Unité des Rétrovirus Endogènes et Eléments Rétroïdes des Eukaryotes Supérieurs, UMR 1573 CNRS, Institut Gustave Roussy, 94805 Villejuif, France1
Author for correspondence: Thierry Heidmann. Fax +33 1 42 11 53 42. e-mail heidmann{at}igr.fr
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Abstract |
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Main text |
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To assay the immunosuppressive activity of the MPMV envelope, we used the procedure that we devised previously (Mangeney & Heidmann, 1998 ), which is illustrated in Fig. 1(a)
. Murine tumour cells (MCA205 and CL8.1 cells; see below) were transfected with an MPMV-env expression vector (pTMO; Brody et al., 1994
) (or an empty expression vector) and antibiotic-resistant cell populations were isolated. These cells expressed the stably transduced envelope vector, as illustrated in the RTPCR analysis shown in Fig. 1(b)
. The cells were then engrafted into immunocompetent mice. In a first series of experiments, we used methylcholanthrene-induced murine fibrosarcoma cells (MCA205; H-2b haplotype). Control tumour cells, when injected into an allogeneic host (five to ten BALB/c mice per group; H-2d haplotype), led to the development of small tumours, in only a limited number of engrafted animals (025%; Fig. 1c
, bottom). Under the same conditions, the MCA205 cells expressing the MPMV envelope were able to form easily detectable tumours that persisted for at least 2 weeks in a large proportion of the engrafted animals (>80%; Fig. 1c
, top). This enhancement of tumour cell growth was not observed, under identical experimental conditions, with irrelevant expression vectors encoding transmembrane proteins unrelated to retroviral envelopes (the murine CD2 and erythropoietin receptor proteins; data not shown) (Mangeney & Heidmann, 1998
). Induction of tumour formation was not due to any difference in intrinsic growth rates between the control and MPMV envelope-transduced cells, as tumour development induced by the two cell populations was identical when engrafted into a syngeneic host (C57BL/6, H-2b haplotype) (Fig. 1c
, insets).
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Previous experiments, using synthetic peptides in vitro (Cianciolo et al., 1985 ; reviewed in Oostendorp et al., 1993
; Haraguchi et al., 1997
), have suggested that a well-conserved domain of 17 amino acids, located within the transmembrane (TM) subunit of retroviral envelopes, might be responsible for the immunosuppressive effects. This domain can also be found in the MPMV envelope, at a position and with a sequence related to that of the well-characterized MoMLV sequence (yet with a five amino acid difference, see Fig. 2a
). In order to analyse the possible role of this domain in the in vivo immunosuppressive effect of the MPMV envelope, we used the D33 mutant MPMV envelope, constructed and characterized by Brody & Hunter (1992)
, which has 11 amino acids deleted from this domain (Fig. 2a
). A remarkable feature of this construct is that the D33 deletion has no deleterious effect on the levels of protein synthesis and processing, the only major difference from the wild-type envelope being a loss of interaction between the surface (SU) and TM subunits of the envelope, resulting in the release of SU from the cell surface into the culture medium (Brody & Hunter, 1992
; Brody et al., 1994
; and data not shown). Accordingly, an expression vector identical to the one for the wild-type MPMV envelope of the assay shown in Fig. 1
was used to transduce murine tumour cells with the mutant D33 envelope and to assay for tumour growth in mice. Interestingly, as illustrated in Fig. 2(b)
, the D33 mutant envelope still showed an immunosuppressive effect in the in vivo assay, although to a reduced extent compared with the wild-type envelope. This result indicates clearly that the so-called immunosuppressive domain is not the sole domain implicated in the in vivo immunosuppressive effect. This favours the involvement of several domains within the envelope protein, possibly dependent upon the overall structure of the protein. This would be consistent with the previously mentioned importance of peptide conformation for reported in vitro effects (Monell & Strand, 1994
), as well as for the much higher efficiency of complete envelope proteins versus peptides in these assays (Oostendorp et al., 1993
). Delineation of the effective domains involved in the immunosuppressive effect will probably be difficult, taking into account also that mutations within the TM moiety of retroviral envelopes are often deleterious for protein synthesis, stability or export (e.g. Brody & Hunter, 1992
; Brody et al., 1994
).
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Acknowledgments |
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References |
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Received 25 August 2000;
accepted 7 March 2001.