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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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Virus-host junction sequences. (A) The junctions detected by LM-PCR in Hs578T cells infected with MMTV(GR). Terminal sequence of MMTV LTR (small letters) and 18 nucleotides of host flanking sequence (capital letters) are shown. Determined host sequence was mapped using a BLAT search at the UCSC Genome Bioinformatics group web page. The exact position of the host sequence amplified in LM-PCR on the chromosome is numbered according to Human Mar. 2006 (hg18) assembly. (B) Duplications of 6 bp long host provirus flanking sequences were determined. MMTV proviral sequences are boxed, inverted repeats at the end of LTRs are underlined. Duplications of host flanking sequences are indicated by large bold letters. Schematic diagram of an integrated MMTV provirus is shown below. Direct repeats of the host sequence are indicated by open arrows. Inverted repeats terminating the LTR are shown as inverted solid triangles.
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Figure 7: Virus-host junction sequences. (A) The junctions detected by LM-PCR in Hs578T cells infected with MMTV(GR). Terminal sequence of MMTV LTR (small letters) and 18 nucleotides of host flanking sequence (capital letters) are shown. Determined host sequence was mapped using a BLAT search at the UCSC Genome Bioinformatics group web page. The exact position of the host sequence amplified in LM-PCR on the chromosome is numbered according to Human Mar. 2006 (hg18) assembly. (B) Duplications of 6 bp long host provirus flanking sequences were determined. MMTV proviral sequences are boxed, inverted repeats at the end of LTRs are underlined. Duplications of host flanking sequences are indicated by large bold letters. Schematic diagram of an integrated MMTV provirus is shown below. Direct repeats of the host sequence are indicated by open arrows. Inverted repeats terminating the LTR are shown as inverted solid triangles.

Mentions: An essential step in the retroviral replication cycle is the integration of the provirus into the host genome. To investigate whether the MMTV(GR) proviral DNA is inserted in the genome of the infected human cells, thereby providing conclusive evidence of infection of human cells, we performed LM-PCR as described previously by Wu et al. [15]. Genomic DNA harvested from human and feline cells transduced in the second infection cycle was used for digestion with MseI, ligation with linker and subsequent amplification of virus-host junction sequences. A number of LM-PCR products were readily detected in both human and feline infected cell lines. Of these, five integration sites determined in the infected Hs578T cells were characterized further. All five human integrants could be unambiguously mapped to a locus in the human genome (Figure 7A).


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

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

Virus-host junction sequences. (A) The junctions detected by LM-PCR in Hs578T cells infected with MMTV(GR). Terminal sequence of MMTV LTR (small letters) and 18 nucleotides of host flanking sequence (capital letters) are shown. Determined host sequence was mapped using a BLAT search at the UCSC Genome Bioinformatics group web page. The exact position of the host sequence amplified in LM-PCR on the chromosome is numbered according to Human Mar. 2006 (hg18) assembly. (B) Duplications of 6 bp long host provirus flanking sequences were determined. MMTV proviral sequences are boxed, inverted repeats at the end of LTRs are underlined. Duplications of host flanking sequences are indicated by large bold letters. Schematic diagram of an integrated MMTV provirus is shown below. Direct repeats of the host sequence are indicated by open arrows. Inverted repeats terminating the LTR are shown as inverted solid triangles.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 7: Virus-host junction sequences. (A) The junctions detected by LM-PCR in Hs578T cells infected with MMTV(GR). Terminal sequence of MMTV LTR (small letters) and 18 nucleotides of host flanking sequence (capital letters) are shown. Determined host sequence was mapped using a BLAT search at the UCSC Genome Bioinformatics group web page. The exact position of the host sequence amplified in LM-PCR on the chromosome is numbered according to Human Mar. 2006 (hg18) assembly. (B) Duplications of 6 bp long host provirus flanking sequences were determined. MMTV proviral sequences are boxed, inverted repeats at the end of LTRs are underlined. Duplications of host flanking sequences are indicated by large bold letters. Schematic diagram of an integrated MMTV provirus is shown below. Direct repeats of the host sequence are indicated by open arrows. Inverted repeats terminating the LTR are shown as inverted solid triangles.
Mentions: An essential step in the retroviral replication cycle is the integration of the provirus into the host genome. To investigate whether the MMTV(GR) proviral DNA is inserted in the genome of the infected human cells, thereby providing conclusive evidence of infection of human cells, we performed LM-PCR as described previously by Wu et al. [15]. Genomic DNA harvested from human and feline cells transduced in the second infection cycle was used for digestion with MseI, ligation with linker and subsequent amplification of virus-host junction sequences. A number of LM-PCR products were readily detected in both human and feline infected cell lines. Of these, five integration sites determined in the infected Hs578T cells were characterized further. All five human integrants could be unambiguously mapped to a locus in the human genome (Figure 7A).

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