5062-5072. 2. Arnheiter, H., and 0. Haller. 1988. Antiviral state against influenza virus neutralized by microinjection of antibodies to interferon-induced Mx proteins. EMBO J. 7:1315-1320. 3. Belkowski, L. S., and G. C. Sen. 1987. Inhibition of vesicular stomatitis viral mRNA synthesis by interferons. J. Virol. 61:
15.
16. 17.
18.
19.
20. 21.
653-660. 4. Bernstein, H. D., M. A. Poritz, K. Strub, P. J. Hoben, S. Brenner, and P. Walter. 1989. Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle. Nature (London) 340:482-486. 5. Chebath, J., P. Benech, M. Revel, and M. Vigneron. 1987. Constitutive expression of (2'-5') oligo A synthetase confers resistance to picornavirus infection. Nature (London) 330:587-
588. 6. Connoily, T., and R. Gilmore. 1989. The signal-recognition particle receptor mediates the GTP-dependent displacement of SRP from the signal sequence of the nascent polypeptide. Cell 57:599-610. 7. Dever, T. E., M. J. Glynias, and W. C. Merrick. 1987. GTPbinding domain: three consensus sequence elements with distinct spacing. Proc. Natl. Acad. Sci. USA 84:1814-1818. 8. Dreiding, P., P. Staeheli, and 0. Haller. 1985. Interferoninduced protein Mx accumulates in nuclei of mouse cells expressing resistance to influenza viruses. Virology 140:192-
22. 23.
24. 25.
26. 27.
196. 9. Gautier, C., M. Mehtali, and R. Lathe. 1989. A ubiquitous mammalian expression vector, pHMG, based on a housekeeping gene promoter. Nucleic Acids Res. 17:8389. 10. Gresser, I., M. G. Tovey, M.-T. Bandu, C. Maury, and D. Brouty-Boye. 1976. Role of interferon in the pathogenesis of virus diseases in mice as demonstrated by the use of antiinterferon serum. I. Rapid evolution of encephalomyocarditis virus infection. J. Exp. Med. 144:1305-1315. 11. Horisberger, M. A., and H. K. Hochkeppel. 1987. IFN-a induced human 78 kD protein: purification and homologies with the mouse Mx protein, production of monoclonal antibodies, and potentiation effect of IFN--y. J. Interferon Res. 7:331-343. 12. Horisberger, M. A., G. K. McMaster, H. Zeller, M. G. Wathelet, J. Dellis, and J. Content. 1990. Cloning and sequence analyses of cDNAs for interferon- and virus-induced human Mx proteins reveal that they contain putative guanine nucleotide-binding sites: functional study of the corresponding gene promoter. J. Virol. 64:1171-1181. 13. Israel, A. 1979. Preliminary characterisation of the particles from productive and abortive infections of L cells by fowl plague virus. Ann. Microbiol. (Paris) 130B:85-100. 14. Kitajewski, J., R. J. Schneider, B. Safer, S. M. Munemitsu, C. E.
28.
29. 30.
31.
32. 33.
3375
Samuel, B. Thimmappaya, and T. Shenk. 1986. Adenovirus VAI RNA antagonizes the antiviral action of interferon by preventing activation of the interferon-induced eIF-2a kinase. Cell 45:195-200. Krug, R. M., M. Shaw, B. Broni, G. Shapiro, and 0. Haller. 1985. Inhibition of influenza viral mRNA synthesis in cells expressing the interferon-induced Mx gene product. J. Virol. 56:201-206. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. Lauffer, L., P. D. Garcia, R. N. Harkins, L. Coussens, A. Ulirich, and P. Walter. 1985. Topology of signal recognition particle receptor in endoplasmic reticulum membrane. Nature (London) 318:334-338. Masters, P. S., and C. E. Samuel. 1983. Mechanism of interferon action: inhibition of vesicular stomatitis virus replication in human amnion U cells by cloned human leukocyte interferon. J. Biol. Chem. 258:12026-12033. Meier, E., J. Fah, M. S. Grob, R. End, P. Staeheli, and 0. Hailer. 1988. A family of interferon-induced Mx-related mRNAs encodes cytoplasmic and nuclear proteins in rat cells. J. Virol. 62:2386-2393. Meyer, T., and M. A. Horisberger. 1984. Combined action of mouse a and ,B interferons in influenza virus-infected macrophages carrying the resistance gene Mx. J. Virol. 49:709-716. Noteborn, M., H. Arnheiter, L. Richter-Mann, H. Browning, and C. Weissmann. 1987. Transport of the murine Mx protein into the nucleus is dependent on a basic carboxy-terminal sequence. J. Interferon Res. 7:657-669. Revel, M., and J. Chebath. 1986. Interferon-activated genes. Trends Biochem. Sci. 11:166-170. Romisch, K., J. Webb, J. Herz, S. Prehn, R. Frank, M. Vingron, and B. Dobberstein. 1989. Homology of 54K protein of signalrecognition particle, docking protein and two E. coli proteins with putative GTP-binding domains. Nature (London) 340: 478-482. Samuel, C. E. 1988. Mechanisms of the antiviral action of interferons. Prog. Nucleic Acid Res. Mol. Biol. 35:27-72. Southern, P. J., and P. Berg. 1982. Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. J. Mol. Appl. Genet. 1:327-341. Staeheli, P. 1990. Interferon-induced proteins and the antiviral state. Adv. Virus. Res. 38:147-200. Staeheli, P., P. Dreiding, 0. Haller, and J. Lindenmann. 1985. Polyclonal and monoclonal antibodies to the interferon-inducible protein Mx of influenza virus-resistant mice. J. Biol. Chem. 260:1821-1825. Staeheli, P., R. Grob, E. Meier, J. G. Sutcliffe, and 0. Haller. 1988. Influenza virus-susceptible mice carry Mx genes with a large deletion or a nonsense mutation. Mol. Cell. Biol. 8: 4518-4523. Staeheli, P., and 0. Haller. 1985. Interferon-induced human protein with homology to protein Mx of influenza virus-resistant mice. Mol. Cell. Biol. 5:2150-2153. Staeheli, P., and 0. Haller. 1987. Interferon-induced Mx protein: a mediator of cellular resistance to influenza virus, p. 1-23. In I. Gresser (ed.), Interferon, vol. 8. Academic Press, Inc. (London), Ltd., London. Staeheli, P., 0. Haller, W. Boll, J. Lindenmann, and C. Weissmann. 1986. Mx protein: constitutive expression in 3T3 cells transformed with cloned Mx cDNA confers selective resistance to influenza virus. Cell 44:157-158. Wagner, R. 1987. Rhabdovirus biology and infection, p. 9-74. In R. Wagner (ed.), The rhabdoviruses. Plenum Publishing Corp., New York. Weitz, G., J. Bekisz, K. Zoon, and H. Arnheiter. 1989. Purification and characterization of a human Mx protein. J. Interferon Res. 9:679-689.