Structural components of mouse mammary tumor virus. II. Isolation and purification of virion polypeptides

Yagi, Mary Jane; Stutzman, R E; Robertson, Betty H.; Compans, Richard W.

doi:10.1128/jvi.26.2.448-456.1978

Cited by 6 publications

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References 17 publications

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“…Immunodiffusion assays. Two-dimensional, double-diffusion assays were performed with 0.3% Noble agar and 0.05% agarose in 0.05 M barbital-sodium buffer, pH 8.6, containing 0.035% EDTA as previously described (25). Rabbit anti-BALB/cfC3H milk-derived MMTV was applied to the plate, and readings were made for a period of up to 7 days.…”

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“…Virus samples were disrupted for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by boiling for 1 min at 100°C with 1% SDS and 1% 13-mercaptoethanol. Electrophoresis was carried out as previously described with 10-20% gradient gels and a discontinuous buffer system (24,25). Gels were processed for determination of radioactivity as described by Compans (7).…”

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Modulation of mouse mammary tumor virus production in the MJY-alpha cell line

Yagi¹,

Blair²,

Lane³

1978

J Virol

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Implantation of the mouse mammary tumor virus (MMTV)-producing mammary tumor cell line MJY-alpha into isogeneic mice elicited both humoral and Tcell responses against MMTV virion antigens. The carcinosarcomas which developed from the implanted cells showed a significant decrease in MMTV synthesis, compared with cells remaining in culture, which was detectable as early as 7 days after implantation and for five transplant generations. Electron microscopic examination of thin sections of the tumors revealed that intracytoplasmic A particles, budding particles, and cell-free MMTV B particles were all affected. However, immunofluorescence assays of tumor sections demonstrated the presence of MMTV viral antigens in the cells. Cell cultures initiated from first-, third-, and fourth-generation tumors were morphologically identical to the original in vitro cell line, although virus production was barely detectable. Analysis of the cultures by electron microscopy revealed a significant increase in MMTV virions after in vitro passage 3. Polypeptide profiles obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of virions purified from these cultures were identical to MMTV. Immunodiffusion demonstrated the cross-reactivity between these virions and MMTV particles obtained from mouse milk. In vitro treatment of MJY-alpha cell cultures with rabbit anti-MMTV antiserum resulted in a reduction of extracellular MMTV virions, as well as alterations in their sodium dodecyl sulfate-polyacrylamide gel electrophoretic polypeptide patterns.Techniques are now available for the propagation of primary and short-term murine mammary tumor cell cultures releasing mouse mammary tumor virus (MMTV;13,15,17,21,23). The levels of MMTV expression and of synthesis of particles in vitro are highly variable; stimulation of virus production in cells with preexisting MMTV expression can be obtained by addition of hydrocortisone or dexamethasone (8,13,(16)(17)(18)(19). However, synthesis of MMTV antigens and virions frequently decreases permanently during in vitro passage, and this has led to difficulties in establishing MMTV-producing cell lines. The reasons for this decline in MMTV replication are unknown, but it demonstrates the fragile nature of this host cell-virus relationship. In this study we present evidence of a transient repression of MMTV production in the mouse mammary tumor cell line MJY-alpha t Present address:MA 02115. after in vivo transplantation or in vitro treatment with antisera. The data suggest that replication of MMTV virions "modulates" in response to antisera and/or T-cell reactivity against MMTV antigens.MATERIALS AND METHODS Cells. The in vitro MJY-alpha cell line was grown as stationary cultures in T-flasks or petri dishes as previously described (21). After in vivo transplantation, primary cell cultures were initiated by using pools of MJY-alpha cell-induced tumors from transplant generations 1, 3, and 4. Tumors were minced, dissociated with saline A-trypsin-EDTA, filtered, and pelleted as previously describe...

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confidence: 99%