Mouse mammary tumor virus (MMTV) is a betaretrovirus that infects rodent cells and usesAlthough the betaretrovirus mouse mammary tumor virus (MMTV) was identified as a transmissible agent that causes breast cancer in mice more than 70 years ago, an understanding of how the virus infects cells has lagged behind that for other retroviruses (32). This was primarily due to a lack of cell lines that produced high-titer infectious virus and of a measurable assay to determine infection. Several years ago, murine leukemia virus (MLV) pseudotypes bearing the MMTV Env protein were developed, and these have greatly facilitated the study of MMTV interaction with cells (12, 18). For example, we recently used MMTV pseudotypes to identify mouse transferrin receptor 1 (TfR1) as the cell entry receptor for this virus (40).The use of TfR1 for virus entry is the first reported case of an enveloped virus attachment protein having a primary function that is directly related to endocytosis. In normal cell physiology, binding of transferrin to the TfR1 results in endocytosis of the complex via clathrin-coated pits that traffic to the acidic endosomal compartment, where iron is released; this is the predominant pathway used by this receptor after endocytosis (28,36). It was previously shown that treatment of MMTVinfected mammary gland cells with acid induced cell-cell fusion (39), and we have found that MMTV pseudovirus infection is dependent on acidified endosomes for infection (40). Although neutralization of the endosomal compartment does not affect infection by most retroviruses, it does inhibit infection by avian leukosis virus (31) as well as other viruses such as influenza virus, Semliki Forest virus, and vesicular stomatitis virus (20). At least for influenza virus, it is this pH change and not receptor binding that induces the conformational change leading to fusion of the virus and plasma membranes (43). Given that MMTV uses a receptor that traffics to the acidic endosome upon ligand binding and that acid pH triggers MMTV-mediated cell-cell fusion, it may be acid pH that triggers the conformational change in the MMTV Env protein as well, thereby allowing virus-cell membrane fusion.Although the TfR1s of different species are highly homologous, MMTV shows clear species tropism. MMTV pseudotypes infect only rodent, not hamster or human, tissue culture cells (12,40). This tropism resides at the level of TfR1, since human and hamster cells expressing the mouse TrfR cDNA are highly susceptible to MMTV pseudotype infection (40). However, there have been reports that continued passage of MMTV on human breast cancer cell lines resulted in adapted viruses that could infect human cells (22,26,45). In addition, several groups have recently identified MMTV-like Env sequences in human breast cancer specimens (14, 46). The Env sequences of different MMTV strains are highly homologous to each other and to the MMTV-like env genes cloned from human breast cancer samples (see Fig. 3). Thus, any sequence differences between these various Envs, e...