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 type-specific proteins to form a functional structure.
Highlights
Previous experiments indicated that mammary tumor virus (MMTV) is a complex retrovirus that encodes the regulator of export/expression of MMTV mRNA (Rem) regulatory protein from a doubly spliced mRNA [1, 2]
Rem is required for optimal export of full-length MMTV RNA from the nucleus [1], and for a post-export function that depends on sequences within the 3Ј MMTV long terminal repeat (LTR) [24] as well as the junction with the envelope gene [11, 24]
We have mapped the limits of the MMTV Rem-responsive element (RmRE) using a reporter vector based on the 3Ј-end of the MMTV genome [1, 24]
Summary
Cell Culture and Transfections—XC rat fibroblast cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal calf serum, gentamicin sulfate (50 g/ml), penicillin (100 units/ml), and streptomycin (50 g/ml). A firefly luciferase reporter vector lacking MMTV sequences was added to each transfection, and activities showed that different transfections within the same experiment had similar DNA uptake. Designed based on our previously described pHMRluc vector [1, 24] This vector contains the 3Ј-end of the MMTV genome, including part of the envelope gene and the 3Ј LTR, downstream of the cytomegalovirus promoter (Fig. 1). Previous data showed that reporter gene activity from this vector is induced by co-expression of the MMTV export protein, Rem, in trans [1]. Rem-induced luciferase activity required the presence of the envelope-3Ј LTR junction in the reporter vector, suggesting that these sequences contain the RmRE [1]. Various MMTV sequences at the envelope-LTR border were reinserted into the pHM⌬eLTRluc vector to determine the region necessary for Rem responsiveness
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