No abstract

Expression of immunoreceptor tyrosine-based activation motif (ITAM)-containing signaling proteins is normally restricted to hematopoietic tissues. The basal activity of ITAM-containing proteins is mediated through negative regulation by coreceptors restricted to hematopoietic tissues. We have identified an ITAM signaling domain encoded within the env gene of murine mammary tumor virus (MMTV). Three-dimensional structures derived in vitro from murine cells stably transfected with MMTV env display a depolarized morphology in comparison with control mammary epithelial cells. This effect is abolished by Y>F substitution within the Env ITAM, as well as inhibitors of Syk and Src protein tyrosine kinases. Env-expressing cells bear hallmarks of cell transformation such as sensitivity to apoptosis induced by tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL) or TNFα, as well as down-regulation of E-cadherin and Keratin-18. Human normal mammary epithelial cells expressing MMTV Env also develop transformed phenotype, as typified by growth in soft agar and Matrigel invasion. These disruptions are abrogated by Y>F substitutions. We conclude that ITAM-dependent signals are generated through MMTV Env and trigger early hallmarks of transformation of mouse and human mammary epithelial cells. Therefore, these data suggest a heretofore unappreciated potential mechanism for the initiation of breast cancer and identify MMTV Env and ITAM-containing proteins in human breast tumors as probable oncoproteins.

Mouse mammary tumor virus (MMTV) is

The paramyxovirus gene end U tracts are thought to serve as templates for the addition of a 3' polyA tail to viral mRNAs. The goal of the work described here was to determine the function in transcription of the naturally occurring variability in length of the gene end U tracts of the paramyxovirus simian virus 5 (SV5). An anchored RT-PCR assay was developed to test the hypothesis that the variable U tracts template the addition of variable lengths of polyA tails to mRNAs. The results showed that although the SV5 NP, M, and SH genes encode U tracts of seven, four, and six U residues, respectively, their mRNAs contain similar polyA tails of approximately 250-290 bases. These results indicate that the variable gene end U tracts are functionally equivalent in directing polyadenylation. A reverse genetics system based on a dicistronic minigenome containing the SH-HN gene junction was used to test the hypothesis that the variable U tracks affect the efficiency of transcription termination. Minigenome templates containing an SH gene end with a long U tract of six residues (U6) directed efficient transcription termination and reinitiation at the downstream HN start site with no nucleotide preference for the downstream intergenic region. Surprisingly, truncating the SH gene end U tract to four residues (U4) did not affect SH termination but, rather, reduced downstream HN reinitiation to 20-30% of wild-type levels. Efficient HN reinitiation could be restored to mutant U4 templates in either of two ways: by increasing the U-tract length from four to six residues or by increasing the length of the intergenic region. Efficient HN reinitiation required a minimum of six bases between the last nucleotide in SH and the first nucleotide in HN. We propose that for some paramyxoviruses, the gene end U tract serves a previously unrecognized role as a spacer region between the gene end and gene start sites.

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...