Requirements for Mouse Mammary Tumor Virus Rem Signal Peptide Processing and Function

Byun, Hyewon; Halani, Nimita; Gou, Yongqiang; Nash, Andrea K.; Lozano, Mary M.; Dudley, Jaquelin P.

doi:10.1128/jvi.06197-11

Cited by 20 publications

(40 citation statements)

References 50 publications

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“…HEK 293 cells were transfected with a Rem expression vector in the presence or absence of two different proteasome inhibitors, MG-132 and lactacystin. Consistent with our published results (27, 32), Western blotting assays using SP-specific antibody showed that levels of the uncleaved Rem of 33 kDa, but not SP, increased in the presence of either inhibitor (Fig. 4A).…”

Section: Resultssupporting

confidence: 92%

“…Previous experiments indicated that uncleaved Rem is stabilized in the presence of proteasome inhibitors, whereas, after cleavage, SP levels are relatively unaffected by such inhibitors (27, 32). We now tested whether ubiquitylated forms of Rem or SP were detectable.…”

Section: Resultsmentioning

confidence: 99%

“…3B) (27). Similarly, GFP-SP expression induced pHM Rluc reporter activity by 9-fold (32), and this induction was inhibited 2.4-fold by the DN p97. Western blotting confirmed that SP was produced from both vectors, although the Rem expression vector yielded smaller amounts of SP, presumably due to increased ERAD of this precursor (27).…”

Section: Resultsmentioning

confidence: 99%

“…We have shown that a cis -acting Rem-responsive element (RmRE) in the reporter vector is necessary for SP-induced activity (31). Furthermore, Rem cleavage is required for SP function in this assay since mutations of the consensus signal peptidase recognition site prevent SP function and localization to the nucleolus (27, 29, 32, 33). SP release from microsomal membranes is independent of signal peptide peptidase (SPP) (29), which affects membrane release of some other viral signal peptides (34–36).…”

Section: Introductionmentioning

confidence: 99%

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Mouse Mammary Tumor Virus Signal Peptide Uses a Novel p97-Dependent and Derlin-Independent Retrotranslocation Mechanism To Escape Proteasomal Degradation

Byun

Das

et al. 2017

mBio

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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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Section: Resultssupporting

confidence: 92%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Mouse Mammary Tumor Virus Signal Peptide Uses a Novel p97-Dependent and Derlin-Independent Retrotranslocation Mechanism To Escape Proteasomal Degradation

Byun

Das

et al. 2017

mBio

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“…Conditions for growth of cell lines and transfections have been previously reported (30). Detailed methods and reagents are given in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Retroviral vectors elevate coexpressed protein levels in trans through cap-dependent translation

Gou

Byun

Zook

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Retroviruses cause immunodeficiency and cancer but also are used as vectors for the expression of heterologous genes. Nevertheless, optimal translation of introduced genes often is not achieved. Here we show that transfection into mammalian cells of lentiviral or gammaretroviral vectors, including those with specific shRNAs, increased expression of a cotransfected gene relative to standard plasmid vectors. Levels of most endogenous cellular proteins were unchanged. Transfer of lentiviral vector sequences into a standard plasmid conferred the ability to give increased expression of cotransfected genes (superinduction). Superinduction by the retroviral vector was not dependent on the cell type or species, the type of reporter gene, or the method of transfection. No differences were detected in the IFN, unfolded protein, or stress responses in the presence of retroviral vectors. RT-PCRs revealed that RNA levels of cotransfected genes were unchanged during superinduction, yet Western blotting, pulse labeling, and the use of bicistronic vectors showed increased cap-dependent translation of cointroduced genes. Expression of the mammalian target of rapamycin (mTOR) kinase target 4E-BP1, but not the mTOR inhibitor Torin 1, preferentially inhibited superinduction relative to basal protein expression. Furthermore, transcription of lentiviral vector sequences from a doxycycline-inducible promoter eliminated superinduction, consistent with a DNA-triggered event. Thus, retroviral DNA increased translation of cointroduced genes in trans by an mTORindependent signaling mechanism. Our experiments have broad applications for the design of retroviral vectors for transfections, DNA vaccines, and gene therapy.ranslational control is critical for mammalian cells and the viruses that infect them. For example, picornaviruses and some flaviviruses have an internal ribosomal entry site (IRES) (1) that allows cap-independent translation of viral RNAs and provides preferential translation over most host cell mRNAs (2). Structural modifications at the 5′ ends of viral RNAs, such as differences in cap methylation or absence of a cap, often distinguish viral mRNAs from their cellular counterparts (3). Multiple cellular proteins recognize foreign RNAs or DNAs, which trigger specific signaling pathways that lead to general translational arrest and/or selective viral RNA degradation (4). Many viral RNAs trigger the IFN signaling pathway and translational inhibition, but viruses encode various proteins or RNAs that mute this response (5). Recognition of foreign nucleic acids by cellular surface or cytosolic receptors also leads to signaling events that provide an innate antiviral response (6).Retroviruses are positive-sense RNA viruses that have been used extensively for introduction of genes or small hairpin RNAs (shRNAs) into cells, both in culture and for gene therapy. Unlike most RNA viruses, retroviruses replicate through a DNA intermediate and use RNA polymerase II to produce mRNAs with structures that are very similar to those of host mRN...

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Revisiting a role for a mammary tumor retrovirus in human breast cancer

Salmons¹,

Günzburg

2013

Intl Journal of Cancer

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There remains great controversy as to whether mouse mammary tumor virus (MMTV), the etiological agent of mammary cancer in mice, or a closely related human retrovirus, plays a role in the development of breast cancer in humans. On one hand, retroviruses such as human T-cell lymphotropic virus and human immunodeficiency virus (HIV) are known causative agents of cancer (in the case of HIV, albeit, indirectly), but attempts to associate other retroviruses with human cancers have been difficult. A recent, high profile, example has been the postulated involvement of another mouse virus, xenotropic murine leukemia virus-related virus, in human prostate cancer, which is now thought to be due to contamination. Here, we review some of the more recent evidence for and against the involvement of MMTV in human breast cancer and suggest future studies that may allow a definitive answer to this conundrum.Mouse mammary tumor virus (MMTV) is the etiological agent of mammary cancer in mice, 1,2 and it has long been postulated that this or a similar, MMTV-related, retrovirus is involved in breast cancer in humans. However, this remains controversial. [3][4][5][6] Indeed, this controversy was one of the reasons for a meeting held in Pisa, Italy organized by Generoso Bevilacqua and his colleagues entitled "viruses and breast cancer" last year. Although retroviruses such as human T-cell lymphotropic virus (HTLV) and human immunodeficiency virus (HIV) are known to be directly or indirectly involved in the causation of cancer, the association of other retroviruses with human cancers has been fraught with difficulties, the most notable and recent of which has been the evidence for xenotropic murine leukemia virus-related virus (XMRV) being associated with prostate cancer, where now the evidence seems to be explainable by the use of contaminated cell's DNA and even contaminated commercially available reagents. 7 The resurgence of interest in a potential role of MMTV in human breast cancer over the last 15 years has been driven by consistent reports that sequences highly homologous to MMTV can be found in up to 40% of human breast cancers, and this data have recently been summarized 8 with some groups even referring to the virus from human tumorderived cells and tissue as HMTV rather than MMTV. Moreover, virus protein expression has been reportedly detected in ten primary cultures of human breast cancer containing MMTV sequences, 9 a putative virus has been isolated and the virus cDNA partially sequenced. 10 MMTV has been shown to be able to infect human cells, 11 where it can randomly integrate its genomic information into the genome of the infected cell, a characteristic step in the retrovirus life cycle, 12 as well as produce infectious progeny capable of spreading. 13 These bona fide integrated proviruses can be detected and isolated from 298 unique integration sites isolated from infected human cells and the integrated proviruses are flanked by a duplicated, five base pair sequence, which is characteristic of MMTV. Moreover, the fla...

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Requirements for Mouse Mammary Tumor Virus Rem Signal Peptide Processing and Function

Cited by 20 publications

References 50 publications

Mouse Mammary Tumor Virus Signal Peptide Uses a Novel p97-Dependent and Derlin-Independent Retrotranslocation Mechanism To Escape Proteasomal Degradation

Mouse Mammary Tumor Virus Signal Peptide Uses a Novel p97-Dependent and Derlin-Independent Retrotranslocation Mechanism To Escape Proteasomal Degradation

Retroviral vectors elevate coexpressed protein levels in trans through cap-dependent translation

Revisiting a role for a mammary tumor retrovirus in human breast cancer

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