Cotranscriptional regulation of MMTV gag translation

Mouse mammary tumor virus (MMTV) has been extensively studied as a model for human breast cancer and for glucocorticoid hormone regulation of transcription. But, very little is known about MMTV viral gene expression and assembly due to lack of a tractable expression system. To study the molecular aspects of MMTV gag expression we cloned three different gag alleles into various mammalian expression vectors. We found that a silently mutated gag (SM) expressed in human and murine cells whereas MMTV gag (Mtv-1) derived from an infectious molecular clone, and human betaretroviral gag (HBRV) derived from human samples did not express. By providing viral cis-elements in the heterologous expression system, we show that MMTV requires the Rem response element and, surprisingly, splice recognition sites along with Rem in trans in order to express Gag. Since splicing of the message does not occur, our data implies that Rem interacts with the mRNA cotranscriptionally. This is the first time that retroviral expression has been shown to require both 5' and 3' splice recognition sites in addition to a viral regulator in trans.;Through chimeras between the expressed SM gag and that of Mtv-1 or HBRV, we were able to delineate a 54-nucleotide sequence in the pp21 domain of gag that is necessary and sufficient to inhibit Gag expression. By comparing different steps in the mRNA metabolism for the inhibited versus the expressed gags we show that transcription, nuclear-to-cytoplasmic transport, and stability are equivalent for all alleles, but translation does not occur. Since gag inhibition also blocked expression from a downstream IRES-GFP cassette, we conclude that the RNA is the target for inhibition. Nevertheless, direct transfection of in vitro synthesized mRNA from all three gags resulted in equivalent protein expression, suggesting a nuclear event drives inhibition. We propose a model where the inhibitory sequence in gag is recognized and bound by a nuclear factor leading to mRNA mislocalization resulting in an inability to interact with the translation machinery, and inefficient protein expression.