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Mouse mammary tumor virus (MMTV), a member of the betaretrovirus family, primarily induces mammary carcinomas in mice. Like human immunodeficiency virus (HIV), MMTV is a complex retrovirus that encodes a viral regulatory protein, Rem. Rem is a 33 kDa glycosylated protein containing an unusually long ER signal peptide (SP). MMTV SP contains all of the functional motifs for the nuclear export of MMTV unspliced/genomic RNA. SP activity requires binding to MMTV RNA. To characterize the minimal Rem-responsive element (RmRE) that overlaps the 3’ LTR, several deletion mutations were introduced in the MMTV-based reporter plasmid, pHMRluc. Results from these mutants in transient transfections revealed a 476-nt RmRE at the junction of the envelope gene and the 3’ LTR. RmRE function was not cell-type specific. The RmRE is predicted to have a complex secondary structure, similar to the Rev-responsive element (RRE) of HIV. Unlike the HIV RRE, the 3’ LTR RmRE occurs in all MMTV mRNAs, and Rem does not increase the export of unspliced RNA of the pHMRluc reporter vector. These results suggest that another RmRE near the 5’-end participates in export of MMTV genomic RNA, whereas the RmRE overlapping the 3’ LTR supports different Rem functions, such as translational regulation. Recent research has shown that SP directs Rem translation to the ER where Rem is cleaved and released into the cytoplasm. Rem mutants with ER signal peptidase cleavage site mutations completely lost function, and mutant proteins were highly unstable and mislocalized. Dominant-negative AAA ATPase p97 and Derlin-1 proteins, which are involved in the ER-associated degradation (ERAD) pathway, inhibited Rem function. Therefore, Rem is a precursor protein that is processed by ER signal peptidases. Rem then manipulates the ERAD system to retrotranslocate SP to the cytoplasm prior to nuclear entry and MMTV RNA binding. Unexpectedly, a commercial control shRNA expression vector, LK0.1, induced additional Rem, HIV-1 Rev and human T-cell leukemia virus type 1 Rex activity (called super-induction). Also, the LK0.1 vector increased protein expression levels of co-transfected genes, and the target of the shRNA was not critical. When the hairpin segment was deleted from LK0.1, the super-induction of Rem activity was greatly reduced. Deletion of cis-acting lentiviral segments also decreased protein expression levels. Although LK0.1 did not affect the levels of interferon-induced genes or eIF-2α phosphorylation, LK0.1 reduced the number of stress granules significantly. Therefore, LK0.1 may induce several cellular signaling pathways, leading to Rem super-induction. This study characterizes the minimal RmRE overlapping the 3’ MMTV LTR and reveals the unique processing of Rem and SP trafficking prior to nucleolar localization. Additional functions of MMTV Rem and other retroviruses may be discovered using studies of cellular events induced by LK0.1.

Mouse mammary tumor virus (MMTV), a member of the betaretrovirus family, primarily induces mammary carcinomas in mice. Like human immunodeficiency virus (HIV), MMTV is a complex retrovirus that encodes a viral regulatory protein, Rem. Rem is a 33 kDa glycosylated protein containing an unusually long ER signal peptide (SP). MMTV SP contains all of the functional motifs for the nuclear export of MMTV unspliced/genomic RNA. SP activity requires binding to MMTV RNA. To characterize the minimal Rem-responsive element (RmRE) that overlaps the 3’ LTR, several deletion mutations were introduced in the MMTV-based reporter plasmid, pHMRluc. Results from these mutants in transient transfections revealed a 476-nt RmRE at the junction of the envelope gene and the 3’ LTR. RmRE function was not cell-type specific. The RmRE is predicted to have a complex secondary structure, similar to the Rev-responsive element (RRE) of HIV. Unlike the HIV RRE, the 3’ LTR RmRE occurs in all MMTV mRNAs, and Rem does not increase the export of unspliced RNA of the pHMRluc reporter vector. These results suggest that another RmRE near the 5’-end participates in export of MMTV genomic RNA, whereas the RmRE overlapping the 3’ LTR supports different Rem functions, such as translational regulation. Recent research has shown that SP directs Rem translation to the ER where Rem is cleaved and released into the cytoplasm. Rem mutants with ER signal peptidase cleavage site mutations completely lost function, and mutant proteins were highly unstable and mislocalized. Dominant-negative AAA ATPase p97 and Derlin-1 proteins, which are involved in the ER-associated degradation (ERAD) pathway, inhibited Rem function. Therefore, Rem is a precursor protein that is processed by ER signal peptidases. Rem then manipulates the ERAD system to retrotranslocate SP to the cytoplasm prior to nuclear entry and MMTV RNA binding. Unexpectedly, a commercial control shRNA expression vector, LK0.1, induced additional Rem, HIV-1 Rev and human T-cell leukemia virus type 1 Rex activity (called super-induction). Also, the LK0.1 vector increased protein expression levels of co-transfected genes, and the target of the shRNA was not critical. When the hairpin segment was deleted from LK0.1, the super-induction of Rem activity was greatly reduced. Deletion of cis-acting lentiviral segments also decreased protein expression levels. Although LK0.1 did not affect the levels of interferon-induced genes or eIF-2α phosphorylation, LK0.1 reduced the number of stress granules significantly. Therefore, LK0.1 may induce several cellular signaling pathways, leading to Rem super-induction. This study characterizes the minimal RmRE overlapping the 3’ MMTV LTR and reveals the unique processing of Rem and SP trafficking prior to nucleolar localization. Additional functions of MMTV Rem and other retroviruses may be discovered using studies of cellular events induced by LK0.1.