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

Indik S, G├╝nzburg 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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Related in: MedlinePlus

Alignment of a partial env gene sequences from the third infection cycle of Hs578T cells. Genomic DNA extracted from the third-round infected Hs578T cells, was used for amplification and cloning of MMTV env sequences. Sequences of sixteen clones were aligned. Mtv-2 and Mtv-17 (accession number AF263910, sequence is shaded) sequences were included in the alignment. Putative heparin binding domain (HBD) and receptor binding site (RBS) are boxed and amino acid residues representing these regions are shown above the boxes. Non-synonymous mutations in proviral sequences from infected cells resulting in an amino acid exchange are indicated. The coordinates of the Mtv2 nucleotide sequence are according to the MMTV reference strain (accession number M15122).
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Figure 9: Alignment of a partial env gene sequences from the third infection cycle of Hs578T cells. Genomic DNA extracted from the third-round infected Hs578T cells, was used for amplification and cloning of MMTV env sequences. Sequences of sixteen clones were aligned. Mtv-2 and Mtv-17 (accession number AF263910, sequence is shaded) sequences were included in the alignment. Putative heparin binding domain (HBD) and receptor binding site (RBS) are boxed and amino acid residues representing these regions are shown above the boxes. Non-synonymous mutations in proviral sequences from infected cells resulting in an amino acid exchange are indicated. The coordinates of the Mtv2 nucleotide sequence are according to the MMTV reference strain (accession number M15122).

Mentions: The sequences obtained from sixteen different plasmid clones were aligned and compared with the Mtv-2 as well as Mtv-17 env coding regions (Figure 9). Although several point mutations were detected, all the sequences could be identified as Mtv-2 sequences. Interestingly, most of the mutations were found between the putative receptor binding site (RBS) and heparin binding domains (HBD), the regions that are thought to play a role in the interaction with the receptor and hence are most likely located at the outer part of the protein [19]. Additionally, one non-synonymous mutation leading to a transition from the basic amino acid lysine to the acidic glutamic acid was identified in the putative HBD.


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

Indik S, G├╝nzburg WH, Kulich P, Salmons B, Rouault F - Retrovirology (2007)

Alignment of a partial env gene sequences from the third infection cycle of Hs578T cells. Genomic DNA extracted from the third-round infected Hs578T cells, was used for amplification and cloning of MMTV env sequences. Sequences of sixteen clones were aligned. Mtv-2 and Mtv-17 (accession number AF263910, sequence is shaded) sequences were included in the alignment. Putative heparin binding domain (HBD) and receptor binding site (RBS) are boxed and amino acid residues representing these regions are shown above the boxes. Non-synonymous mutations in proviral sequences from infected cells resulting in an amino acid exchange are indicated. The coordinates of the Mtv2 nucleotide sequence are according to the MMTV reference strain (accession number M15122).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Alignment of a partial env gene sequences from the third infection cycle of Hs578T cells. Genomic DNA extracted from the third-round infected Hs578T cells, was used for amplification and cloning of MMTV env sequences. Sequences of sixteen clones were aligned. Mtv-2 and Mtv-17 (accession number AF263910, sequence is shaded) sequences were included in the alignment. Putative heparin binding domain (HBD) and receptor binding site (RBS) are boxed and amino acid residues representing these regions are shown above the boxes. Non-synonymous mutations in proviral sequences from infected cells resulting in an amino acid exchange are indicated. The coordinates of the Mtv2 nucleotide sequence are according to the MMTV reference strain (accession number M15122).
Mentions: The sequences obtained from sixteen different plasmid clones were aligned and compared with the Mtv-2 as well as Mtv-17 env coding regions (Figure 9). Although several point mutations were detected, all the sequences could be identified as Mtv-2 sequences. Interestingly, most of the mutations were found between the putative receptor binding site (RBS) and heparin binding domains (HBD), the regions that are thought to play a role in the interaction with the receptor and hence are most likely located at the outer part of the protein [19]. Additionally, one non-synonymous mutation leading to a transition from the basic amino acid lysine to the acidic glutamic acid was identified in the putative HBD.

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