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Mouse mammary tumor virus (MMTV) is a complex murine retrovirus that encodes an HIV Rev-like export protein, Rem, from a doubly spliced version of envelope (Env) mRNA. Previously, the Nterminal 98-amino acid sequence of Rem, which is identical to Env signal peptide (SP), and full-length Rem were shown to be functional in a reporter assay that measures a postexport function. Here we show that MMTV-infected cells or cells transfected with rem or env cDNAs express SP, which is the active component in the reporter assay. Uncleaved Rem was partially glycosylated, but mutations in both glycosylation sites within the C terminus prevented Rem function. Mutations that reduced Rem or Env cleavage by signal peptidase greatly reduced SP levels and functional activity in the reporter assay and allowed accumulation of the uncleaved protein. Fluorescence microscopy revealed that GFP-tagged cleavagesite mutants are unstable and lack fluorescence compared with wild-type Rem, suggesting improper folding. Proteasome inhibitors allowed accumulation of uncleaved Rem relative to SP and increased reporter activity, consistent with SP retrotranslocation and proteasome escape before nuclear entry. Expression of a dominant-negative p97 ATPase did not alter levels of unprocessed Rem and SP but decreased reporter activity, suggesting p97-facilitated retrotranslocation of SP. Our results provide an example of a SP that is processed by signal peptidase and retrotranslocated to allow nuclear localization and function. mouse mammary tumor virus | retrotranslocation | retrovirus | signal peptide | ERAD M ouse mammary tumor virus (MMTV) has provided many insights into cell and cancer biology. MMTV is a complex mouse retrovirus with regulatory features similar to those found in human complex retroviruses, such as HIV and human T-cell leukemia virus (HTLV) (1). These features include the ability to manipulate the immune system and to produce a doubly spliced mRNA encoding a protein (Rem) functionally similar to HIV Rev (1, 2). Rem mRNA is translated in the same reading frame as the MMTV envelope (env) gene but has a deletion of internal sequences encoding the surface (SU) and transmembrane (TM) proteins (1, 2) (Fig. 1A).MMTV Rem is a 301-amino acid protein that regulates expression and export of full-length viral RNA from the nucleus to the cytoplasm using the Crm1 export pathway (1, 2). The Rem protein contains several Rev-like motifs, including a nuclear localization signal (NLS), a nucleolar localization sequence, an arginine-rich RNA-binding motif, and a leucine-rich nuclear export sequence (Fig. 1A). These motifs map to the N-terminal 98 amino acids, which are identical in sequence to the signal peptide (SP) of the MMTV Env protein (1, 2). Similar to HIV Rev or HTLV-1 Rex (3, 4), GFP fusions to Rem or the SP are localized to nucleoli. 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-re...

Endoplasmic reticulum (ER)-associated degradation (ERAD) is a universally important process among eukaryotic cells. ERAD is necessary to preserve cell integrity since the accumulation of defective proteins results in diseases associated with neurological dysfunction, cancer, and infections. This process involves recognition of misfolded or misassembled proteins that have been translated in association with ER membranes. Recognition of ERAD substrates leads to their extraction through the ER membrane (retrotranslocation or dislocation), ubiquitination, and destruction by cytosolic proteasomes. This review focuses on ERAD and its components as well as how viruses use this process to promote their replication and to avoid the immune response.

Complex human-pathogenic retroviruses cause high morbidity and mortality worldwide, but resist antiviral drugs and vaccine development due to evasion of the immune response. A complex retrovirus, mouse mammary tumor virus (MMTV), requires replication in B and T lymphocytes for mammary gland transmission and is antagonized by the innate immune restriction factor murine Apobec3 (mA3). To determine whether the regulatory/accessory protein Rem affects innate responses to MMTV, a splice-donor mutant (MMTV-SD) lacking Rem expression was injected into BALB/c mice. Mammary tumors induced by MMTV-SD had a lower proviral load, lower incidence, and longer latency than mammary tumors induced by wild-type MMTV (MMTV-WT). MMTV-SD proviruses had many G-to-A mutations on the proviral plus strand, but also C-to-T transitions within WRC motifs. Similarly, a lymphomagenic MMTV variant lacking Rem expression showed decreased proviral loads and increased WRC motif mutations relative to those in wild-type-virus-induced tumors, consistent with activation-induced cytidine deaminase (AID) mutagenesis in lymphoid cells. These mutations are typical of the Apobec family member AID, a B-cell-specific mutagenic protein involved in antibody variable region hypermutation. In contrast, mutations in WRC motifs and proviral loads were similar in MMTV-WT and MMTV-SD proviruses from tumors in AID-insufficient mice. AID was not packaged in MMTV virions. Rem coexpression in transfection experiments led to AID proteasomal degradation. Our data suggest that rem specifies a human-pathogenic immunodeficiency virus type 1 (HIV-1) Vif-like protein that inhibits AID and antagonizes innate immunity during MMTV replication in lymphocytes. IMPORTANCE Complex retroviruses, such as human-pathogenic immunodeficiency virus type 1 (HIV-1), cause many human deaths. These retroviruses produce lifelong infections through viral proteins that interfere with host immunity. The complex retrovirus mouse mammary tumor virus (MMTV) allows for studies of host-pathogen interactions not possible in humans. A mutation preventing expression of the MMTV Rem protein in two different MMTV strains decreased proviral loads in tumors and increased viral genome mutations typical of an evolutionarily ancient enzyme, AID. Although the presence of AID generally improves antibody-based immunity, it may contribute to human cancer progression. We observed that coexpression of MMTV Rem and AID led to AID destruction. Our results suggest that Rem is the first known protein inhibitor of AID and that further experiments could lead to new disease treatments.

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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 ...

Multiple pathogens, including viruses and bacteria, manipulate endoplasmic reticulum-associated degradation (ERAD) to avoid the host immune response and promote their replication. The betaretrovirus mouse mammary tumor virus (MMTV) encodes Rem, which is a precursor protein that is cleaved into a 98-amino-acid signal peptide (SP) and a C-terminal protein (Rem-CT). SP uses retrotranslocation for ER membrane extraction and yet avoids ERAD by an unknown mechanism to enter the nucleus and function as a Rev-like protein. To determine how SP escapes ERAD, we used a ubiquitin-activated interaction trap (UBAIT) screen to trap and identify transient protein interactions with SP, including the ERAD-associated p97 ATPase, but not E3 ligases or Derlin proteins linked to retrotranslocation, polyubiquitylation, and proteasomal degradation of extracted proteins. A dominant negative p97 ATPase inhibited both Rem and SP function. Immunoprecipitation experiments indicated that Rem, but not SP, is polyubiquitylated. Using both yeast and mammalian expression systems, linkage of a ubiquitin-like domain (UbL) to SP or Rem induced degradation by the proteasome, whereas SP was stable in the absence of the UbL. ERAD-associated Derlin proteins were not required for SP activity. Together, these results suggested that Rem uses a novel p97-dependent, Derlin-independent retrotranslocation mechanism distinct from other pathogens to avoid SP ubiquitylation and proteasomal degradation.

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