Knockout and reconstitution of a functional human type I interferon ...

THEJOURNAL OF BIOLWICAL CHEMISTRY Vol. 269, No. 29, Issue of July 22, pp. 18747-18749, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

Communication

we designate Hu-IFN-aR1 here)exhibited activity in response only t o Hu-IFN-aB2 (also designatedHu-IFN-a8). Results with antibodies to Hu-IFN-aR1 and other Type I interferon receptor components have suggested that HU-IFNaR1 isone of two or more componentsthat constitute thefunctional Hu-IFN-a receptor complex. This conclusion was sup(Received for publication, May 10, 1994) ported by the identification of two separate components Cathleen M. Cleary, RobertJ. Donnelly, following chemical cross-linking and/or immunoprecipitationof Jaemog Soh$, ThomasM. Mariano, and 1251-Hu-IFN-aAreceptor complexes with an anti-IFN-a recepSidney Pestka tor antibody (Raziuddin and Gupta, 1985; Colamonici and PfefFrom the Department of Molecular Genetics and fer, 1991; Colamonici and Domanski, 1993; Colamonici et al., Microbiology, University of Medicine and Dentistry of 1990, 1992). These two components seem t o differ from the New Jersey, Robert Wood Johnson Medical School, cloned Hu-IFN-aR1. The monoclonal antibodies against one Piscataway, New Jersey08854-5635 subunit (110 kDa) and therecombinant Hu-IFN-aR1 block the The functional subunits of the human T h e I inter- biological activity of Type I interferons, while a monoclonal feron (IFN) receptor complex have not been defined. antibody against thesecond subunit (210 kDa) does not (Uze et Using site-specific recombination in a yeast artificial al., 1991; Colamonici and Domanski, 1993; Benoit et al., 1993), chromosome (YAC), we have produced adeletion within suggesting that the Type I IFN receptor consists of at least the humanIFN-a receptor (Hu-IFN-aR1) gene which three different subunits. Furthermore, functional differences eliminates exon I1 of the gene. This deletion effectively among the Hu-IFN-aspecies have been described in a variety eliminates the MHC Class I antigen induction and anti- of assays and havesuggested the existence of multiple compoviral activity previously reported for this fully func- nents in the specific ligand-receptor interactions (Evinger et tional parental YAC clone (Soh, J., Mariano, T. M., Lim, al., 1981; Rehberg et al., 1982; Ortaldo et al., 1983, 1984; J.-K., Izotova, L., Mirochnitchenko, O., Schwartz, B., Pestka, 1983, 1984; Pestka et al., 1987; Hu et al., 1993). All Langer, J., and Pestka, S. (1994~)J. Biol. Chem. 269, these observations suggest that the Hu-IFN-aR1 molecule is 18102-18110).We have successfully reconstituted this acone component of the Type I receptor complex. tivity by expression of the cDNA encoding the Hu-IFNBy functional screening of yeast artificial chromosomes C V R l component(Uz6, G., Lutfalla, G., and Gresser, I. (YACs) (Soh et al., 1993,1994a, 1994b; Cook et al., 1994a, (1990)Cell 60,225-234) in cells containing the YAC with this deletion. The Hu-IFN-aR1 subunit thus plays a criti- 199413) similar tothat which led t o the cloning of the accessory cal role in the functional human Type I IFN receptor chain of the Hu-IFN-y receptor (accessory factor-1, a @-like complex, whose components are encoded on this YAC. In receptor chain), we were able to isolate a functional YAC clone addition, as binding of ligands is retained in the cells that,when expressed inhamster cells, exhibited the full containing the YAC with the deletion, it isclear a second complement of Type I interferon receptor activityin response to subunit encoded on the YAC is responsible for ligand all Type I interferons tested (Soh et al., 1994~).Because this binding activity. This system will now allow the identi- YAC clone (aYAC or cuRy9-2 in figures)contained the gene for fication of additional subunits involved in the response Hu-IFN-aR1 (Uze et al., 1990; Lutfalla et al., 1992; Mariano et to the Type I IFNs and the functional significance of al., 1992), we had the opportunityt o determine if the Hu-IFNeach. aR1 is indeed required for Type I interferon receptor activity. To accomplish this, we used homologous recombination into the aYAC with a vector containing human repetitiveDNA Alu seThe full complement of the components of the humanType I quences to delete regions between Alu sequences (Soh et al., interferon receptor has been sought for several years. In1986, 1993, 1994b) and provide a knockout of all or part of the Hua series of genomic clones were isolated after transfection of IFN-aR1 gene. Aclonewas obtained (AaYAC, noted as CrRylOA) mouse NIH3T3 cells with total humanDNA that responded to in which exon I1 of the Hu-IFN-aR1gene was deleted (Fig. 1). Type I interferons (human interferon a, Hu-IFN-a’; human We then determined the MHC Class I antigen induction and Hu-IFN-@);however, a specific cosmid or cDNA antiviral activity in hamster cells containing this AaYAC in interferon @, clone was not isolated at that time (Jung and Pestka, 1986; response to human Type I interferons. As shown in Fig. 2, cells containing the AaYACdo not reJung, 1991). Using a similar procedure, Uze et al. (1990) reported the cloning of a cDNA they considered to encode the spond to Hu-IFN-olA or Hu-IFN-aB2 in MHC Class I antigen human interferona receptor. However, this cDNA clone (which induction, whereas cells containing the fully functional aYAC do. Parental hamster cells do not respond to these human interferons at theconcentrations tested. As shown in Figs. 3 and * This study was supported in part by United States Public Health 4, hamster cells containing the aYAC exhibit significantly enServices Grant R01 CA46465 from NCI, National Institutes of Health hanced protection against encephalomyocarditis virus (EMCV), (to S . P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby whereas hamster cells containing the AaYAC exhibit no more marked “advertisement” in accordance with 18 U.S.C. Section 1734 protection against EMCV than the parental hamster cells in solely to indicate this fact. response to both of these interferons.We thus conclude that the $ Supported in part by the Becton Dickinson Company as a desig- activity of the Hu-IFN-aR1 is required to construct a fully nated Becton Dickinson Scholar. The abbreviationsused are: Hu-IFN, human interferon;YAC, yeast functional receptor. In order t o reconstitute these biological activities, the Huartificial chromosome; bp, base paifis);kb, kilobase pair(s);MHC, major histocompatibility complex; EMCV, encephalomyocarditis virus. IFN-aR1 cDNA clone (see legendt o Fig. 2) was thenintroduced

Knockout and Reconstitution of a Functional Human Type I Interferon Receptor Complex*

18747

A Functional Human Qpe I IFN Receptor Complex

18748

EEE*

E

E VI1

ALU

NE0

Vlll

E IX

x

XI

ALU

FIG.1.Map of the Hu-IFN-aR1 gene showing the location of the insertionof pJS1.The EcoRI sitesof the 32.9 kb Hu-IFN-aR1 gene are shown above the intron-exon structural map (exons areboxed, introns areheavy lines) (Lutfalla et al., 1992; Mariano et al., 1992). The exons are numbered I-XI. The sizeof the EcoRI fragments determined from theDNA sequence that containexon sequences are 8331bp (exonsI1 and III), 10,699 bp (exonsIV-VIII), 4898 bp (exons M-XI),and a 5’ end fragment greater than 4026 bp (exonwhich I), appears atapproximately 12 kb on a Southern blot of genomic DNA. The insertion vectorpJSl (9.6 kb) containing theneomycin phosphotransferase gene (NEO) (Soh et al., 1994a, 1994b, 1994~) shown is as a linear fragment, as would be the structurefollowing a ClaI digestion resulting in the Alu sequences ( M U ) at the ends of the fragment. The insertionof the vector a t Alu sequences located a t 1890-2135 bp (1)and at 14,507-14,829 bp (2)of the gene results in the introduction of EcoRI sites a t new positions increasing the size of the 12-kb fragmentby 3 kb and reducing the8331-bp fragment to 3.5 kb (exon 111).The shaded area of the gene is the location of the deletion. E , EcoRI; C , ClaI. E* indicates thelocation of several clustered EcoRI sites in the gene.

16-9/aRy9-2 Hu-IFN-aA

Hu-aRl Hu-IFN-cLA

16-9/aRy 1O/ Hu-aR1 Hu-IFN-aB

16-9 Hu-IFN-aB2

300

200

100

0

100

0

100

0

100

0

100

Relative Fluorescence FIG.2. Induction of Class I MHC cell surface antigen expression by interferons. All cell lines were maintained in F-12 medium containing 10% fetalbovine serum. Parental 16-9 cells are Chinese hamster ovaryK1 cells containing the geneencoding the human Class HLA I B7 antigen and humanchromosome 6 (Soh et al., 1993); 16-9/aRy9-2 (aYAC) cells (16-9/9-2) are 16-9 cells transfected with the aYAC containing the IFNAR locus as described (Soh et al., 1994~);16-9/aRylOA are 16-9 cells transfected with theAaYAC containing a deletion of exon I1 of the Hu-IFN-aR1 gene. Cell lines carrying YACs were maintained in medium containing 0.25 mg/ml antibiotic G418. The Hu-IFN-aR1 cDNA was excised from plasmid pYH12 (Y. Hibino and S. Pestka, unpublished data) and cloned into thevector p8942 ( g i f t of G. Mark andB. Daugherty, Merck Sharp & Dohme Research Laboratories) for eukaryotic expression under the control of the adenovilvs 2 major promoter. late This plasmid was then transfected into 16-9/aRylOA cells. Clones were selected in medium containing 0.4 mg/ml hygromycin B and designated 16-9/aRylOA Hu-aR1 (AaYAC reconstituted with the Hu-IFN-aR1 expression vector; see far rightpanels).Isolates were expanded and maintained in medium containing 0.25 mg/ml each antibioticG418 and hygromycin B. To test for induction of Class I MHC antigen expression, cells were treated withHu-IFN-(rA or Hu-IFN-aB2a s indicated in the figure a t 100 unitdm1 in the absence of antibiotic for 3 daysa s described (Hibino et al., 1992; Soh et al., 1994a). Cells were harvested by trypsinization and treated with mouse anti-HLA monoclonal antibody (W6/32), washed, and treated with fluorescein isothiocyanate-conjugatedgoat anti-mouse IgG. Cells were washed of excess antibody and analyzed by cytofluorography. The shaded regions represent MHC Class I antigen expression of IFN-treated cells; unshaded regions represent that of untreated cells.

into thecells containing theAaYAC. The transfectedcells were then able to respond to both Hu-IFN-clA and Hu-IFN-aB2 in both MHC Class I antigen induction (Fig. 2) and antiviral assays (Figs. 3 and 4). These resultsconfirm that Hu-IFN-aR1 is an important component of the human Type I receptor complex. Deletion of this gene from a YAC containing the fully functional receptor results in loss of biological responses to the IFNs tested. Trans-

fection of Hu-IFN-aR1 cDNA reconstitutes these biological activities. As we reported (Soh et al., 1994c), the aYAC contains all the components fora functional Type I interferon receptor complex. The results in this report allow us to conclude that theHu-IFNaR1 protein is one component of this receptor complex. With the useof mice with a deletionof the Mu-IFN-aR1 gene, Muller et al. (1994) have also concluded that the IFN-aR1 protein is

18749

A Functional Human p p e I IFN Receptor Complex

Hu-IFN-otA

2

12

i-

(EMCV) KO+ aR1

ligand binding activity. Additional analysis of this aYAC clone will permit us to identify the remaining components of the functional Type I interferon receptor. \

Acknowledgments-We thank Drs. Daniel Cohen and David Pattersonfor YAC clones,Gilles Uz6 forplasmid pVADN123,OlgaMirochnitchenko and Barbara Schwartz for assistance in the antiviral assays, Eleanor Kells for preparation of this manuscript, Dr. Edward Yurkow forthe cytofluorographicanalyses, and J . Langer and J.-K. Lim for ligand binding assays. REFERENCES

FIG.3. Antiviral activity of Hu-IFN-ak The data in the figure represent the reciprocal of the IFN titer (unitdml)for 50% protection of cells (ED,,) against EMCV. The 16-9 cell line is a human x hamster hybrid containing the long arm of human Chromosome 6 and a transfected HLA-B7 gene (Cook et al., 1994a; Soh et al., 1993, 1994a);aYAC represents 16-9 cellscontaining the aYAC (Soh et al., 1994~); KO represents 16-9 cellscontaining the AaYAC (aRylOA) with exon I1deleted; KO + &l represents the KO cells reconstituted by transfection with the Hu-IFN-aR1 cDNA clone. Antiviral assays were performedas reported (Familletti et al., 1981). The data for Hu-IFN-aA are shown.

Hu-IFN-otB2

(EMCV) KO+

I

FIG.4. Antiviral activity of Hu-IFN-orB2. The experiments were performed as described in the legend to Fig. 3 except that Hu-IFN-aB2 was substituted for Hu-IFN-aA. The data for 16-9 and KO cells are maximal values as the end point titer was >10,000unitdml.

required for Type I interferon receptorfunction.Direct,although weak, participation of the Hu-IFN-aR1 polypeptide in IFN-a ligand binding was suggested by the observation that the human and bovine IFN-aR1 polypeptide expressed in Xenopus Zaeuis oocytes can bind and cross-link radiolabeled HuIFN-(rA and Hu-IFN-aB2 to a small extent.'Nevertheless, when we examined the bindingof Hu-IFN-aA and Hu-IFN-aB2 to pools of hamster cells containing theAaYAC, there was substantial binding of these ligands.' However, there was little binding to hamster cells expressing only Hu-IFN-aR1 (Soh et al., 1994~).We can thusalso conclude that a second subunit of is responsible for the receptor complex other than Hu-IFN-aR1 J.-K. Lim and J . A. Langer, personal communication.

Benoit, P., Maguire, D., Plavec, I., Kocher, H., Tovey, M., and Meyer, F. (1993) J. Immunol. 160,707-716 Colamonici, 0.R., and Domanski, P. (1993) J. Biol. Chem. 268,10895-10899 Colamonici, 0. R., and Pfeffer, L. M. (1991) Pharmacol. Ther 62,227-233 Colamonici, 0.R., DAlessandro, F. Diaz, M. O., Gregory, S. A., Neckers, L. M., and Nordan, R. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 7230-7234 Colamonici, 0. R., Pfeffer, L. M., DAlessandro, F., Platanias, L. C., Gregory, S . A., Rosolen,A., Nordan, R., Cruciani, R. A,, and Diaz, M. 0.(1992)J.Immunol. 148, 2126-2132 Cook, J. R., Emanuel, S. L., Donnelly, R. J., Soh, J., Mariano,T. M., Schwartz, B., Rhee, S., and Pestka, S. (1994a) J. Biol. Chem. 269, 7013-7018 Cook, J . R., Emanuel, S. L., and Pestka, S. (1994b) Gene Anal. Tech. Applic. 10, 109-112 Evinger, M., Rubinstein, M., and Pestka, S . (1981) Arch. Biochem. Biophys. 210, 319-329 Familletti, P.C., Rubinstein, S., and Pestka, S . (1981) MethodsEnzymol. 78, 387-394 Hibino, Y., Kumar, C. S., Mariano, T.M., Lai, D., and Pestka, S . (1992) J. Biol. Chem. 267,3741-3749 Hu, R., Gan, Y., Liu, J., Miller, D., and Zoon, K.C. (1993) J. Biol. Chem. 268, 12591-12595 Jung, V. (1991) The Human Interferon Gamma Receptor and Signal lhnsduction. Ph. D. thesis, Rutgers University, Piscataway, N J Jung, V., and Pestka, S. (1986) Methods Enzymol. 119,597-611 Lutfalla, G. Gardiner, K., Proudhon,D., Vielh, E., and UzB, G. (1992)J . Biol. Chem. 267,2802-2809 Mariano, T. M., Donnelly, R. J., Soh, J., and Pestka,S. (1992) in Interferon: Principlesand Medical Applications (Baron, S., Coppenhaver, D., Dianzani, F., Fleischman, W. R., Hughes, T.K., Klimpel, G. R., Niesel, D. W., Stanton, G. J., and Tynng, S. K., eds) pp. 129-138, University of Texas Medical Branch a t Galveston, Galveston, TX Muller, U., Steinhoff,U., Reis, L. F. L., Hemmi,S., Pavlovic, J., Zinkernagel, R. M., and Aguet, M. (1994) Science, in press Ortaldo, J. R., Mason, A,, Rehberg, E., Moschera, J., Kelder, B., Pestka, S., and Herberman, R. B. (1983) J. Biol. Chem. 268, 15011-15015 Ortaldo, J. R., Herberman, R. B., Harvey, C., Osheroff, P., Pan, Y.-C. E., Kelder, B., and Pestka, S. (1984) Proc. Natl. Acad. Sci. U. S. A . 81, 4926-4929 Pestka, S. (1983) Arch. Biochem. Biophys. 221, 1-37 Pestka, S . (1984)in Biochemical and Biophysical Studies of Proteins and Nucleic Acids (Lo,T.-B., Liu, T.-Y., and Li, C.-H., eds.) pp. 11-58, Elsevier Science Publishing Co., New York Pestka, S., Langer, J.A.,Zoon, K. C.,and Samuel,C. E. (1987)Annu.Reu. Biochem. 66.727-777 Raziuddin, A,, and Gupta, S. L. (1985) in The 2 5 A System: Molecular and Clinical Aspects of the Interferon-regulated Pathway(Williams, B. R. G., and Silverman, R. H., eds) pp. 219-226, Alan R. Liss, New York Rehberg, E., Kelder, B., Hoal, E. G., and Pestka, S. (1982) J. Biol. Chem. 267, 11497-11502 Soh, J. Donnelly, R. J., Mariano, T.M., Cook, J. R., Schwartz, B., and Peatka, S . (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8737-8741 Soh, J., Donnelly, R. J., Kotenko, S., Mariano, T. M., Cook, J. R., Wang, N., Emanuel, S. L., Schwartz, B., Miki, T., and Pestka, S. (1994a) Cell 76, 793-802 Soh, J., Mariano, T. M., Bradshaw, G.,Donnelly, R. J., and Pestka,S. (1994b)DNA Cell Biol. 13, 301-309 Soh, J., Mariano, T.M., Lim, J.-K., Izotova, L., Mirochnitchenko, O., Schwartz, B., Langer, J., and Pestka, S. (1994~) J.Biol. Chem. 269, 18102-18110 UzB, G., Lutfalla, G., and Gresser, I. (1990) Cell 60, 225-234 UzB, G., Lutfalla, G., Eid, P., Maury, C., Bandu, M. T., Gresser, I., and Mogensen, K. (1991) EUKJ. Immunol. 21,447451 ~~