Expression of infectious murine leukemia viruses by RAW264.7 cells, a potential complication for studies with a widely used mouse macrophage cell line
© Hartley et al; licensee BioMed Central Ltd. 2008
Received: 07 November 2007
Accepted: 04 January 2008
Published: 04 January 2008
The mouse macrophage-like cell line RAW264.7, the most commonly used mouse macrophage cell line in medical research, was originally reported to be free of replication-competent murine leukemia virus (MuLV) despite its origin in a tumor induced by Abelson MuLV containing Moloney MuLV as helper virus. As currently available, however, we find that it produces significant levels of ecotropic MuLV with the biologic features of the Moloney isolate and also MuLV of the polytropic or MCF class. Newborn mice developed lymphoma following inoculation with the MuLV mixture expressed by these cells. These findings should be considered in interpretation of increasingly widespread use of these cells for propagation of other viruses, studies of biological responses to virus infection and use in RNA interference and cell signalling studies.
In contrast to most other mouse-derived cell cultures, the macrophage-like cell line RAW264.7  supports replication of murine noroviruses and is widely used for this purpose . Further, in studies of a mouse model of severe respiratory disease, RAW264.7 was found to be uniquely efficient for propagation of the causative agent, pneumonia virus of mice, and for measuring infection-related proinflammatory mediators . In addition, because of ease of cell propagation, high efficiency for DNA transfection, sensitivity to RNA interference , possession of receptors for many relevant ligands, and other properties, RAW264.7 has been chosen by the Alliance for Cellular Signaling as the primary experimental system for their large-scale study of signaling pathways . The RAW264.7 cell line was derived about 30 years ago from a tumor developing in a BAB/14 mouse, a BALB/c IgH congenic strain, inoculated with Abelson murine leukemia virus (MuLV), a defective transforming virus containing the v-abl tyrosine kinase oncogene, and replication-competent Moloney (Mo-MuLV) that served as helper virus . At the time it was described, tests for presence of replication competent virus were negative and cells in the American Type Culture Collection repository (ATCC TIB-71) were so designated until recently. As far as we can determine, ATCC is the major if not sole, commercial source of this cell line. To date, a Pubmed retrieval lists over 1500 publications that have used the RAW264.7 cell line in the research reported.
Results and conclusion
In tests for the presence of infectious MuLV, cell-free harvests of RAW264.7 supernatants from two separate acquisitions from ATCC were positive using the XC plaque assay  in SC-1 cells , indicating the presence of virus of the ecotropic MuLV class. Titers of several independent harvests ranged from 101.2 to 104.2 XC plaque-forming units (pfu) per ml. Lower titers were associated with high density cell growth and consequent low pH (data not shown).
Infection of cell cultures of various mouse strains by most ecotropic MuLVs is regulated by the Fv1 genotype of the mouse and the tropism of the virus. Thus, Fv1 n cells are permissive for N-tropic and partially restrictive for B-tropic MuLVs; the reciprocal pattern is observed for cells of Fv1 b mice. A few isolates with long laboratory passage histories, including Mo-MuLV, are not restricted by either allele and are termed NB-tropic. XC plaque titration of RAW264.7 supernatant in NIH3T3 (Fv1 n ) and BALB3T3 (Fv1 b ) cells gave essentially identical titers (104.1 and 104.2, respectively) indicating NB-tropism. Additionally, focal immunofluoresence assays (IFA)  utilizing monoclonal hybridoma antibody mAb538, specifically reactive with the Mo-MuLV envelope (SU) protein , were positive in NIH3T3 cells and to about 1000-fold lower titer in Mus dunni cells, confirming the presence of a virus with ecotropic Mo-MuLV biological properties. Why earlier tests failed to detect ecotropic MuLV in RAW264.7 cells is unknown but possible explanations include differences in culture conditions and the health of the cells when assayed or in sensitivity of the tests used in different laboratories at different times.
It is well established that mouse-passaged ecotropic MuLV stocks, like the Mo-MuLV helper virus used in establishing RAW264.7 , frequently contain MuLV of other classes – xenotropic and recombinant MuLVs that result from interaction between ecotropic sequences and polytropic, also referred to as MCF, proviral sequences. Indeed, isolates of these classes were reported in a stock of Abelson MuLV complex . Comparative IFA titrations of a RAW264.7 supernatant were performed in NIH3T3 cells using anti-Mo-MuLV mAb538 and two mAbs that specifically detect polytropic MuLV SU antigen: 514, reactive with all tested polytropic MuLVs , and HY7, reactive with certain polytropic subsets [12, 13]. Titer estimates of 104 and 104.5 ffu per ml were obtained for polytropic and Moloney ecotropic MuLVs, respectively. Negative results with mAbs reactive with xenotropic MuLVs indicated no significant population of this class in RAW264.7 supernatants (data not shown). Thus, RAW264.7 cells express approximately equal levels of ecotropic Moloney-like and polytropic MCF MuLVs, with some variation in titer probably depending on culture conditions.
Inoculation of neonatal mice of sensitive strains with Mo-MuLV results in development of almost exclusively T cell lymphoblastic lymphomas (LL), mostly of thymic origin. To determine the pathogenic potential of harvests from RAW264.7 cells, we inoculated 1–2 day old Cr:NIH(S) (NIH Swiss) and BALB/cAnNCr (BALB/c) mice, 0.02 ml intraperitoneally and in the region of the thymus with a filtered supernatant of RAW264.7 cells or 264.7 SC-1, a harvest of SC-1 cells infected with RAW264.7 supernatant and passaged twice. For comparison, mice were similarly inoculated with Mo-MuLV (molecularly cloned and propagated in SC-1 cells). Controls were uninoculated mice of the same strains. Mice were obtained from the Division of Cancer Treatment, NCI, Frederick, MD and studied under NIAID Animal Care and Use Committee approved protocols and housing. Mice were observed for 8 to 12 months and necropsied when signs of splenomegaly, lymphadenopathy, labored breathing or lethargy were noted or the experiment was terminated at 12 months. Diagnosis was based on gross findings, microscopic examination of H&E stained formalin fixed, paraffin embedded tissues or studied by IHC using the anti-p30 antibody, anti-CD3 for T-cell lineage identification (DAKO Corporation, Carpinteria, CA Catalog # A452), and anti-PAX5 for B-cell lineage (Goat anti-Pax 5, Santa Cruz Biotechnology, Santa Cruz, CA, Catalog #sc-1974) . Criteria for histopathological diagnosis were as described .
Comparative Pathogenicity of 264.7MuLVs and Mo-MuLV in Mice
87 +/- 16
93 +/- 13
RAW264.7 cell-free supernatant
259 +/- 68
163 +/- 68
264.7-MuLV, SC-1 passage
146 +/- 24
98 +/- 14
RAW264.7 cell-free supernatant
168 +/- 53
264.7-MuLV SC-1 passage
216 +/- 84
Six of 34 264.7MuLVmix- and 2 of 16 MoMuLV-induced tumors were non-lymphoid and diagnosed as erythroleukemia based on splenomegaly with a high frequency of erythroid cells and lack of reactivity with CD3 and PAX5. These neoplasms, not usually seen following Mo-MuLV infection, may be related to the generalized hyperplasia of hematopoietic lineages, including erythroid, reported in pre-leukemic Mo-MuLV-infected mice .
A further unusual finding was a mast cell tumor, a rarely seen mouse neoplasm that cannot unequivocally be considered related to the virus inoculation.
As shown in this report, RAW264.7 cells as currently available from ATCC express ecotropic and polytropic MuLVs. The ecotropic virus has biological properties of the Mo-MuLV helper virus of the Abelson virus complex that induced the tumor from which the cell line derived. Cell-free culture supernatants containing the mixed virus population induced hematopoietic disease in newborn mice, primarily LL that were mostly of T cell type, as is characteristic of Mo-MuLV lymphomagenesis, but also some of B cell origin. This tumor-inducing potential may confound pathogenicity testing of unrelated viruses propagated in RAW264.7, especially in newborn mice. Adult mice are susceptible to infection by Mo- and other MuLVs, however [22, 23], and mixed infection of MuLVs with related and unrelated viruses may have effects not directly attributable to lymphomagenicity. For example, radiation-induced RadLV enhances expression of MHC Class I genes  as does Mo-MuLV in cell culture . Synergism between different retroviruses of low pathogenicity induces a rapidly fatal neurological disease , ecotropic MuLV potentiates LDV-related paralytic disease  and Mo-MuLV potentiates polyomavirus-induced runting syndrome .
Ecotropic MuLV infection of adult mice has been shown to increase B-cell proliferation, serum immunoglobulin M levels and expression of transcripts associated with B cell activation . Further, infection of bone marrow cells with MoMuLV or bone marrow and primary B cells by Abelson MuLV induces expression of activation-induced cytidine deaminase (AID) by activating NFκB [29, 30]. Ectopic expression of AID can result in generalized somatic hypermutation .
Such illustrations of unanticipated consequences of MuLV infection as well as conceivable disruptive effects of MuLV replication, including integration into cellular DNA and cell surface expression of MuLV antigens, suggest caution in experimental design and data interpretation in studies utilizing RAW264.7 cells.
Murine leukemia virus
Immunofluorescence focus assay
This work was supported in part by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases and Divisions of Research Services, Office of the Director, and, in part, by a NIAID contract to SoBran, Inc. We thank Dr. Torgny N. Fredrickson for helpful diagnostic consultations and discussions. We are also thankful for the excellent assistance of Elizabeth M. Williams, Lawrence J. Faucette, Frank Malik and Dr. Lily I. Cheng. Dr. Sandra Ruscetti, NCI, kindly provided the anti-Rauscher p30 goat antiserum. Dr. Bruce Chesebro, NIAID, kindly provided MAb 538.
- Raschke WC, Baird S, Ralph P, Nakoinz I: Functional macrophage cell lines transformed by Abelson leukemia virus. Cell. 1978, 15: 261-267. 10.1016/0092-8674(78)90101-0.View ArticlePubMedGoogle Scholar
- Wobus CE, Karst SM, Thackray LB, Chang K-O, Sosnovtsev SV, Belliot G, Krug JM, Green KY, Virgin HWIV: Replication of Norovirus in cell culture reveals a tropsim for dendritic cells and macrophages. PloS Biology. 2004, 2: 2076-2084. 10.1371/journal.pbio.0020432.View ArticleGoogle Scholar
- Dyer KD, Schellens IMM, Bonville CA, Martin BV, Domachowske JB, Rosenberg HF: Efficient replication of pneumonia virus of mice (PVM) in a mouse macrophage cell line. Virol Journal. 2007, 4: 48-51. 10.1186/1743-422X-4-48.View ArticleGoogle Scholar
- Shin J-J, Wall EA, Zavzavadjian JR, Santat LA, Liu J, Hwang J-I, Rebres R, Roach T, Seaman W, Simon MI, Fraser IDC: A single lentiviral vector platform for microRNA-based conditional RNA interference and coordinated gene expression. Proc Natl Acad Sci USA. 2006, 103: 13759-13764. 10.1073/pnas.0606179103.PubMed CentralView ArticlePubMedGoogle Scholar
- Evans LH, Morrison RP, Malik FG, Portis J, Britt WJ: A neutralizable epitope common to the envelope glycoproteins of ecotropic, polytropic, xenotropic, and amphotropic murine leukemia viruses. J Virol. 1990, 64 (12): 6176-6183.PubMed CentralPubMedGoogle Scholar
- Rowe WP, Pugh WE, Hartley JW: Plaque assay techniques for murine leukemia viruses. Virology. 1970, 42: 1136-1139. 10.1016/0042-6822(70)90362-4.View ArticlePubMedGoogle Scholar
- Hartley JW, Rowe WP: Clonal cell lines from a feral mouse embryo which lack host-range restriction for murine leukemia viruses. Virology. 1975, 65: 128-134. 10.1016/0042-6822(75)90013-6.View ArticlePubMedGoogle Scholar
- Sitbon M, Nishio J, Wehrly D, Lodmell D, Chesebro B: Use of a focal immunofluorescence assay on live cells for quantitation of retroviruses: distinction of host range classes in virus mixtures and biological cloning of dual-tropic murine leukemia viruses. Virology. 1985, 141: 110-118. 10.1016/0042-6822(85)90187-4.View ArticlePubMedGoogle Scholar
- Lavignon M, Evans LH: A multistep process of leukemogenesis in Moloney murine leukemia virus-infected mice that is modulated by retroviral pseudotyping and interference. J Virol. 1996, 70: 3852-3862.PubMed CentralPubMedGoogle Scholar
- Chang KSS, Log T, Bandyopadhyay AK: Characterization of xenotropic and dual-tropic type C retroviruses isolated from Abelson tumour. J Gen Virol. 1982, 58: 115-125.View ArticlePubMedGoogle Scholar
- Chesebro B, Britt W, Evans L, Wehrly K, Nishio J, Cloyd M: Characterization of monoclonal antibodies reactive with murine leukemia viruses: use in analysis of strains of Friend MCF and Friend ecotropic murine leukemia virus. Virology. 1983, 127: 134-148. 10.1016/0042-6822(83)90378-1.View ArticlePubMedGoogle Scholar
- Cloyd MW, Chesebro B, Portis JL, Weir M: MCF-specific murine monoclonal antibodies made against AKR-247 MCF virus recognize a unique determinant associated with the gp70-p15(E) complex. J Virol. 1982, 41: 1112-1117.PubMed CentralPubMedGoogle Scholar
- Ward JM, Erexson CR, Faucette LJ, Foley JF, Diikstra C, Cattoretti G: Immunohistochemical markers for the rodent immune system. Toxicol Pathol. 2006, 34: 616-630. 10.1080/01926230600941340.View ArticlePubMedGoogle Scholar
- Morse HC, Anver MR, Fredrickson TN, Haines DC, Harris AW, Harris NL, Jaffe ES, Kogan SC, MacLennan IC, Pattengale P, Ward JM: Bethesda proposals for classification of lymphoid neoplasms in mice. Blood. 2002, 100: 246-258. 10.1182/blood.V100.1.246.View ArticlePubMedGoogle Scholar
- Vasmel WLE, Zijlstra M, Radaszkiewicz T, Leupers CJM, de Goede REY, Melief CJM: Major histocompatibility complex class II-regulated immunity to murine leukemia virus protects against early T-cell but not late B-cell lymphomas. J Virol. 1988, 62: 3156-3166.PubMed CentralPubMedGoogle Scholar
- Lovmand J, Sorensen AB, Schmidt J, Ostergaard M, Luz A, Pedersen FS: B-cell lymphoma induction by Akv murine leukemia viruses harboring one or both copies of the tandem repeat in the U3 enhancer. J Virol. 1998, 72: 5745-5756.PubMed CentralPubMedGoogle Scholar
- van Lohuizen M, Verbeek S, Scheijen B, Wientjens E, van der Guiden H, Berns A: Identification of cooperating oncogenes in E μ-myc transgenic mice by provirus tagging. Cell. 1991, 65: 737-752. 10.1016/0092-8674(91)90382-9.View ArticlePubMedGoogle Scholar
- Rosenberg N, Baltimore D: A quantitative assay for transformation of bone marrow cells by Abelson leukemia virus. J Exp Med. 1976, 143: 1453-1463. 10.1084/jem.143.6.1453.View ArticlePubMedGoogle Scholar
- Tang Y, Chattopadhyay SK, Hartley JW, Fredrickson TN, Morse HC: Clonal outgrowths of T and B cells in SCID mice reconstituted with cells from mice with MAIDS. In Vivo. 1994, 8: 953-960.PubMedGoogle Scholar
- Davis BR, Brightman BK, Chandy KG, Fan H: Characterization of a preleukemic state induced by Moloney murine leukemia virus: Evidence for two infection events during leukemogenesis. Proc Natl Acad Sci. 1987, 84: 4875-4879. 10.1073/pnas.84.14.4875.PubMed CentralView ArticlePubMedGoogle Scholar
- Gisselbrecht S, Pozo F, Debre P, Hurot MA, Lacombe MJ, Levy JP: Genetic control of sensitivity to Moloney-virus-induced leukemias in mice. I. Demonstration of multigenic control. Int J Cancer. 1978, 21: 626-634. 10.1002/ijc.2910210513.View ArticlePubMedGoogle Scholar
- Lee JS, Giese NA, Elkins KL, Yetter RA, Holmes KL, Hartley JW, Morse HC: Effects of exogenous, nonleukemogenic, ecotropic leukemia virus infections on the immune systems of adult C57BL/6 mice. J Virol. 1995, 69: 4182-4188.PubMed CentralPubMedGoogle Scholar
- Meruelo D, Nimelstein SH, Jones PP, Lieberman M, McDevitt HO: Increased synthesis and expression of H-2 antigens on thymocytes as a result of radiation leukemia virus infection: A possible mechanism for H-2 linked control of virus-induced neoplasia. J Exp Med. 1978, 147: 470-487. 10.1084/jem.147.2.470.View ArticlePubMedGoogle Scholar
- Wilson LD, Flyer DC, Faller DV: Murine retroviruses control Class 1 major histocompatibility antigen gene expression via a trans effect at the transcriptional level. Mol Cell Bio. 1987, 7: 2406-2415.View ArticleGoogle Scholar
- Evans LH, Lavignob M, Peterson K, Hasenkrug K, Robertson S, Malik F, Virtaneva K: In vivo interactions of ecotropic and polytropic murine leukemia viruses in mixed virus infections. J Virol. 2006, 80: 4748-4757. 10.1128/JVI.80.10.4748-4757.2006.PubMed CentralView ArticlePubMedGoogle Scholar
- Anderson GW, Palmer GA, Rowland RRR, Even C, Plagemann PGW: Infection of central nervous system cells by ecotropic murine leukemia virus in C58 and AKR mice and in in utero-infected CE/J mice predisposes mice to paralytic infection by lactate dehydrogenase-elevating virus. J Virol. 1995, 69: 308-319.PubMed CentralPubMedGoogle Scholar
- Atencio IA, Belli B, Hobbs M, Cheng SF, Villarreal LP, Fan H: A model for mixed virus disease: co-infection with Moloney murine leukemia virus potentiates runting induced by polyomavirus (A2 strain) in BALB/c and NIH Swiss mice. Virology. 1995, 212: 356-366. 10.1006/viro.1995.1493.View ArticlePubMedGoogle Scholar
- Gourzi P, Leonova T, Papavasiliou FN: Viral induction of AID is independent of the interferon and the Toll-like receptor signalling pathways but requires NF-κB. J Exp Med. 2007, 204: 259-265. 10.1084/jem.20061801.PubMed CentralView ArticlePubMedGoogle Scholar
- Gourzi P, Leonova T, Papavasiliou FN: A role for activation-induced cytidine deaminase in the host response against a transforming retrovirus. Immunity. 2006, 24: 779-786. 10.1016/j.immuni.2006.03.021.View ArticlePubMedGoogle Scholar
- Yoshikawa K, Okazaki IM, Eto T, Kinoshita K, Muramatsu M, Nagaoka H, Honjo T: AID enzyme-induced hypermutation in an actively transcribed gene in fibroblasts. Science. 2002, 296: 2033-2036. 10.1126/science.1071556.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.