Browsing by Subject "MMTV"

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Characterization of the MMTV-encoded Rem protein
(2010-05) Ali, Almas Fatima, 1986-; Dudley, Jaquelin; Huibregtse, Jon M.
Mouse mammary tumor virus (MMTV) is a betaretrovirus that causes mammary tumors in mice. MMTV is the only known complex murine retrovirus and encodes Rem, an HIV-1 Rev-like protein. Rem is a 301-amino-acid (33 kDa) protein that is cotranslationally targeted to the ER, where the first 98 amino acids constitute the signal peptide (SP). The SP is cleaved and retrotranslocated to the cytoplasm prior to nuclear entry. In this thesis, the results show that the presence of a leucine at position 71 allows more efficient cleavage of SP and increases Rem activity. Further, in Rem-transfected cells, the majority of SP appears in the nuclear fraction, consistent with fluorescent microscopy data. The C-terminal fragment of Rem (RemCT) is glycosylated in the ER and, although glycosylation sites are present outside the SP, mutations of both these sites abolish SP activity in a reporter assay. Indirect evidence suggests that unglycosylated RemCT is degraded by the proteasome, whereas glycosylated RemCT is likely secreted out of the cell. A variant of MMTV (TBLV) that lacks functional Sag and RemCT has been prepared and will be studied in mice to elucidate the role of RemCT in vivo. Development of an antibody to RemCT will allow tracking of the protein in virus-producing cells. Although there are many other similarities between complex retroviruses like HIV-1 and MMTV, current evidence suggests that Rem lacks an HIV Tat-like transactivator function.
Function and trafficking of the MMTV-encoded Rem gene product
(2011-05) Byun, Hyewon; Dudley, Jaquelin
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.
MMTV encodes a protein antagonist of multiple Apobec family members
(2018-12-04) Bhupindersingh, Gurvani; Dudley, Jaquelin; Tucker, Haley; Sullivan, Christopher; Vasquez, Karen; Ehrlich, Lauren
Mouse Mammary Tumor Virus (MMTV) is a member of the genus Betaretrovirus in the family Retroviridae. MMTV, which primarily induces mammary carcinomas, has been extensively used as a model system to study human breast cancers. Due to its complex organization similar to human retroviruses, such as human immunodeficiency virus type 1(HIV-1) [12], studies of MMTV pathogenesis greatly aid in understanding viral interactions with the natural host immune system. MMTV encodes a 33 kDa regulatory protein, Rem, which is synthesized from a doubly spliced form of viral genomic RNA [23, 48]. The precursor Rem is targeted to the endoplasmic reticulum (ER) for translation and is cleaved by signal peptidase into an N-terminal Signal Peptide (SP) and a C-terminal protein (Rem-CT) [50]. Previous studies have demonstrated that SP (also cleaved from envelope precursor protein) can manipulate the ER-associated degradation pathway (ERAD) and retrotranslocate to the cytoplasm prior to nuclear entry to facilitate export of MMTV RNA [24]. SP thus has an analogous function to HIV-encoded Rev protein. The role of Rem-CT remains unknown. Cytidine deaminases belonging to the apolipoprotein B messenger RNA editing catalytic polypeptide (APOBEC/Apobec) family are restriction factors for human and mouse retroviruses, respectively, as well as for retrotransposons and some DNA viruses [129, 132, 141-150, 162-166]. Murine Apobec3 (mA3)-deficient mice are more susceptible to MMTV infection [179], yet no known MMTV-encoded antagonist of mA3 has been identified. MMTV requires replication through B and T lymphocytes [33, 34, 37], both of which express Apobec family proteins, prior to mammary gland infection and tumorigenesis [93, 97]. Experiments were performed to address the role of Rem C-terminal sequences to antagonize Apobec enzymes and facilitate virus replication in vivo. Previous results have shown that mice infected with Rem-null MMTV developed mammary tumors with a lower incidence and increased latency compared to those infected with wild-type MMTV. Absence of Rem during in vivo infection resulted in lower proviral loads and increased mutations of the viral genome in mammary tumors. These differences were abolished in mice deficient for Activation-Induced Cytidine Deaminase (AID), a primordial member of the Apobec family that plays a role in shaping the adaptive immune response and development of human B-cell lymphomas. Tissue culture experiments revealed that Rem expression targets AID for proteasomal degradation. This study provides evidence that Rem is an HIV-Vif like protein and the first protein inhibitor of AID. In addition, these results suggest that Rem may antagonize other Apobec enzymes
Unique trafficking of mouse mammary tumor virus rem protein and its effects on ERAD and adaptive immunity
(2017-08) Aleman, Alex, Jr.; Dudley, Jaquelin; Upton, Jason
Protein quality control is of the utmost importance within the cell. Misfolded proteins and misassembled protein complexes lead to protein aggregates that have been associated with disease states such as Alzheimer’s, Parkinson’s, and cancer. Studies of the retrovirus MMTV have revealed a novel protein, Rem, with a unique trafficking scheme that subverts host endoplasmic reticulum-associated degradation (ERAD). At least a third of cellular proteins are synthesized in association with the ER and mishaps in ERAD further contribute to the formation of disease-associated protein aggregates. Utilizing a novel protein technology, ubiquitin- activated interaction traps or UBAITs, additional evidence for a novel, p97-dependent, Derlin- independent ERAD mechanism has been attained. These studies have potential application for manipulation of ERAD and protein quality control for therapeutic and anti-viral benefits. Additional studies of Rem have revealed a possible antagonistic function on the adaptive immune system. AID is the enzyme responsible for somatic hypermutation and class switch recombination at immunoglobulin loci in activated B cells. Studies in our lab have shown that RemCT, a cleaved product of Rem with a unique trafficking scheme, results in degradation AID in cell culture. Further, the lack of RemCT in virally-infected mice leads to the accumulation of AID-like mutations within proviral sequences from MMTV-induced mammary and T-cell tumors. Here I present a novel screen utilizing the new UBAIT technology, which identified a direct interaction between MMTV-encoded SP and the ERAD component, p97. The UBAIT strategy also suggested host proteins affecting AID stability in the presence and absence of the Rem C-terminus.