Mapping of the Functional Boundaries and Secondary Structure of the Mouse Mammary Tumor Virus Rem-responsive Element

Abstract

Mouse mammary tumor virus (MMTV) is a complex retrovirus that encodes at least three regulatory and accessory proteins, including Rem. Rem is required for nuclear export of unspliced viral RNA and efficient expression of viral proteins. Our previous data indicated that sequences at the envelope-3 long terminal repeat junction are required for proper export of viral RNA. To further map the Rem-responsive element (RmRE), reporter vectors containing various portions of the viral envelope gene and the 3 long terminal repeat were tested in the presence and absence of Rem in transient transfection assays. A 476-bp fragment that spans the envelope-long terminal repeat junction had activity equivalent to the entire 3-end of the mouse mammary tumor virus genome, but further deletions at the 5-or 3-ends reduced Rem responsiveness. RNase structure mapping of the full-length RmRE and a 3-truncation suggested multiple domains with local base pairing and intervening single-stranded segments. A secondary structure model constrained by these data is reminiscent of the RNA response elements of other complex retroviruses, with numerous local stem-loops and long-range base pairs near the 5-and 3-boundaries, and differs substantially from an earlier model generated without experimental constraints. Covariation analysis provides limited support for basic features of our model. Reporter assays in human and mouse cell lines revealed similar boundaries, suggesting that the RmRE does not require cell typespecific proteins to form a functional structure. Mouse mammary tumor virus (MMTV)3 has multiple regulatory and accessory genes (1, 2). The known accessory genes specify a dUTPase (3), which is believed to be involved in retroviral replication in non-dividing cells (4), as well as superantigen (Sag). Sag is a transmembrane glycoprotein that is involved in the lymphocyte-mediated transmission of MMTV from maternal milk in the gut to susceptible epithelial cells in the mammary gland (5, 6). The Sag protein expressed by endogenous (germline) MMTV proviruses has been reported to provide susceptibility to infection by exogenous MMTVs or the bacterial pathogen, Vibrio cholerae (7). These results suggest a role for MMTV Sag in the host innate immune response.MMTV recently was shown to be a complex retrovirus (1). Complex retroviruses encode RNA-binding proteins that facilitate nuclear export of unspliced viral RNA by using a leucinerich nuclear export sequence (8), which binds to chromosome region maintenance 1 (Crm1)(9), whereas simple retroviruses have a cis-acting constitutive transport element that directly interacts with components of the Tap/NXF1 pathway (10). Similar to other complex retroviruses, MMTV encodes a Revlike protein, regulator of export/expression of MMTV mRNA (Rem) (1). Rem is translated from a doubly spliced mRNA into a 33-kDa protein that contains nuclear and nucleolar localization signals as well as a predicted RNA-binding motif and leucine-rich nuclear export sequence (1, 2). Our previous experiments indicated that Rem ...

Retroviral Rem protein requires processing by signal peptidase and retrotranslocation for nuclear function

“…Both GFPRem and GFP-SP fusions are functional in a reporter assay that measures a postexport function (e.g., translation) (5). Rem-dependent reporter activity requires the NLS as well as the presence of a Rem-responsive element (RmRE) in the reporter construct (1,6). Together, these data suggest that SP binds to MMTV RNA in the nucleolus for Rem-dependent reporter activity.…”

“…1E). Previous results have shown that Rem activity in the pHMRluc assay measures a postexport function that requires both a nuclear localization signal and a binding site (RmRE) localized at the env-U3 border of MMTV RNA (1,6). Both the NLS and the RNA-binding motif are localized within SP.…”

“…S1) or in 293T cells at several different plasmid concentrations. Because SP alone is functional in the luciferase assay (6), these data indicate that both rem and env mRNAs generate functional SP.…”

“…Reporter levels were assessed per 100 μg of protein using the Dual Luciferase Assay Kit (Promega). Firefly luciferase levels were used to normalize Renilla luciferase activity for general effects on transfection efficiency and expression as previously described (6). The average of triplicate assays ± SDs containing expression constructs is reported relative to transfections containing only reporter vectors (assigned a relative value of 1).…”

“…Growth of the HEK cells (293T), human T lymphoma cells (Jurkat), GR-B2 mouse mammary tumor cells, and HC11 normal mouse mammary cell lines has been described previously (6,27). HC11 cells were infected by coculture with Jurkat cells producing the cloned provirus HYB-MTV (13).…”