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Rapid spread of mouse mammary tumor virus in cultured human breast cells.

Indik S, Günzburg WH, Kulich P, Salmons B, Rouault F - Retrovirology (2007)

Bottom Line: The infected human cells expressed, upon cultivation with dexamethasone, MMTV structural proteins and released spiked B-type virions, the infectivity of which could be neutralized by anti-MMTV antibody.Replication of the virus was efficiently blocked by an inhibitor of reverse transcription, 3'-azido-3'-deoxythymidine.The human origin of the infected cells was confirmed by determining a number of integration sites hosting the provirus, which were unequivocally identified as human sequences.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Institute for Virology and Biomedicine, University of Veterinary Medicine Vienna, Vienna, A-1210, Austria. stanislav.indik@vu-wien.ac.at

ABSTRACT

Background: The role of mouse mammary tumor virus (MMTV) as a causative agent in human breast carcinogenesis has recently been the subject of renewed interest. The proposed model is based on the detection of MMTV sequences in human breast cancer but not in healthy breast tissue. One of the main drawbacks to this model, however, was that until now human cells had not been demonstrated to sustain productive MMTV infection.

Results: Here, we show for the first time the rapid spread of mouse mammary tumor virus, MMTV(GR), in cultured human mammary cells (Hs578T), ultimately leading to the infection of every cell in culture. The replication of the virus was monitored by quantitative PCR, quantitative RT-PCR and immunofluorescence imaging. The infected human cells expressed, upon cultivation with dexamethasone, MMTV structural proteins and released spiked B-type virions, the infectivity of which could be neutralized by anti-MMTV antibody. Replication of the virus was efficiently blocked by an inhibitor of reverse transcription, 3'-azido-3'-deoxythymidine. The human origin of the infected cells was confirmed by determining a number of integration sites hosting the provirus, which were unequivocally identified as human sequences.

Conclusion: Taken together, our results show that human cells can support replication of mouse mammary tumor virus.

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Electron microscopy of viral particles released from the infected Hs578T (A) and GR cells (B).
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Figure 6: Electron microscopy of viral particles released from the infected Hs578T (A) and GR cells (B).

Mentions: The production of MMTV particles by the third-round infected Hs578T cells was confirmed by electron microscopy. Cells cultured for five weeks in medium containing 10-6 M DEX and expressing MMTV-specific antigen as shown by immunostaining, were used as a source of the virus for electron microscopy. Negatively stained, high-speed centrifugation pellets contained particles morphologically resembling those of MMTV (Figure 6A and 6B). Characteristic prominent glycoprotein knobs on the surface of the virions, a hallmark of MMTV, were clearly visible. The eccentrically placed nucleoid, another characteristic of B-type viruses as well as the expected diameter of the particles (~130 nm), together with the fact that no comparable virus-like particles were found in non-infected cell supernatants, provided further evidence that the observed structure is MMTV. The presence of spiked virions in culture medium several months after the initial inoculation of the cells with MMTV(GR) virus is unlikely to be due to carry-over of residual virus but rather is indicative of virus replication in human cells.


Rapid spread of mouse mammary tumor virus in cultured human breast cells.

Indik S, Günzburg WH, Kulich P, Salmons B, Rouault F - Retrovirology (2007)

Electron microscopy of viral particles released from the infected Hs578T (A) and GR cells (B).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2169256&req=5

Figure 6: Electron microscopy of viral particles released from the infected Hs578T (A) and GR cells (B).
Mentions: The production of MMTV particles by the third-round infected Hs578T cells was confirmed by electron microscopy. Cells cultured for five weeks in medium containing 10-6 M DEX and expressing MMTV-specific antigen as shown by immunostaining, were used as a source of the virus for electron microscopy. Negatively stained, high-speed centrifugation pellets contained particles morphologically resembling those of MMTV (Figure 6A and 6B). Characteristic prominent glycoprotein knobs on the surface of the virions, a hallmark of MMTV, were clearly visible. The eccentrically placed nucleoid, another characteristic of B-type viruses as well as the expected diameter of the particles (~130 nm), together with the fact that no comparable virus-like particles were found in non-infected cell supernatants, provided further evidence that the observed structure is MMTV. The presence of spiked virions in culture medium several months after the initial inoculation of the cells with MMTV(GR) virus is unlikely to be due to carry-over of residual virus but rather is indicative of virus replication in human cells.

Bottom Line: The infected human cells expressed, upon cultivation with dexamethasone, MMTV structural proteins and released spiked B-type virions, the infectivity of which could be neutralized by anti-MMTV antibody.Replication of the virus was efficiently blocked by an inhibitor of reverse transcription, 3'-azido-3'-deoxythymidine.The human origin of the infected cells was confirmed by determining a number of integration sites hosting the provirus, which were unequivocally identified as human sequences.

View Article: PubMed Central - HTML - PubMed

Affiliation: Research Institute for Virology and Biomedicine, University of Veterinary Medicine Vienna, Vienna, A-1210, Austria. stanislav.indik@vu-wien.ac.at

ABSTRACT

Background: The role of mouse mammary tumor virus (MMTV) as a causative agent in human breast carcinogenesis has recently been the subject of renewed interest. The proposed model is based on the detection of MMTV sequences in human breast cancer but not in healthy breast tissue. One of the main drawbacks to this model, however, was that until now human cells had not been demonstrated to sustain productive MMTV infection.

Results: Here, we show for the first time the rapid spread of mouse mammary tumor virus, MMTV(GR), in cultured human mammary cells (Hs578T), ultimately leading to the infection of every cell in culture. The replication of the virus was monitored by quantitative PCR, quantitative RT-PCR and immunofluorescence imaging. The infected human cells expressed, upon cultivation with dexamethasone, MMTV structural proteins and released spiked B-type virions, the infectivity of which could be neutralized by anti-MMTV antibody. Replication of the virus was efficiently blocked by an inhibitor of reverse transcription, 3'-azido-3'-deoxythymidine. The human origin of the infected cells was confirmed by determining a number of integration sites hosting the provirus, which were unequivocally identified as human sequences.

Conclusion: Taken together, our results show that human cells can support replication of mouse mammary tumor virus.

Show MeSH
Related in: MedlinePlus