Induction of Alpha/Beta Interferon by Myxoma ... - Journal of Virology

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JOURNAL OF VIROLOGY, June 2009, p. 5928–5932 0022-538X/09/$08.00⫹0 doi:10.1128/JVI.02587-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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NOTES Induction of Alpha/Beta Interferon by Myxoma Virus Is Selectively Abrogated When Primary Mouse Embryo Fibroblasts Become Immortalized䌤§ Fuan Wang,1,2† John W. Barrett,1,2† Yiyue Ma,1,2 Gregory A. Dekaban,1,2‡ and Grant McFadden1,3* BioTherapeutics Research Group, Robarts Research Institute,1 and Department of Microbiology and Immunology, The University of Western Ontario,2 London, Ontario, Canada, and Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida3 Received 15 December 2008/Accepted 13 March 2009

Mouse embryo fibroblasts (MEFs) are a widely used cell culture system in life sciences, including virology. Here, we show that although primary MEFs are nonpermissive to myxoma virus replication, the corresponding immortalized MEFs support a highly productive myxoma virus infection. We further demonstrate that this permissive phenotype for myxoma virus in immortalized MEFs is due to the immortalization-associated selective block to the cellular alpha/beta interferon induction machinery involved in responding to myxoma virus challenge. Thus, our report presents a clear example, illustrating that a drastic phenotypic alteration can occur with respect to virus infection between primary cells and their immortalized counterparts. Myxoma virus is a member of the poxvirus family and causes highly lethal infections in rabbits but not in any other species, such as mice (7, 17). During our efforts to elucidate the molecular basis for the myxoma virus species barrier, we demonstrated that primary mouse embryo fibroblasts (pMEFs) are resistant to productive myxoma virus infection, a phenotype congruent with the noninfectivity of the virus in vivo (32). In striking contrast, however, when nonpermissive pMEFs become spontaneously immortalized, the resultant immortalized MEFs (iMEFs) developed a permissive state that supported a highly productive myxoma virus infection. Until now, little if anything was known about the underlying molecular mechanisms responsible for this phenotypic alteration of myxoma virus infectivity in iMEFs. Here, we show that the immortalization of pMEFs causes a selective block to the induction machinery of cellular alpha/beta interferon (IFN-␣/␤), rendering the resultant iMEFs unable to mount an antiviral response to the infecting myxoma virus. Previously, we reported that pMEFs (C57BL/6 or 129Sv/Ev background) do not support permissive myxoma virus infection (32). As demonstrated here in Fig. 1A, left, infection of pMEFs with a recombinant myxoma virus expressing ␤-galactosidase under the control of a late viral promoter (22) produced only isolated blue cells with X-Gal (5-bromo-4-chloro-3-indolyl-␤-

D-galactopyranoside) staining. In contrast, when pMEFs were cultured for multiple passages until they became spontaneously immortalized through the standard 3T3 protocol (30), classical myxoma virus-induced blue foci (Fig. 1A, right) formed in the resulting iMEFs, indicating that a full-fledged permissive state had evolved following pMEF immortalization. Quantitatively, myxoma virus infection of iMEFs resulted in a 3-log amplification of progeny virus in comparison to that of

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, 1600 SW Archer Rd., Academic Research Building, Room R4295, P.O. Box 100266, Gainesville, FL 32610. Phone: (352) 273-6852. Fax: (352) 273-6849. E-mail: [email protected]. † These authors contributed equally to the work. ‡ Co-senior author. § Supplemental material for this article may be found at http://jvi .asm.org/. 䌤 Published ahead of print on 18 March 2009.

FIG. 1. iMEFs are permissive to myxoma virus infection. (A) pMEFs and iMEFs were infected with myxoma virus at a multiplicity of infection of 0.01, and X-Gal staining was performed 48 h after infection. (B) pMEFs and iMEFs were infected with myxoma virus at a multiplicity of infection of 0.01 for 48 h. Myxoma virus yields were determined by titrating infectious viral progeny on permissive BGMK cells. 5928

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FIG. 2. IFN-␣/␤–STAT1 signaling cascade is functional in iMEFs. (A) iMEFs were stimulated with IFN-␣/␤ for various amounts of time, as indicated, and the whole-cell lysates were probed with phosphoSTAT1 antibodies. (B) iMEFs were left untreated (⫺) or were treated with IFN-␣/␤ for 30 min (⫹). Subsequently, the cells were stained with STAT1 antibody (Transduction Laboratories). Nuclear DNA was visualized with DAPI (4⬘,6-diamidino-2-phenylindole dihydrochloride; Molecular Probes). (C) iMEFs were infected with myxoma virus at a multiplicity of infection of 0.01 in the absence (⫺) or presence (⫹) of exogenous IFN-␣/␤, and X-Gal staining was performed 48 h after infection.

pMEFs (Fig. 1B), further confirming that myxoma virus infection in iMEFs was productive. Our previous work shows that the nonpermissiveness of pMEFs to myxoma virus infection is mediated by STAT1dependent type I IFN (IFN-␣/␤) response (32). Therefore, we reasoned that the immortalization-associated permissiveness of iMEFs to myxoma virus was due to their cellular inability to mount appropriate type I IFN responses to myxoma virus challenge. As a first step to test this, we evaluated the integrity of the type I IFN-STAT1 signaling pathway in iMEFs by stimulation with exogenous IFN-␣ and IFN-␤ (50 U/ml each; Calbiochem). Whole-cell lysates were then prepared for Western blot analysis with antibodies against the activated forms of STAT1. As shown in Fig. 2A, IFN-␣/␤ stimulation induced the expected STAT1 phosphorylation at both Y701 and S727 (24, 26). Furthermore, the typical IFN-␣/␤-invoked translocation of STAT1 from the cytoplasm to the nucleus (5, 24, 29) was also observed (Fig. 2B, top). Together, these results clearly demonstrate that IFN-␣/␤ receptor-STAT1 signal transduction was able to proceed normally in iMEFs. Of functional importance, we further observed that this exogenous IFN-␣/␤ treatment rendered the iMEFs completely nonpermissive to myxoma vi-

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FIG. 3. iMEFs fail to express IFN-␣/␤ following myxoma virus infection. (A) iMEFs were infected with myxoma virus at a multiplicity of infection of 1 for various amounts of time, as indicated, and the whole-cell lysates were probed with phosphorylated ERK1/2 (pERK1/2) or ERK1/2 antibodies (Upstate Biotechnology). The positive control was epidermal growth factor-stimulated A431 cell lysate (Upstate Biotechnology). (B) iMEFs were infected with myxoma virus at a multiplicity of infection of 1 for 24 h. Levels of IFN-␤ secreted from the infected iMEFs or pMEFs were determined by standard sandwich ELISA. (C) iMEFs were infected with myxoma virus at a multiplicity of infection of 1 for various amounts of time, as indicated, and total RNA was extracted using an RNeasy kit (Qiagen) for the assessment of IFN-␣/␤ mRNA levels by RT-PCR. The positive control was the total RNA obtained from myxoma virus-infected pMEFs (8 h after infection at a multiplicity of infection of 1). GAPDH (glyceraldehyde phosphodehydrogenase) was used as a housekeeping gene control.

rus infection (Fig. 2C, right). Taken collectively, these data show that the IFN-␣/␤–STAT1 signaling pathway per se is functionally intact in iMEFs and suggest that the cellular machinery involved in myxoma virus-stimulated type I IFN induction, but not IFN action, was somehow compromised following immortalization of pMEFs. Of note in this regard, classical 3T3 cells derived from spontaneous immortalization of primary Swiss MEFs (ATCC) (30) displayed the same susceptibility to myxoma virus infection and IFN response phenotypes as the iMEFs used here (see Fig. S1 in the supplemental material). This observation further suggests that the acquisition of a permissive phenotype for myxoma virus infection in iMEFs appears to be a regular phenomenon associated with the immortalization of pMEFs. One of the initial cellular signaling events related to the type I IFN response invoked by myxoma virus infection in pMEFs was robust phosphorylation of the mitogen-activated protein kinase ERK1/2 (32). Interestingly, no significant phosphoryla-

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tion of ERK1/2 was observed in myxoma virus-infected iMEFs (Fig. 3A, top), despite the fact that total ERK1/2 protein levels remained undiminished following the infection (Fig. 3A, bottom). Next, we examined type I IFN production by myxoma virus infection in iMEFs by utilizing enzyme-linked immunosorbent assay (ELISA) analysis of IFN-␤ released into the culture supernatants (4). As shown in Fig. 3B, little IFN-␤ was observed following myxoma virus infection of iMEFs, whereas a significant increase in IFN-␤ secretion was seen in myxoma virus-infected pMEFs. We then examined IFN-␣/␤ mRNA expression in myxoma virus-infected iMEFs by utilizing reverse transcription-PCR (RT-PCR), with the primers and conditions used as previously described (18). Consistent with the ELISA data, there was essentially no mRNA transcription of either IFN-␣ (Fig. 3C, top) or IFN-␤ (Fig. 3C, middle) in response to myxoma virus infection. Similar results were obtained with myxoma virus-infected 3T3 cells (see Fig. S2 in the supplemental material). Interferon regulatory factor 3 (IRF3) is a critical mediator involved in the transcriptional initiation of IFN-␣/␤ genes (8, 12, 19, 25). Therefore, we sought to examine whether the immortalization of pMEFs had caused a defect in IRF3 activation that might explain the lack of type I IFN production in myxoma virus-infected iMEFs. To this end, we infected iMEFs with myxoma virus for various amounts of time. As shown in Fig. 4A, top, myxoma virus infection did not initiate any observable change in IRF3 band migration patterns, as revealed by an anti-IRF3 antibody (Zymed). In contrast, when poly(I)-poly(C) (Sigma), a synthetic analogue for doublestranded RNA (dsRNA), was added to the cell culture media to stimulate Toll-like receptor 3 (TLR3) signaling (1, 2, 16), appreciable IRF3 band shifts typical of IRF3 activation following its phosphorylation were clearly observed (Fig. 4A, bottom). IRF3 is a preexisting cytoplasmic protein that migrates to the nucleus only after IRF3 phosphorylation (12, 23). Consistent with the Western blot data, immunofluorescence

FIG. 4. Immortalization of pMEFs selectively blocks the cellular IFN␣/␤ induction pathway involved in response to myxoma virus infection. (A) iMEFs were infected with myxoma virus at a multiplicity of infection of 1 or stimulated with dsRNA that was added to the cell culture media at 20 ␮g/ml for various amounts of time, as indicated. Whole-cell lysates were then prepared for Western blotting with an anti-IRF3 protein antibody. (B) iMEFs were left untreated (mock), infected with myxoma virus at a multiplicity of infection of 1 for 6 h, or stimulated with dsRNA that was added to the cell culture media at 20 ␮g/ml for 2 h. Subsequently, the cells were stained with anti-IRF3 antibody followed by a secondary antibody conjugated to Texas Red (Jackson ImmunoResearch Laboratories). Nuclear DNA was visualized with DAPI (4⬘,6-diamidino-2-phenylindole dihydrochloride) staining. (C) iMEFs were left untreated (mock) or

treated with dsRNA that was added to the culture media at 20 ␮g/ml for 6 h. Total RNA was extracted for RT-PCR evaluation of IFN-␣/␤ mRNA levels. GAPDH (glyceraldehyde phosphodehydrogenase) was used as a housekeeping gene control. (D) iMEFs were left untreated (mock) or were transfected with 10 ␮g/ml of dsRNA or various DNAs complexed with Lipofectamine 2000 (Invitrogen) for 6 h. Total RNA was analyzed by RT-PCR for IFN-␣/␤ mRNA. GAPDH was used as a housekeeping gene control. (E) iMEFs were first infected with myxoma virus at a multiplicity of infection of 5 for 10 h. Subsequently, the infected cells were left untreated (mock) or were treated with dsRNA that was added to the culture media at 20 ␮g/ml for 6 h. Total RNA was analyzed by RT-PCR for IFN-␣/␤ mRNA. GAPDH was used as a housekeeping gene control. (F) iMEFs were first infected with myxoma virus at a multiplicity of infection of 5 for 10 h. Subsequently, the infected iMEFs were left untreated (mock) or were transfected with 10 ␮g/ml of dsRNA or various DNAs complexed with Lipofectamine 2000 for 6 h. Total RNA was analyzed by RT-PCR for IFN-␣/␤ mRNA. GAPDH was used as a housekeeping gene control. (G) iMEFs were left untreated (mock), were treated with dsRNA that was added to the cell culture media at 20 ␮g/ml for 12 h, or were transfected with 10 ␮g/ml of dsRNA complexed with Lipofectamine 2000 for 12 h. Thereafter, the treated iMEFs were infected with myxoma virus at a multiplicity of infection of 0.01, and X-Gal staining was performed 48 h after infection.

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microscopy demonstrated that the nuclear translocation of IRF3 occurred only in the iMEFs stimulated with dsRNA added to the cell media (Fig. 4B, top), whereas in both the mock and myxoma virus-infected cells, IRF3 remained in the cytoplasm (Fig. 4B, top). These data indicate that the IRF3 signaling machinery itself was competent in iMEFs, but myxoma virus infection failed to trigger its activation. Next, we performed RT-PCR and observed that mRNA expression of both IFN-␣ (Fig. 4C, top) and IFN-␤ (Fig. 4C, middle) was robustly induced by dsRNA that was added to the cell media. Of note, the IRF3–IFN-␣/␤ induction profiles via the dsRNATLR3 pathway observed in the iMEFs here were comparable to those obtained in the corresponding pMEFs (data not shown). Parallel to the membrane-bound TLRs, cytoplasmic dsRNA/ double-stranded DNA (dsDNA) signaling cascades also play essential roles in the innate immunity to microbial infections (20). Therefore, we sought to deliver dsRNA intracellularly by transfection to activate the cytoplasmic dsRNA signaling through retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) (10, 15, 20, 34, 35). We observed that transfected dsRNA caused a strong induction of both IFN-␣ (Fig. 4D, top) and IFN-␤ (Fig. 4D, middle) in iMEFs. In a similar fashion, dsDNA transfection was conducted to activate the cytoplasmic dsDNA signaling (13, 27), possibly involving the DAI and inflammasome pathways (21, 28, 33). Notably, the synthetic dsDNA poly(dA-dT) (Amersham) induced a potent induction of IFN-␣ (Fig. 4D, top), while Escherichia coli and calf thymus DNAs (Invitrogen) were poor inducers for IFN-␣ (Fig. 4D, top). Nonetheless, transfections in iMEFs of the three different dsDNAs all potently induced IFN-␤ expression (Fig. 4D, middle), a phenotype congruent with the type I IFN response seen in dsDNA-transfected pMEFs (27). Taken together, these data clearly indicate that the immortalization of pMEFs selectively blocks the cellular IFN-␣/␤ induction machinery involved in sensing and responding to myxoma virus infection, whereas signal transduction through the membrane-bound dsRNA-TLR3, the cytoplasmic dsRNA–RIG-I/MDA5, and the dsDNA-DAI/inflammasome signaling modules still remains functionally competent. Innate sensing of viral pathogen-associated molecular patterns is key for the host cell to initiate the cellular antiviral responses (1, 14). In primary human macrophages, but not in primary human fibroblasts, RIG-I has been shown to be a major sensor for myxoma virus infection that triggers the induction of type I IFN and tumor necrosis factor (31). In stark contrast to human macrophages, however, comparable knockdown studies revealed that RIG-I did not appear to be involved in sensing myxoma virus infection in either pMEFs or iMEFs (data not shown). These observations suggest that the innate sensor(s) for myxoma virus infection may instead be composed of multiple cell-type and/or species-specific elements, which remain to be identified for the mouse system. Next, we examined whether or not myxoma virus infection would inhibit dsRNA-TLR3 and/or cytoplasmic dsRNA/ dsDNA signaling. To this end, we infected iMEFs with myxoma virus for 10 h and then challenged the infected cells with dsRNA that was added to the cell media or through dsRNA and dsDNA transfections. As shown in Fig. 4E, myxoma virus infection inhibited mRNA induction of both IFN-␣ (Fig. 4E,

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top) and IFN-␤ (Fig. 4E, middle) via the dsRNA-TLR3 pathway. Remarkably, the expression of IFN-␣ (Fig. 4F, top) and IFN-␤ (Fig. 4F, middle), as induced through the cytoplasmic dsRNA/dsDNA cascades, was also suppressed by myxoma virus infection. Together, these data show that a permissive myxoma virus infection can be highly inhibitory to the innate antiviral signal transduction through both the membrane-associated dsRNA-TLR3 and cytoplasmic dsRNA/dsDNA pathways. Consequently, these observations have direct relevance to the differing efficacies of myxoma virus proliferation seen in various experimental cell systems, as has been demonstrated for an array of other viruses (3, 6, 9, 11, 34). To ascertain the functional importance of endogenous type I IFN for the determination of myxoma virus infection phenotypes in iMEFs, we treated the cells with dsRNA that was added to the cell media or with dsRNA transfection for 12 h to allow for IFN-␣/␤ expression (data not shown). As shown in Fig. 4G, the iMEFs rendered in the IFN-␣/␤-expressing state via the dsRNA-TLR3 (middle) (1, 2, 16) or dsRNA–RIG-I/ MDA5 (right) (10, 15, 34, 35) pathways now became nonpermissive to myxoma virus infection. Thus, these data further demonstrate that induction of IFN-␣/␤ is a truly decisive cellular factor in dictating myxoma virus infectivity in normal mouse fibroblasts. In conclusion, our data reported here have provided insightful evidence that the immortalization of primary fibroblasts in culture can dramatically alter the cellular ability to respond to a given virus infection and suggest that caution needs to be exercised in comparing virus infections of primary cells to their culture-adapted cell line counterparts. We thank Erik Barton, Herbert W. Virgin IV, and Robert D. Schreiber for providing the pMEFs, which made this project possible. We are grateful to Ilda Moniz for her administrative assistance at Robarts Research Institute. This work was supported by the Canadian Institutes of Health Research. REFERENCES 1. Akira, S., S. Uematsu, and O. Takeuchi. 2006. Pathogen recognition and innate immunity. Cell 124:783–801. 2. Alexopoulou, L., A. C. Holt, R. Medzhitov, and R. A. Flavell. 2001. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature 413:732–738. 3. Andrejeva, J., K. S. Childs, D. F. Young, T. S. Carlos, N. Stock, S. Goodbourn, and R. E. Randall. 2004. The V proteins of paramyxoviruses bind the IFN-inducible RNA helicase, mda-5, and inhibit its activation of the IFNbeta promoter. Proc. Natl. Acad. Sci. USA 101:17264–17269. 4. Asselin-Paturel, C., A. Boonstra, M. Dalod, I. Durand, N. Yessaad, C. Dezutter-Dambuyant, A. Vicari, A. O’Garra, C. Biron, F. Briere, and G. Trinchieri. 2001. Mouse type I IFN-producing cells are immature APCs with plasmacytoid morphology. Nat. Immunol. 2:1144–1150. 5. Biron, C. A., and G. C. Sen. 2001. Interferons and other cytokines, p. 321–351. In D. M. Knipe and P. M. Howley (ed.), Fields virology, 4th ed., vol. 1. Lippincott Williams & Wilkins, Philadelphia, PA. 6. Bowie, A. G., and I. R. Haga. 2005. The role of Toll-like receptors in the host response to viruses. Mol. Immunol. 42:859–867. 7. Fenner, F., and F. N. Ratcliffe. 1965. Myxomatosis. Cambridge University Press, Cambridge, United Kingdom. 8. Fitzgerald, K. A., S. M. McWhirter, K. L. Faia, D. C. Rowe, E. Latz, D. T. Golenbock, A. J. Coyle, S. M. Liao, and T. Maniatis. 2003. IKKepsilon and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 4:491–496. 9. Foy, E., K. Li, R. Sumpter, Jr., Y. M. Loo, C. L. Johnson, C. Wang, P. M. Fish, M. Yoneyama, T. Fujita, S. M. Lemon, and M. Gale, Jr. 2005. Control of antiviral defenses through hepatitis C virus disruption of retinoic acidinducible gene-I signaling. Proc. Natl. Acad. Sci. USA 102:2986–2991. 10. Gitlin, L., W. Barchet, S. Gilfillan, M. Cella, B. Beutler, R. A. Flavell, M. S. Diamond, and M. Colonna. 2006. Essential role of mda-5 in type I IFN

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