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Interferon-induced proteins: Identification of Mx proteins in various mammalian species
VIROLOGY
180, 185-190 (1991)
Interferon-Induced Proteins : Identification of Mx Proteins in Various Mammalian Species MICHEL A . HORISBERGER 1 AND MARIE C . GUNST Crba-Geigy Ltd., Biotechnology, CH-4002 Baste, Switzerland Received June 27, 1990, accepted September 13, 1990 Mx protein controls influenza virus pathogenicity in mice in vivo. It is an abundant protein synthesized in response to interferon-a/a . Searches for homolog proteins in various animal species have been conducted using several methods : (1) radioactive labeling of proteins induced by interferon and analysis by 2-D gel electrophoresis, (2) immunoprecipitation, (3) protoblot-ELISA analysis of cell-protein extracts, and (4) immunostaining of fixed cells . All mammalian species tested so far (including human, horse, and pig, which are prone to influenza virus infection) responded to interferon-a by synthesizing one or two Mx proteins differing in relative molecular weight and p1 . The expression of Mx proteins within one species differed sometimes between primary cultures of diploid cells and established cell lines . Mx proteins were detected in all species in the cytoplasm and, in some rodents only, also in the nucleus . This study indicates that the site of action of the Mx gene family might be cytoplasmic . The conservation and ubiquity of this family of proteins reflects a more general and essential cellular function than was initially considered . © 1991 Academic area s , Inc .
INTRODUCTION
1988 . Annual Meeting of the International Society for Interferon Research, Kyoto, Japan . Abstract 3-25) .
Mx protein, a protein originally discovered in A2G mice (Horisberger et al., 1983) controls influenza virus pathogenicity in mice in vivo . Mice expressing the Mx gene resist infection with influenza A and B viruses (Lindenmann, 1964), whereas mice lacking a functional Mx gene succumb to infection . The natural function of the Mx system in wild mice is not yet clear . Bovines are short-term carriers of influenza virus with no epidemiological consequences, and we have shown evidence that an efficient Mx system mediates this natural resistance (Horisberger, 1988) . Influenza A viruses infect a variety of avian and mammalian hosts, including humans, horses, and pigs . Among these species, Mx proteins have been described so far only in man (Staeheli and Haller, 1987 ; Horisberger and Hochkeppel, 1987) . Moreover Mx proteins can be used as markers for monitoring the biological activity of IFN in vivo (Horisberger and de Staritzky, 1989 ; Horisberger et at, 1990b ; Jakschies et al., 1990 ; von Wussow et al ., 1990) . A systematic survey of mammalian species for the identification of Mx proteins was therefore of general interest, The Mx proteins that we describe in the present communication have been characterized by inducibility with IFN-a, by immunological relatedness, relative mol wt, p1, and intracellular distribution . The intracellular distribution is discussed in relation to the possible functions of Mx proteins . A preliminary description of some of these results has previously been presented (Horisberger, M . A .
MATERIALS AND METHODS Interferon and cells The recombinant human interferon-a B/D hybrid (rHuIFN-a B/D) was produced in yeast by recombinant DNA technology, and purified by immunoaffinity chromatography to a purity of >95% . It comprises amino acids 1 to 60 from HuIFN-aB, 61 to 92 from HuIFNaD, and 93 to 166 from HuIFN-aB (and not 61 to 166 from HuIFN-aD as previously published ; Horisberger and de Staritzky, 1987) . The rHuIFN-a B/D has a broad host range, and its specific activity, as determined by plaque inhibition of vesicular stomatitis virus grown in human diploid cells, was 5 X 10 7 IU/mg (Horisberger and de Staritzky, 1987) . Chicken IFN was a generous gift from C . Jungwirth (WOrzburg) . Whenever possible, the target cells were diploid cells with a finite life span in tissue culture . They were obtained by digestion of tissues from embryonic or newborn animals with a neutral protease (Dispase, Boehringer-Mannheim) at pH 6 .7 in phosphate-buffered saline . All cells were propagated in Eagle's MEM supplemented with 10% fetal calf serum and antibiotics . Primary cells and established cell lines were subcultured using a solution of trypsin (1 :250) in EDTA (GIBCO) . A2G mice, homozygous for the gene Mx, were obtained from Olac (Bicester, England) . Immunological procedures Immunoprecipitation and immunostaining were performed as described previously (Horisberger, 1988),
To whom requests for reprints should be sent . 185
0042-6822/91 $3 .00 Copyright m 1991 by Academic
Press, Inc . All rights of reproduction in any form reserved .
1 86
HORISBERGER AND GUNST TABLE 1 CRITERIA FOR IDENTIFICATION OF
Identification of Mx hornologs MX
PROTEINS
1 . Induction by interferon-a 2 . Detection on 2-D gels (M,, p1) from radiolabeled cellular extracts 3 . Antigenic crossreaction (a) Immunoprecipitation (b) Western blot-ELISA 4 . Immunostaining of fixed cells
using either monospecific rabbit polyclonal antibodies to the murine Mx1 protein or monospecific mouse polyclonal antibodies to the human Mxl (or MxA) protein (Horisberger and Hochkeppel, 1985, 1987) .
Cultures were maintained in the continuous presence of the hybrid IFN-B/D (1000-3000 IU/ml) for 5 hr . Cultures were then washed with Hanks' balanced salt solution and incubated for 60 min with [ 35 S]methionine (25 ,Ci/ml) in the same buffer supplemented with 20 TM HEPES, pH 7 .4 . The labeling was terminated by washing the culture with cold phosphate saline buffer, the cells were lysed in 19 66 SDS, and the proteins were precipitated with ethanol . Analysis of 35 S-labeled proteins in the two-dimensional system was performed exactly as described (Horisberger et al., 1983) . Cultures not exposed to IFN were run in parallel as controls . Proteins accumulated
TABLE 2 IDENTIFICATION AND INTRACELLULAR LOCALIZATION
OF
Mx proteins
Cell species Human Embryonic foreskin diploid cells (Flow 4000) Glioblastoma T98G cell line Monkey Vero cell line (ATCC No . CCL 81) Marmoset newborn kidney diploid cells Pig Kidney diploid cells Kidney cell line PK-1 5 (ATCC No . CCL 33) Cattle Calf kidney diploid cells MDBK cell line (ATCC No . CCL 22) Sheep Choroid plexus cell line Horse Kidney diploid cells Dermis cell line NBL-6 (ATCC No . CCL 57) Dog Dog kidney diploid cells Cat Embryonic lung diploid cells Rabbit Embryonic diploid cells Hamster Embryonic diploid cells CHO ovary cell line (ATCC No . CCL 61) Guinea pig Embryonic diploid cells Rat Embryonic diploid cells Mouse A2G (Mx i-) embryonic diploid cells BALB/c (Mx-) embryonic diploid cells Birds Chick embryonic diploid cells a
From Aebi et al., 1989 .
IFN-a-INDUCED
MX
PROTEINS IN MAMMALS Localization
I (MxA) 2° (MxA, MxB)
Cytoplasm Cytoplasm
1
Cytoplasm Cytoplasm
2 2
Cytoplasm Cytoplasm
2 2
Cytoplasm Cytoplasm
1
Cytoplasm
2 2
Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm
1 (MIA2) 1 (MxA1)
Cytoplasm and nucleus Cytoplasm
2
Cytoplasm and nucleus
3
Cytoplasm and nucleus Cytoplasm and nucleus
0 ? (Not detected)
1 87
IFN-INDUCED Mx PROTEINS
RAT
HAMSTER CHO
embryonic cells
FIG. 1 . Induction of Mx proteins in rat and hamstercells . Monolayers of cells treated with 3000 IUiml of rIFN-a8/D for 5 hr (a, c, e) and of control cells (b, d, f) were pulse-labeled with [ 35 S]methlonine for 60 min . The fluorographs represent the radioactive proteins of whole-cell extracts separated in the two-dimensional system . The direction of separations were as indicated in the upper right corner of Fig . 2 . Arrows indicate the position of the IFN-induced Mx proteins . a, b, rat embryonic diploid cells ; c,d, Chinese hamster ovary (CHO) cells showing the induction of the acidic MxA1 protein, e,f, Chinese embryonic diploid cells showing the induction of the acidic MxA2 protein .
after 18 hr in the continuous presence of IFN were extracted as described above . They were separated by one-dimensional or two-dimensional gel electrophoresis, transferred onto nitrocellulose (Western blotting), and tested for their binding by monospecific polyclonal antibodies directed against the human or the murine Mx proteins, as described elsewhere (Horisberger and Hochkeppel, 1987) .
RESULTS AND DISCUSSION Members of a given family should share several features, and the criteria that we have used to identify Mx proteins are shown in Table 1 . Radioactive pulse labeling of cells treated for 5 hr with IFN and analysis of cell extracts by 2-D gel electrophoresis are a powerful method to detect de novo synthesis of IFN-induced Mx proteins . Immunoprecipitation of radioactive Mx proteins was used to confirm that no Mx protein had been masked by comigration with another cellular protein on
2-D gels . Results of immunoprecipitation are not shown in this paper, but examples can be found in our previous publications (Horisberger and Hochkeppel, 1987 ; Horisberger, 1988 ; Horisberger et al., 1 990b) . Western blot-ELISA analysis was used to detect minor Mx proteins which accumulated over a period of 18 hr and which would have been missed by the pulse-labeling analysis . Examples of this technique are not shown, but also can be found in the previous publications listed above . The primary cells and established cell lines from various mammalian species that we have used for this study are listed in Table 2 . The main characteristics of Mx proteins are also summarized in Table 2 . The mouse polyclonal antibodies to the human Mxt (or MxA) protein showed a broad interspecies cross-reactivity recognizing all Mx proteins described here, whereas the rabbit polyclonal antibodies to the mouse Mx1 protein had a limited cross-reactivity . Figure 1 shows typical protein patterns on 2-D gel electrophoresis, and Fig . 2 is a composite representa-
1 88
HORISBERGFR AND GUNST 92 .5 kD r
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M6
basic
• 0 0
62 .5 kD~
45 kD human
® A
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7 .0 T
•
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©
B
o o
0
0 horse
Q
O
Q A O
dog o
Q :o
0
0
cat
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Q j, eA1 0 o
•a
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0 guinea pig Mz1 •
0 rat
Q
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A2
0
0
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m
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bovine
C3 0
Al :
0
b
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•0 0
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_
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actin g
0
O
monkey
v
a
B
o
hamster
6 .4 5 .8 r r A
NEPHGE w , acidic o O w 0 O m
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Q
FIG . 2 . Relative position of IFN-induced Mx proteins from various mammals, separated in the two-dimensional system . The patterns are restricted to the region to where Mx proteins migrate . The direchon of separations by nonequllibrium pH gradient electrophoresis (NEPHGE) and by SDS-polyacrylamide gel electrophoresis (SDSPAGE) are shown in the upper right corner . tho positions of cellular proteins invariably found in all mammalian species are shown . Actin is indicated by a star . MxA refers to acidic Mx proteins . MxB refers to Mx proteins with a pl ; 7 .
tion of patterns restricted to the region to where Mx proteins migrated . In all mammalian species tested so far IFN-a induced the synthesis of one of two Mx proteins differing in relative mot wt (range 70 to 80 kDa) and in p/ (range 5 .8 to 7 .6) (Fig . 2) . The only exceptions were mice from strains sensitive to influenza virus infection (e .g ., Balb/c mice) . These mice fail to synthesize Mx proteins since either their Mx transcripts encode truncated proteins or the reading frame is interrupted by a nonsense mutation (Staeheli et al ., 1988) . We have been unable to identify an Mx protein in chick embryonic cells by the criteria listed in Table 1 . Other bird species have not been tested . Acidic Mx proteins were designated MxA1, MxA2, in the order of increasing p/ . Basic Mx proteins (p/ , 7) were designated MxB . This identification based on pl will help to relate Mx proteins described here with deduced amino
acid sequences from cDNA clones . All species had at least one MxA protein . Rat was the only species expressing three Mx proteins corresponding to three distinct mRNAs that have been cloned (Meier et al., 1990) . The two acidic proteins differ in only eight positions and by three charges, and they are clearly separated on 2-D gels (Fig . 1) . They are designated MxA1 and MxA2, corresponding respectively to the designation Mx3 and Mx2 by Meier etaL (1990) . We have observed that the rat MxA2 separated into two distinct spots on two-dimensional gels, indicating proteins of similar me[ wt, but differing in p/ (Fig . 1) . This could be explained by alternative splicing of the mRNA, alternative initiation of translation, or by post-translational modification of the rat MxA2 protein . The expression of Mx proteins within one species differed sometimes between primary cultures of diploid cells and established cell lines . For instance, primary cultures of hamster embryonic cells expressed only the MxA2 protein in response to IFN-a, whereas the CHO cell line expressed mainly the MxA1 protein (Fig . 1 and Table 2) . Expression of MxA2 in the CHO cell line was detected only on immunoblots of proteins accumulated during 18 hr of IFN treatment (not shown) . In human diploid cells IFN-a induces two distinct mRNAs, but only the MxA protein is expressed in fibroblasts, lymphocytes, and monocytes (Aebi etal., 1989 ; Horisberger et al ., 1990a) . The second mRNA encodes an MxB protein which has been shown to be expressed, together with MxA, in the glioblastoma cell line T98G (Aebi et al., 1989) . The position of MxB on 2-D gel (Fig . 2) has been determined after in vitro transcription and translation of the corresponding cDNA (Horisberger et al., 1990a) . A similar type of Mx gene regulation could occur in several mammals in which only one Mx protein has been detected to date . These examples indicate that the list in Table 2 is probably not complete . The intracellular distribution was examined by immunostaining of fixed cells using monospecific polyclonal antibodies . In all species Mx proteins were observed as a diffuse staining within the cytoplasm . The mouse Mx1 protein gave a distinct nuclear staining as well as a weaker but readily detectable cytoplasmic staining (Fig . 3) . Occasionally the mouse Mxl protein was found exclusively in the cytoplasm . Intranuclear localization of Mx proteins was observed only in some rodents, as a diffuse, rarely punctate, appearance (hamster MxA2, Fig . 3), or as a speckled appearance (mouse, rat, guinea pig) . This speckled appearance reflects perhaps an aggregated form of Mx proteins, as suggested earlier (Horisberger and Hochkeppel, 1985) . Nucleolar staining was excluded from these cells . Mx proteins may participate in a cellular defense
IFN-INDUCED Mx PROTEINS
-I
189
g-k- , ,
mouse cells
hamster diploid cells
FIG . 3 . Intracellular localization of IFN-induced Mx proteins n A2(3 Mouse and Chinese hamster diploid cells . Primary cultures of embryonic cells were either pretreated for 18 hr with 3000 IU/ml of rlFN-aB/D (a, c), or kept as control cells (b, d) . Fixation and permeabillzatlon of cells are described in the text . The immunostaining was performed using cross-reacting polyclonal antibodies directed against the human MxA protein, and a rabbit anti-mouse IgG conjugated witn percxidase as a second antibody .
mechanism against viruses other than influenza virus (Meier et al., 1990 ; Pavlovic et al., 1990) . All Mx proteins cloned so far have a putative GTP-binding site with a consensus sequence suggesting a role of these proteins in the vectorial transport of proteins (Horisberger et al., 1990a ; Meier et al., 1990) . Moreover, yeasts encode a protein that may be functionally equivalent to mammalian Mx proteins, and that is involved in vacuolar protein sorting (Rothman et al., 1990) . Mx proteins may therefore have a pleiotropic function localized in the cytoplasm . This would be in agreement with the proposed action of the mouse Mx protein against influenza virus which has been suggested to occur at the post-transcriptional/translational level (Meyer and
Horisberger, 1984) . The abundance of Mx protein upon IFN stimulation (Horisberger and Hochkeppel, 1987) suggests a stoichiometric interaction with cellular components . No Mx protein has yet been identified in chicken . Other birds which represent an important reservoir for influenza viruses should be tested . We have found no special features of Mx proteins in human, horse, and pig that might explain why these species are susceptible hosts for influenza virus . It will be important to understand the function of these proteins in susceptible hosts to design a strategy to prevent the spread of influenza viruses . There are conflicting reports as to whether the human MxA promotes resistance to influ-
190
HORISBERGER AND GUNST
enza infections . The MxA protein may have no or only a weak protective effect (Weitz of al., 1989) . Alternatively, the MxA protein may have a strong activity against influenza virus (Pavlovic et al., 1990) ; if this is indeed the case, it will be important to uncover the factors which allow the virus to break this mechanism of cellular resistance . ACKNOWLEDGMENTS We thank K . de Staritzkyfor technical assistance, and T . E . Bilham for critical reading of this manuscript .
REFERENCES AEBI, M ., FAH, J ., HURT, N ., SAMUEL, C . E ., THOMIS, D ., BAaIGHER, L ., PAVLOVIC, J ., HALLER, 0 ., and STAEHELI, P . (1989) . CDNA structure and regulation of two interferon-induced human Mx proteins . Mol. Cell. Biol. 9, 5062-5072 . HORISBERGER, M . A . (1988) . The action of recombinant bovine interforons on influenza virus replication correlates with the induction of two Mx-related proteins in bovine cells . Virology 162, 181-186 . HORISBERGER, M . A ., and DE STARITZKY, K . (1987) . A recombinant human interferon-a B/D hybrid with a broad host-range . J. Gen . Viral. 68,945-948 . HORISBERGER, M . A ., and HOCHKEPPEL, H . K . (1985) . The IFN-induced mouse protein Mx involved in the mechanism of resistance to influenza viruses : Purification and characterization . In "The Biology of the Interferon System 1984'' (H . Kirchner and H . Schellekens, Eds ), pp. 313--316 . Elsevier Science, Amsterdam . HORISOERGER, M . A ., and HOCHKEPPEL, H . K . (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 . HORISBERGER, M . A ., MCMASTER, G . K ., ZELLER, H ., WATHELET, M . G ., DELLIS, J ., and CONTENT, J . (1990a) . 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. Viral. 64, 1171-1181 . HORISBERGER, M . A., SCHRENK, R ., STAIGER, S ., LEYVRAZ, A . R ., and MARTINOO, S . (1990b) . Induction of Mx-related protein in cat peripheral blood mononuclear cells after administration of recombinant human interferon hybrid . Antiviral Res. 13, 53-60 .
HORISBERGER, M . A ., STAEHELI, P ., and HALLER, 0 . (1983) . Interferon induces a unique protein in mouse cells bearing a gene for resistance to influenza virus . Proc. Nail. Aced. Sci. USA 80, 19101914 . HORISBERGER, M . A ., and DE STARITZKY, K . (1989) . Expression and stability of the Mx protein in different tissues of mice, in response to interferon inducers or to influenza virus infection . .J. Interferon Res . 9, 583-590 . JAKSCHIES, D ., HOCHKEPPEL, H . K ., HORISBERGER, M . A ., DEICHER, H ., and voN Wussow, P . (1990) . Emergence and decay of the human Mx homolog in cancer patients during and after interferon-alpha therapy. J. Biol. Response Modif. 9, 305-312 . LINDENMANN, J . (1964) . Inheritance of resistance to influenza virus in mice . Pron . Sec. Exp . Biol . Med. 116, 506-509 . MEIER, E ., FAH, J ., GROB, M . S ., END, R ., STAEHELI, P ., and HALLER, 0 . (1988) . A family of interferon-induced Mx-related mRNAs encodes cytoplasmic and nuclear proteins in rat cells . J. Vlrol. 62, 23862393 . MEIER, E ., KuNZ, G ., HALLER, 0 ., and ARNHEITER, H . (1990) . Unexpected activity of rat Mx proteins against a rhabdovirus, submitted for publication . MEYER, T ., and HORISBERGER, M . A . (1984) . Combined action of mouse a and 0 interferons in influenza virus-infected macrophages carrying the resistance gene Mx . J. Viral. 49, 709-716 . PAVLOVIC, J ., ZURCHER, T., HALLER, C ., and STAEHELI, P. (1990) . Resistance to influenza and vesicular stomatitis virus conferred by expression of human MxA protein . 1. Viral. 64, 3370-3375 . ROTHMAN, J . H ., RAYMOND, C . K ., GILBERT, T ., O'HARA, P . J ., and STEVENS, T. H . (1990) . A putative GTP binding protein homologous to interferon-inducible Mx proteins performs an essential function in yeast protein sorting . Cell 61 1063-1074 . STAEHELI, P ., and HALLER, 0 . (1987) . Interferon-induced human protein with homology to protein Mx of influenza-resistant mice . Me[ Cell . Biol. 5, 2150-2153 . STAEHELI, P ., GROB, R ., MEIER, E ., SUTCLIFFE, J . G., and HALLER, 0 . (1988) . Influenza virus-susceptible mice carry Mx genes with a large deletion or a nonsense mutation . Mot. Cell. Riot 8, 45184523 . VON WUSSOW, P ., JAKSCHIES, D ., BLOCK, B ., TSCHECHNE, B ., SCHEDEL, I ., HORISBERGER, M . A ., HOCHKEPPEL, H . K ., and DEICHER, H . (1990) . The interferon-induced Mx-homologous protein in people with symptomatic HIV-1 infection . AIDS 4, 119-124 . WEITZ, G ., BEKISZ, J ., ZOON . K., and ARNHEITER, H . (1989) . Purification and characterization of a human Mx protein . .J. Interferon Res. 9, 679-689 .
180, 185-190 (1991)
Interferon-Induced Proteins : Identification of Mx Proteins in Various Mammalian Species MICHEL A . HORISBERGER 1 AND MARIE C . GUNST Crba-Geigy Ltd., Biotechnology, CH-4002 Baste, Switzerland Received June 27, 1990, accepted September 13, 1990 Mx protein controls influenza virus pathogenicity in mice in vivo. It is an abundant protein synthesized in response to interferon-a/a . Searches for homolog proteins in various animal species have been conducted using several methods : (1) radioactive labeling of proteins induced by interferon and analysis by 2-D gel electrophoresis, (2) immunoprecipitation, (3) protoblot-ELISA analysis of cell-protein extracts, and (4) immunostaining of fixed cells . All mammalian species tested so far (including human, horse, and pig, which are prone to influenza virus infection) responded to interferon-a by synthesizing one or two Mx proteins differing in relative molecular weight and p1 . The expression of Mx proteins within one species differed sometimes between primary cultures of diploid cells and established cell lines . Mx proteins were detected in all species in the cytoplasm and, in some rodents only, also in the nucleus . This study indicates that the site of action of the Mx gene family might be cytoplasmic . The conservation and ubiquity of this family of proteins reflects a more general and essential cellular function than was initially considered . © 1991 Academic area s , Inc .
INTRODUCTION
1988 . Annual Meeting of the International Society for Interferon Research, Kyoto, Japan . Abstract 3-25) .
Mx protein, a protein originally discovered in A2G mice (Horisberger et al., 1983) controls influenza virus pathogenicity in mice in vivo . Mice expressing the Mx gene resist infection with influenza A and B viruses (Lindenmann, 1964), whereas mice lacking a functional Mx gene succumb to infection . The natural function of the Mx system in wild mice is not yet clear . Bovines are short-term carriers of influenza virus with no epidemiological consequences, and we have shown evidence that an efficient Mx system mediates this natural resistance (Horisberger, 1988) . Influenza A viruses infect a variety of avian and mammalian hosts, including humans, horses, and pigs . Among these species, Mx proteins have been described so far only in man (Staeheli and Haller, 1987 ; Horisberger and Hochkeppel, 1987) . Moreover Mx proteins can be used as markers for monitoring the biological activity of IFN in vivo (Horisberger and de Staritzky, 1989 ; Horisberger et at, 1990b ; Jakschies et al., 1990 ; von Wussow et al ., 1990) . A systematic survey of mammalian species for the identification of Mx proteins was therefore of general interest, The Mx proteins that we describe in the present communication have been characterized by inducibility with IFN-a, by immunological relatedness, relative mol wt, p1, and intracellular distribution . The intracellular distribution is discussed in relation to the possible functions of Mx proteins . A preliminary description of some of these results has previously been presented (Horisberger, M . A .
MATERIALS AND METHODS Interferon and cells The recombinant human interferon-a B/D hybrid (rHuIFN-a B/D) was produced in yeast by recombinant DNA technology, and purified by immunoaffinity chromatography to a purity of >95% . It comprises amino acids 1 to 60 from HuIFN-aB, 61 to 92 from HuIFNaD, and 93 to 166 from HuIFN-aB (and not 61 to 166 from HuIFN-aD as previously published ; Horisberger and de Staritzky, 1987) . The rHuIFN-a B/D has a broad host range, and its specific activity, as determined by plaque inhibition of vesicular stomatitis virus grown in human diploid cells, was 5 X 10 7 IU/mg (Horisberger and de Staritzky, 1987) . Chicken IFN was a generous gift from C . Jungwirth (WOrzburg) . Whenever possible, the target cells were diploid cells with a finite life span in tissue culture . They were obtained by digestion of tissues from embryonic or newborn animals with a neutral protease (Dispase, Boehringer-Mannheim) at pH 6 .7 in phosphate-buffered saline . All cells were propagated in Eagle's MEM supplemented with 10% fetal calf serum and antibiotics . Primary cells and established cell lines were subcultured using a solution of trypsin (1 :250) in EDTA (GIBCO) . A2G mice, homozygous for the gene Mx, were obtained from Olac (Bicester, England) . Immunological procedures Immunoprecipitation and immunostaining were performed as described previously (Horisberger, 1988),
To whom requests for reprints should be sent . 185
0042-6822/91 $3 .00 Copyright m 1991 by Academic
Press, Inc . All rights of reproduction in any form reserved .
1 86
HORISBERGER AND GUNST TABLE 1 CRITERIA FOR IDENTIFICATION OF
Identification of Mx hornologs MX
PROTEINS
1 . Induction by interferon-a 2 . Detection on 2-D gels (M,, p1) from radiolabeled cellular extracts 3 . Antigenic crossreaction (a) Immunoprecipitation (b) Western blot-ELISA 4 . Immunostaining of fixed cells
using either monospecific rabbit polyclonal antibodies to the murine Mx1 protein or monospecific mouse polyclonal antibodies to the human Mxl (or MxA) protein (Horisberger and Hochkeppel, 1985, 1987) .
Cultures were maintained in the continuous presence of the hybrid IFN-B/D (1000-3000 IU/ml) for 5 hr . Cultures were then washed with Hanks' balanced salt solution and incubated for 60 min with [ 35 S]methionine (25 ,Ci/ml) in the same buffer supplemented with 20 TM HEPES, pH 7 .4 . The labeling was terminated by washing the culture with cold phosphate saline buffer, the cells were lysed in 19 66 SDS, and the proteins were precipitated with ethanol . Analysis of 35 S-labeled proteins in the two-dimensional system was performed exactly as described (Horisberger et al., 1983) . Cultures not exposed to IFN were run in parallel as controls . Proteins accumulated
TABLE 2 IDENTIFICATION AND INTRACELLULAR LOCALIZATION
OF
Mx proteins
Cell species Human Embryonic foreskin diploid cells (Flow 4000) Glioblastoma T98G cell line Monkey Vero cell line (ATCC No . CCL 81) Marmoset newborn kidney diploid cells Pig Kidney diploid cells Kidney cell line PK-1 5 (ATCC No . CCL 33) Cattle Calf kidney diploid cells MDBK cell line (ATCC No . CCL 22) Sheep Choroid plexus cell line Horse Kidney diploid cells Dermis cell line NBL-6 (ATCC No . CCL 57) Dog Dog kidney diploid cells Cat Embryonic lung diploid cells Rabbit Embryonic diploid cells Hamster Embryonic diploid cells CHO ovary cell line (ATCC No . CCL 61) Guinea pig Embryonic diploid cells Rat Embryonic diploid cells Mouse A2G (Mx i-) embryonic diploid cells BALB/c (Mx-) embryonic diploid cells Birds Chick embryonic diploid cells a
From Aebi et al., 1989 .
IFN-a-INDUCED
MX
PROTEINS IN MAMMALS Localization
I (MxA) 2° (MxA, MxB)
Cytoplasm Cytoplasm
1
Cytoplasm Cytoplasm
2 2
Cytoplasm Cytoplasm
2 2
Cytoplasm Cytoplasm
1
Cytoplasm
2 2
Cytoplasm Cytoplasm Cytoplasm Cytoplasm Cytoplasm
1 (MIA2) 1 (MxA1)
Cytoplasm and nucleus Cytoplasm
2
Cytoplasm and nucleus
3
Cytoplasm and nucleus Cytoplasm and nucleus
0 ? (Not detected)
1 87
IFN-INDUCED Mx PROTEINS
RAT
HAMSTER CHO
embryonic cells
FIG. 1 . Induction of Mx proteins in rat and hamstercells . Monolayers of cells treated with 3000 IUiml of rIFN-a8/D for 5 hr (a, c, e) and of control cells (b, d, f) were pulse-labeled with [ 35 S]methlonine for 60 min . The fluorographs represent the radioactive proteins of whole-cell extracts separated in the two-dimensional system . The direction of separations were as indicated in the upper right corner of Fig . 2 . Arrows indicate the position of the IFN-induced Mx proteins . a, b, rat embryonic diploid cells ; c,d, Chinese hamster ovary (CHO) cells showing the induction of the acidic MxA1 protein, e,f, Chinese embryonic diploid cells showing the induction of the acidic MxA2 protein .
after 18 hr in the continuous presence of IFN were extracted as described above . They were separated by one-dimensional or two-dimensional gel electrophoresis, transferred onto nitrocellulose (Western blotting), and tested for their binding by monospecific polyclonal antibodies directed against the human or the murine Mx proteins, as described elsewhere (Horisberger and Hochkeppel, 1987) .
RESULTS AND DISCUSSION Members of a given family should share several features, and the criteria that we have used to identify Mx proteins are shown in Table 1 . Radioactive pulse labeling of cells treated for 5 hr with IFN and analysis of cell extracts by 2-D gel electrophoresis are a powerful method to detect de novo synthesis of IFN-induced Mx proteins . Immunoprecipitation of radioactive Mx proteins was used to confirm that no Mx protein had been masked by comigration with another cellular protein on
2-D gels . Results of immunoprecipitation are not shown in this paper, but examples can be found in our previous publications (Horisberger and Hochkeppel, 1987 ; Horisberger, 1988 ; Horisberger et al., 1 990b) . Western blot-ELISA analysis was used to detect minor Mx proteins which accumulated over a period of 18 hr and which would have been missed by the pulse-labeling analysis . Examples of this technique are not shown, but also can be found in the previous publications listed above . The primary cells and established cell lines from various mammalian species that we have used for this study are listed in Table 2 . The main characteristics of Mx proteins are also summarized in Table 2 . The mouse polyclonal antibodies to the human Mxt (or MxA) protein showed a broad interspecies cross-reactivity recognizing all Mx proteins described here, whereas the rabbit polyclonal antibodies to the mouse Mx1 protein had a limited cross-reactivity . Figure 1 shows typical protein patterns on 2-D gel electrophoresis, and Fig . 2 is a composite representa-
1 88
HORISBERGFR AND GUNST 92 .5 kD r
MxA
M6
basic
• 0 0
62 .5 kD~
45 kD human
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FIG . 2 . Relative position of IFN-induced Mx proteins from various mammals, separated in the two-dimensional system . The patterns are restricted to the region to where Mx proteins migrate . The direchon of separations by nonequllibrium pH gradient electrophoresis (NEPHGE) and by SDS-polyacrylamide gel electrophoresis (SDSPAGE) are shown in the upper right corner . tho positions of cellular proteins invariably found in all mammalian species are shown . Actin is indicated by a star . MxA refers to acidic Mx proteins . MxB refers to Mx proteins with a pl ; 7 .
tion of patterns restricted to the region to where Mx proteins migrated . In all mammalian species tested so far IFN-a induced the synthesis of one of two Mx proteins differing in relative mot wt (range 70 to 80 kDa) and in p/ (range 5 .8 to 7 .6) (Fig . 2) . The only exceptions were mice from strains sensitive to influenza virus infection (e .g ., Balb/c mice) . These mice fail to synthesize Mx proteins since either their Mx transcripts encode truncated proteins or the reading frame is interrupted by a nonsense mutation (Staeheli et al ., 1988) . We have been unable to identify an Mx protein in chick embryonic cells by the criteria listed in Table 1 . Other bird species have not been tested . Acidic Mx proteins were designated MxA1, MxA2, in the order of increasing p/ . Basic Mx proteins (p/ , 7) were designated MxB . This identification based on pl will help to relate Mx proteins described here with deduced amino
acid sequences from cDNA clones . All species had at least one MxA protein . Rat was the only species expressing three Mx proteins corresponding to three distinct mRNAs that have been cloned (Meier et al., 1990) . The two acidic proteins differ in only eight positions and by three charges, and they are clearly separated on 2-D gels (Fig . 1) . They are designated MxA1 and MxA2, corresponding respectively to the designation Mx3 and Mx2 by Meier etaL (1990) . We have observed that the rat MxA2 separated into two distinct spots on two-dimensional gels, indicating proteins of similar me[ wt, but differing in p/ (Fig . 1) . This could be explained by alternative splicing of the mRNA, alternative initiation of translation, or by post-translational modification of the rat MxA2 protein . The expression of Mx proteins within one species differed sometimes between primary cultures of diploid cells and established cell lines . For instance, primary cultures of hamster embryonic cells expressed only the MxA2 protein in response to IFN-a, whereas the CHO cell line expressed mainly the MxA1 protein (Fig . 1 and Table 2) . Expression of MxA2 in the CHO cell line was detected only on immunoblots of proteins accumulated during 18 hr of IFN treatment (not shown) . In human diploid cells IFN-a induces two distinct mRNAs, but only the MxA protein is expressed in fibroblasts, lymphocytes, and monocytes (Aebi etal., 1989 ; Horisberger et al ., 1990a) . The second mRNA encodes an MxB protein which has been shown to be expressed, together with MxA, in the glioblastoma cell line T98G (Aebi et al., 1989) . The position of MxB on 2-D gel (Fig . 2) has been determined after in vitro transcription and translation of the corresponding cDNA (Horisberger et al., 1990a) . A similar type of Mx gene regulation could occur in several mammals in which only one Mx protein has been detected to date . These examples indicate that the list in Table 2 is probably not complete . The intracellular distribution was examined by immunostaining of fixed cells using monospecific polyclonal antibodies . In all species Mx proteins were observed as a diffuse staining within the cytoplasm . The mouse Mx1 protein gave a distinct nuclear staining as well as a weaker but readily detectable cytoplasmic staining (Fig . 3) . Occasionally the mouse Mxl protein was found exclusively in the cytoplasm . Intranuclear localization of Mx proteins was observed only in some rodents, as a diffuse, rarely punctate, appearance (hamster MxA2, Fig . 3), or as a speckled appearance (mouse, rat, guinea pig) . This speckled appearance reflects perhaps an aggregated form of Mx proteins, as suggested earlier (Horisberger and Hochkeppel, 1985) . Nucleolar staining was excluded from these cells . Mx proteins may participate in a cellular defense
IFN-INDUCED Mx PROTEINS
-I
189
g-k- , ,
mouse cells
hamster diploid cells
FIG . 3 . Intracellular localization of IFN-induced Mx proteins n A2(3 Mouse and Chinese hamster diploid cells . Primary cultures of embryonic cells were either pretreated for 18 hr with 3000 IU/ml of rlFN-aB/D (a, c), or kept as control cells (b, d) . Fixation and permeabillzatlon of cells are described in the text . The immunostaining was performed using cross-reacting polyclonal antibodies directed against the human MxA protein, and a rabbit anti-mouse IgG conjugated witn percxidase as a second antibody .
mechanism against viruses other than influenza virus (Meier et al., 1990 ; Pavlovic et al., 1990) . All Mx proteins cloned so far have a putative GTP-binding site with a consensus sequence suggesting a role of these proteins in the vectorial transport of proteins (Horisberger et al., 1990a ; Meier et al., 1990) . Moreover, yeasts encode a protein that may be functionally equivalent to mammalian Mx proteins, and that is involved in vacuolar protein sorting (Rothman et al., 1990) . Mx proteins may therefore have a pleiotropic function localized in the cytoplasm . This would be in agreement with the proposed action of the mouse Mx protein against influenza virus which has been suggested to occur at the post-transcriptional/translational level (Meyer and
Horisberger, 1984) . The abundance of Mx protein upon IFN stimulation (Horisberger and Hochkeppel, 1987) suggests a stoichiometric interaction with cellular components . No Mx protein has yet been identified in chicken . Other birds which represent an important reservoir for influenza viruses should be tested . We have found no special features of Mx proteins in human, horse, and pig that might explain why these species are susceptible hosts for influenza virus . It will be important to understand the function of these proteins in susceptible hosts to design a strategy to prevent the spread of influenza viruses . There are conflicting reports as to whether the human MxA promotes resistance to influ-
190
HORISBERGER AND GUNST
enza infections . The MxA protein may have no or only a weak protective effect (Weitz of al., 1989) . Alternatively, the MxA protein may have a strong activity against influenza virus (Pavlovic et al., 1990) ; if this is indeed the case, it will be important to uncover the factors which allow the virus to break this mechanism of cellular resistance . ACKNOWLEDGMENTS We thank K . de Staritzkyfor technical assistance, and T . E . Bilham for critical reading of this manuscript .
REFERENCES AEBI, M ., FAH, J ., HURT, N ., SAMUEL, C . E ., THOMIS, D ., BAaIGHER, L ., PAVLOVIC, J ., HALLER, 0 ., and STAEHELI, P . (1989) . CDNA structure and regulation of two interferon-induced human Mx proteins . Mol. Cell. Biol. 9, 5062-5072 . HORISBERGER, M . A . (1988) . The action of recombinant bovine interforons on influenza virus replication correlates with the induction of two Mx-related proteins in bovine cells . Virology 162, 181-186 . HORISBERGER, M . A ., and DE STARITZKY, K . (1987) . A recombinant human interferon-a B/D hybrid with a broad host-range . J. Gen . Viral. 68,945-948 . HORISBERGER, M . A ., and HOCHKEPPEL, H . K . (1985) . The IFN-induced mouse protein Mx involved in the mechanism of resistance to influenza viruses : Purification and characterization . In "The Biology of the Interferon System 1984'' (H . Kirchner and H . Schellekens, Eds ), pp. 313--316 . Elsevier Science, Amsterdam . HORISOERGER, M . A ., and HOCHKEPPEL, H . K . (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 . HORISBERGER, M . A ., MCMASTER, G . K ., ZELLER, H ., WATHELET, M . G ., DELLIS, J ., and CONTENT, J . (1990a) . 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. Viral. 64, 1171-1181 . HORISBERGER, M . A., SCHRENK, R ., STAIGER, S ., LEYVRAZ, A . R ., and MARTINOO, S . (1990b) . Induction of Mx-related protein in cat peripheral blood mononuclear cells after administration of recombinant human interferon hybrid . Antiviral Res. 13, 53-60 .
HORISBERGER, M . A ., STAEHELI, P ., and HALLER, 0 . (1983) . Interferon induces a unique protein in mouse cells bearing a gene for resistance to influenza virus . Proc. Nail. Aced. Sci. USA 80, 19101914 . HORISBERGER, M . A ., and DE STARITZKY, K . (1989) . Expression and stability of the Mx protein in different tissues of mice, in response to interferon inducers or to influenza virus infection . .J. Interferon Res . 9, 583-590 . JAKSCHIES, D ., HOCHKEPPEL, H . K ., HORISBERGER, M . A ., DEICHER, H ., and voN Wussow, P . (1990) . Emergence and decay of the human Mx homolog in cancer patients during and after interferon-alpha therapy. J. Biol. Response Modif. 9, 305-312 . LINDENMANN, J . (1964) . Inheritance of resistance to influenza virus in mice . Pron . Sec. Exp . Biol . Med. 116, 506-509 . MEIER, E ., FAH, J ., GROB, M . S ., END, R ., STAEHELI, P ., and HALLER, 0 . (1988) . A family of interferon-induced Mx-related mRNAs encodes cytoplasmic and nuclear proteins in rat cells . J. Vlrol. 62, 23862393 . MEIER, E ., KuNZ, G ., HALLER, 0 ., and ARNHEITER, H . (1990) . Unexpected activity of rat Mx proteins against a rhabdovirus, submitted for publication . MEYER, T ., and HORISBERGER, M . A . (1984) . Combined action of mouse a and 0 interferons in influenza virus-infected macrophages carrying the resistance gene Mx . J. Viral. 49, 709-716 . PAVLOVIC, J ., ZURCHER, T., HALLER, C ., and STAEHELI, P. (1990) . Resistance to influenza and vesicular stomatitis virus conferred by expression of human MxA protein . 1. Viral. 64, 3370-3375 . ROTHMAN, J . H ., RAYMOND, C . K ., GILBERT, T ., O'HARA, P . J ., and STEVENS, T. H . (1990) . A putative GTP binding protein homologous to interferon-inducible Mx proteins performs an essential function in yeast protein sorting . Cell 61 1063-1074 . STAEHELI, P ., and HALLER, 0 . (1987) . Interferon-induced human protein with homology to protein Mx of influenza-resistant mice . Me[ Cell . Biol. 5, 2150-2153 . STAEHELI, P ., GROB, R ., MEIER, E ., SUTCLIFFE, J . G., and HALLER, 0 . (1988) . Influenza virus-susceptible mice carry Mx genes with a large deletion or a nonsense mutation . Mot. Cell. Riot 8, 45184523 . VON WUSSOW, P ., JAKSCHIES, D ., BLOCK, B ., TSCHECHNE, B ., SCHEDEL, I ., HORISBERGER, M . A ., HOCHKEPPEL, H . K ., and DEICHER, H . (1990) . The interferon-induced Mx-homologous protein in people with symptomatic HIV-1 infection . AIDS 4, 119-124 . WEITZ, G ., BEKISZ, J ., ZOON . K., and ARNHEITER, H . (1989) . Purification and characterization of a human Mx protein . .J. Interferon Res. 9, 679-689 .