We have identified a novel, multiple spliced, subgenomic mRNA species in MMTV producing cells of different origin containing an open reading frame encoding a 39-kDa Rev-like protein, Rem (regulator of expression of MMTV). An EGFP-Rem fusion protein is shown to be predominantly in the nucleolus. Further leptomycin B inhibits the nuclear export of nonspliced MMTV transcripts, implicating Rem in nuclear export by the Crm1 pathway in MMTV. Rem is thus reminiscent of the Rec protein from the related endogenous human retrovirus, HERV-K.

Based on integration site preferences, retroviruses can be placed into three groups. Viruses that comprise the first group, murine leukemia virus and foamy virus, integrate preferentially near transcription start sites. The second group, notably human immunodeficiency virus and simian immunodeficiency virus, preferentially targets transcription units. Avian sarcoma-leukosis virus (ASLV) and human T-cell leukemia virus (HTLV), forming the third group, show little preference for any genomic feature. We have previously shown that some human cells sustain mouse mammary tumor virus (MMTV) infection; therefore, we infected a susceptible human breast cell line, Hs578T, and, without introducing a species-specific bias, compared the MMTV integration profile to those of other retroviruses. Additionally, we infected a mouse cell line, NMuMG, and thus we could compare MMTV integration site selection in human and mouse cells. In total, we examined 468 unique MMTV integration sites. Irrespective of whether human or mouse cells were infected, no integration bias favoring transcription start sites was detected, a profile that is reminiscent of that of ASLV and HTLV. However, in contrast to ASLV and HTLV, not even a modest tendency in favor of integration within genes was observed. Similarly, repetitive sequences and genes that are frequently tagged by MMTV in mammary tumors were not preferentially targeted in cell culture either in mouse or in human cells; hence, we conclude that MMTV displays the most random dispersion of integration sites among retroviruses determined so far.

Mouse mammary tumor virus (MMTV) has long been speculated to be involved in human breast cancer and more recently in human primary biliary cirrhosis. Despite complete proviral sequences markedly homologous to MMTV being identified in human breast cancer tissue, no convincing evidence has been presented to date that MMTV can infect human cells. Using both wild-type and a genetically marked virus (MMTV-EGFP), we show here the successful infection of a number of different human cells by MMTV. Furthermore, infection of human cells is shown to be almost as efficient as the infection of murine mammary epithelial cells. Sequencing of PCR products from integrated proviruses reveals that reverse transcription and integration of the viral genome has occurred as expected. Furthermore, sequencing of two independent MMTV proviral integration sites reveal them to be present only in the human and not in the mouse genome. Infection requires an intact MMTV envelope protein and is blocked either by heat inactivation of the virus or by specific neutralizing anti-MMTV serum, ruling out a nonspecific mechanism of viral transfer. Thus, MMTV can infect human cells and this finding provides a possible explanation for the detection by others of MMTV sequences in human breast cancer patients.

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