Publications by authors named "Jaquelin P Dudley"

31 Publications

Unconventional p97/VCP-Mediated ER to Endosomal Trafficking of a Retroviral Protein.

J Virol 2021 May 5. Epub 2021 May 5.

Department of Molecular Biosciences, Center for Infectious Disease, and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX

Mouse mammary tumor virus (MMTV) encodes a Rem precursor protein that specifies both regulatory and accessory functions. Rem is cleaved at the ER membrane into a functional N-terminal signal peptide (SP) and the C-terminus (Rem-CT). Rem-CT lacks a membrane-spanning domain and a known ER retention signal, yet was not detectably secreted into cell supernatants. Inhibition of intracellular trafficking by the drug Brefeldin A (BFA), which interferes with the ER to Golgi secretory pathway, resulted in dramatically reduced intracellular Rem-CT levels that were not rescued by proteasomal or lysosomal inhibitors. A Rem mutant lacking glycosylation was cleaved into SP and Rem-CT, but was insensitive to BFA, suggesting that unglycosylated Rem-CT does not reach this BFA-dependent compartment. Treatment with Endoglycosidase H indicated that Rem-CT does not traffic through the Golgi. Analysis of wild-type Rem-CT and its glycosylation mutant by confocal microscopy revealed that both were primarily localized to the ER lumen. A small fraction of wild-type Rem-CT, but not the unglycosylated mutant, was co-localized with Rab5+ early endosomes. Expression of a dominant-negative (DN) form of ADP ribosylation factor 1 (Arf1) (T31N) mimicked the effects of BFA by reducing Rem-CT levels and increased Rem-CT association with early and late endosomes. Inhibition of the AAA ATPase, p97/VCP, rescued Rem-CT in the presence of BFA or DN Arf1 and prevented localization to Rab5+ endosomes. Thus, Rem-CT uses an unconventional p97-mediated scheme for trafficking to early endosomes.Mouse mammary tumor virus is a complex retrovirus that encodes a regulatory/accessory protein, Rem. Rem is a precursor protein that is processed at the endoplasmic reticulum (ER) membrane by signal peptidase. The N-terminal SP uses the p97/VCP ATPase to elude ER-associated degradation to traffic to the nucleus and serve a human immunodeficiency virus Rev-like function. In contrast, the function of the C-terminal glycosylated cleavage product (Rem-CT) is unknown. Since localization is critical for protein function, we used mutants, inhibitors, and confocal microscopy to localize Rem-CT. Surprisingly, Rem-CT, which lacks a transmembrane domain or an ER retention signal, was detected primarily within the ER and required glycosylation and the p97 ATPase for early endosome trafficking without passage through the Golgi. Thus, Rem-CT uses a novel intracellular trafficking pathway, potentially impacting host anti-viral immunity.
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http://dx.doi.org/10.1128/JVI.00531-21DOI Listing
May 2021

The Role of APOBECs in Viral Replication.

Microorganisms 2020 Nov 30;8(12). Epub 2020 Nov 30.

Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712, USA.

Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) proteins are a diverse and evolutionarily conserved family of cytidine deaminases that provide a variety of functions from tissue-specific gene expression and immunoglobulin diversity to control of viruses and retrotransposons. APOBEC family expansion has been documented among mammalian species, suggesting a powerful selection for their activity. Enzymes with a duplicated zinc-binding domain often have catalytically active and inactive domains, yet both have antiviral function. Although APOBEC antiviral function was discovered through hypermutation of HIV-1 genomes lacking an active Vif protein, much evidence indicates that APOBECs also inhibit virus replication through mechanisms other than mutagenesis. Multiple steps of the viral replication cycle may be affected, although nucleic acid replication is a primary target. Packaging of APOBECs into virions was first noted with HIV-1, yet is not a prerequisite for viral inhibition. APOBEC antagonism may occur in viral producer and recipient cells. Signatures of APOBEC activity include G-to-A and C-to-T mutations in a particular sequence context. The importance of APOBEC activity for viral inhibition is reflected in the identification of numerous viral factors, including HIV-1 Vif, which are dedicated to antagonism of these deaminases. Such viral antagonists often are only partially successful, leading to APOBEC selection for viral variants that enhance replication or avoid immune elimination.
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http://dx.doi.org/10.3390/microorganisms8121899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760323PMC
November 2020

A Protein Antagonist of Activation-Induced Cytidine Deaminase Encoded by a Complex Mouse Retrovirus.

mBio 2019 08 13;10(4). Epub 2019 Aug 13.

Dept. of Molecular Biosciences, LaMontagne Center for Infectious Disease, and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA

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 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. 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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http://dx.doi.org/10.1128/mBio.01678-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692512PMC
August 2019

Methods for detecting Zika virus in feces: A case study in captive squirrel monkeys (Saimiri boliviensis boliviensis).

PLoS One 2018 20;13(12):e0209391. Epub 2018 Dec 20.

Department of Molecular Biosciences, Institute for Cellular and Molecular Biology and LaMontagne Center for Infectious Disease, University of Texas at Austin, Austin, Texas, United States of America.

A strain of Zika virus (ZIKV) of Asian origin associated with birth defects and neurological disorders has emerged and spread through the Americas. ZIKV was first isolated in the blood of nonhuman primates in Africa and has been detected in the blood, saliva, and urine of a few catarrhine species in both Africa and Asia, suggesting that nonhuman primates may serve as both a source and a reservoir of the virus. The recent introduction of ZIKV to human populations in the Americas presents the potential for the virus to spread into nonhuman primate reservoirs. Thus, it is critical to develop efficient and noninvasive detection methods to monitor the spread of the virus in wild nonhuman primate populations. Here, we describe a method for ZIKV detection in noninvasively collected fecal samples of a Neotropical primate. Fecal samples were collected from two captive squirrel monkeys (Saimiri boliviensis boliviensis) that were experimentally infected with ZIKV (Strain Mexico_1_44) and an additional two uninfected squirrel monkeys. Nucleic acids were extracted from these samples, and RT-qPCR was used to assay for the presence of ZIKV using primers flanking a 101 bp region of the NS5 gene. In both ZIKV-inoculated animals, ZIKV was detected 5-11 days post-infection, but was not detected in the uninfected animals. We compare the fecal results to ZIKV detection in serum, saliva, and urine samples from the same individuals. Our results indicate that fecal detection is a cost-effective, noninvasive method for monitoring wild populations of Neotropical primates as possible ZIKV reservoirs.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0209391PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6301608PMC
May 2019

A cis-Acting Element Downstream of the Mouse Mammary Tumor Virus Major Splice Donor Critical for RNA Elongation and Stability.

J Mol Biol 2018 10 1;430(21):4307-4324. Epub 2018 Sep 1.

Department of Biochemistry, College of Medicine and Health Sciences, UAE University, Tawam Hospital Complex, P.O. Box 17666, Al Ain, United Arab Emirates. Electronic address:

Background: The mouse mammary tumor virus (MMTV) encodes a functional signal peptide, a cleavage product of envelope and Rem proteins. Signal peptide interacts with a 3' cis-acting RNA element, the Rem-responsive element (RmRE), to facilitate expression of both unspliced genomic (gRNA) and spliced mRNAs. An additional RmRE has been proposed at the 5' end of the genome, facilitating nuclear export of the unspliced gRNA, whereas the 3' RmRE could facilitate translation of all other mRNAs, including gRNA.

Results: To address this hypothesis, a series of mutations were introduced into a 24-nt region found exclusively in the unspliced gRNA. Mutant clones using MMTV or human cytomegalovirus promoters were tested in both transient and stable transfections to determine their effect on gRNA nuclear export, stability, and translation. Nuclear export of the gRNA was affected only in a small mutant subset in stably transfected Jurkat T cells. Quantitative real-time RT-PCR of actinomycin D-treated cells expressing MMTV revealed that multiple mutants were severely compromised for RNA expression and stability. Both genomic and spliced nuclear RNAs were reduced, leading to abrogation of Gag and Env protein expressed from unspliced and spliced mRNAs, respectively. RT-PCRs with multiple primer pairs indicated failure to elongate genomic MMTV transcripts beyond ~500 nt compared to the wild type in a cell line-dependent manner.

Conclusions: MMTV contains a novel cis-acting element downstream of the major splice donor critical for facilitating MMTV gRNA elongation and stability. Presence of a mirror repeat within the element may represent important viral/host factor binding site(s) within MMTV gRNA.
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http://dx.doi.org/10.1016/j.jmb.2018.08.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6193448PMC
October 2018

MMTV does not encode viral microRNAs but alters the levels of cancer-associated host microRNAs.

Virology 2018 01 5;513:180-187. Epub 2017 Nov 5.

Department of Biochemistry, College of Medicine and Health Sciences, UAE University, Tawam Hospital Complex, P.O. Box 17666, Al Ain, United Arab Emirates. Electronic address:

Mouse mammary tumor virus (MMTV) induces breast cancer in mice in the absence of known virally-encoded oncogenes. Tumorigenesis by MMTV is thought to occur primarily through insertional mutagenesis, leading to the activation of cellular proto-oncogenes and outgrowth of selected cells. Here we investigated whether MMTV encodes microRNAs (miRNAs) and/or modulates host miRNAs that could contribute to tumorigenesis. High throughput small RNA sequencing analysis of MMTV-infected cells and MMTV-induced mammary tumors demonstrates that MMTV does not encode miRNAs. However, infected tissues have altered levels of several host miRNAs, including increased expression of members of the oncogenic miRNA cluster, miR-17-92. Notably, similar changes in miRNA levels have been previously reported in human breast cancers. Combined, our results demonstrate that virally encoded miRNAs do not contribute to MMTV-mediated tumorigenesis, but that changes in specific host miRNAs in infected cells may contribute to virus replication and tumor biology.
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http://dx.doi.org/10.1016/j.virol.2017.09.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258203PMC
January 2018

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

mBio 2017 03 28;8(2). Epub 2017 Mar 28.

Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas, USA

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. Bacterial and viral infections produce pathogen-specific proteins that interfere with host functions, including the immune response. Mouse mammary tumor virus (MMTV) is a model system for studies of human complex retroviruses, such as HIV-1, as well as cancer induction. We have shown that MMTV encodes a regulatory protein, Rem, which is cleaved into an N-terminal signal peptide (SP) and a C-terminal protein (Rem-CT) within the endoplasmic reticulum (ER) membrane. SP function requires ER membrane extraction by retrotranslocation, which is part of a protein quality control system known as ER-associated degradation (ERAD) that is essential to cellular health. Through poorly understood mechanisms, certain pathogen-derived proteins are retrotranslocated but not degraded. We demonstrate here that MMTV SP retrotranslocation from the ER membrane avoids degradation through a unique process involving interaction with cellular p97 ATPase and failure to acquire cellular proteasome-targeting sequences.
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http://dx.doi.org/10.1128/mBio.00328-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5371415PMC
March 2017

Lessons Learned from Mouse Mammary Tumor Virus in Animal Models.

ILAR J 2016 ;57(1):12-23

Jaquelin P. Dudley, PhD, is a professor in the Department of Molecular Biosciences, Center for Infectious Disease and Institute for Cellular and Molecular Biology at the University of Texas at Austin. Tatyana V. Golovkina, PhD, is a professor in the Department of Microbiology at the University of Chicago in Chicago, Illinois. Susan R. Ross, PhD, is a professor in the Department of Microbiology in the Perelman School of Medicine of the University of Pennsylvania in Philadelphia, Pennsylvania.

Mouse mammary tumor virus (MMTV), which was discovered as a milk-transmitted, infectious, cancer-inducing agent in the 1930s, has been used as an animal model for the study of retroviral infection and transmission, antiviral immune responses, and breast cancer and lymphoma biology. The main target cells for MMTV infection in vivo are cells of the immune system and mammary epithelial cells. Although the host mounts an immune response to the virus, MMTV has evolved multiple means of evading this response. MMTV causes mammary tumors when the provirus integrates into the mammary epithelial and lymphoid cell genome during viral replication and thereby activates cellular oncogene expression. Thus, tumor induction is a by-product of the infection cycle. A number of important oncogenes have been discovered by carrying out MMTV integration site analysis, some of which may play a role in human breast cancer.
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http://dx.doi.org/10.1093/ilar/ilv044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5007637PMC
January 2017

APOBECs and virus restriction.

Virology 2015 May 26;479-480:131-45. Epub 2015 Mar 26.

Department of Molecular Biosciences, Center for Infectious Disease, and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712, United States. Electronic address:

The APOBEC family of single-stranded DNA cytosine deaminases comprises a formidable arm of the vertebrate innate immune system. Pre-vertebrates express a single APOBEC, whereas some mammals produce as many as 11 enzymes. The APOBEC3 subfamily displays both copy number variation and polymorphisms, consistent with ongoing pathogenic pressures. These enzymes restrict the replication of many DNA-based parasites, such as exogenous viruses and endogenous transposable elements. APOBEC1 and activation-induced cytosine deaminase (AID) have specialized functions in RNA editing and antibody gene diversification, respectively, whereas APOBEC2 and APOBEC4 appear to have different functions. Nevertheless, the APOBEC family protects against both periodic viral zoonoses as well as exogenous and endogenous parasite replication. This review highlights viral pathogens that are restricted by APOBEC enzymes, but manage to escape through unique mechanisms. The sensitivity of viruses that lack counterdefense measures highlights the need to develop APOBEC-enabling small molecules as a new class of anti-viral drugs.
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http://dx.doi.org/10.1016/j.virol.2015.03.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4424171PMC
May 2015

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

Proc Natl Acad Sci U S A 2015 Mar 3;112(11):3505-10. Epub 2015 Mar 3.

Department of Molecular Biosciences, Center for Infectious Disease, and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, TX 78712-1191

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 mTOR-independent signaling mechanism. Our experiments have broad applications for the design of retroviral vectors for transfections, DNA vaccines, and gene therapy.
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http://dx.doi.org/10.1073/pnas.1420477112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4371913PMC
March 2015

ERAD and how viruses exploit it.

Front Microbiol 2014 3;5:330. Epub 2014 Jul 3.

Department of Molecular Biosciences, Center for Infectious Diseases and Institute for Cellular and Molecular Biology, The University of Texas at Austin Austin, TX, USA.

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.
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http://dx.doi.org/10.3389/fmicb.2014.00330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4080680PMC
July 2014

IL-2-independent and TNF-α-dependent expansion of Vβ5+ natural regulatory T cells during retrovirus infection.

J Immunol 2013 Jun 3;190(11):5485-95. Epub 2013 May 3.

Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840, USA.

Friend virus infection of mice induces the expansion and activation of regulatory T cells (Tregs) that dampen acute immune responses and promote the establishment and maintenance of chronic infection. Adoptive transfer experiments and the expression of neuropilin-1 indicate that these cells are predominantly natural Tregs rather than virus-specific conventional CD4(+) T cells that converted into induced Tregs. Analysis of Treg TCR Vβ chain usage revealed a broadly distributed polyclonal response with a high proportionate expansion of the Vβ5(+) Treg subset, which is known to be responsive to endogenous retrovirus-encoded superantigens. In contrast to the major population of Tregs, the Vβ5(+) subset expressed markers of terminally differentiated effector cells, and their expansion was associated with the level of the antiviral CD8(+) T cell response rather than the level of Friend virus infection. Surprisingly, the expansion and accumulation of the Vβ5(+) Tregs was IL-2 independent but dependent on TNF-α. These experiments reveal a subset-specific Treg induction by a new pathway.
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http://dx.doi.org/10.4049/jimmunol.1202951DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3739475PMC
June 2013

Requirements for mouse mammary tumor virus Rem signal peptide processing and function.

J Virol 2012 Jan 9;86(1):214-25. Epub 2011 Nov 9.

Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712, USA.

Mouse mammary tumor virus (MMTV) encodes a Rev-like protein, Rem, which is involved in the nuclear export and expression of viral RNA. Previous data have shown that all Rev-like functions are localized to the 98-amino-acid signal peptide (SP) at the N terminus of MMTV Rem or envelope proteins. MMTV-SP uses endoplasmic reticulum-associated degradation (ERAD) for protein trafficking. Rem cleavage by signal peptidase in the ER is necessary for MMTV-SP function in a reporter assay, but many requirements for trafficking are not known. To allow detection and localization of both MMTV-SP and the C-terminal cleavage product, we prepared plasmids expressing green fluorescent protein (GFP) tags. N-terminal Rem tagging led to protein accumulation relative to untagged Rem and allowed signal peptidase cleavage but reduced its specific activity. C-terminal tagging also led to Rem accumulation yet dramatically reduced cleavage, GFP fluorescence, and activity relative to N-terminally tagged Rem (GFPRem). Substitutions of an invariant leucine at position 71 between the known RNA-binding and nuclear export sequences interfered with GFPRem accumulation and activity but not cleavage. Similarly, deletion of 100 or 150 C-terminal amino acids from GFPRem dramatically reduced both Rem and MMTV-SP levels and function. Removal of the entire C terminus (203 amino acids) restored both protein levels and activity of MMTV-SP. Only C-terminal GFP tagging, and not other modifications, appeared to trap Rem in the ER membrane. Thus, Rem conformation in both the ER lumen and cytoplasm determines cleavage, retrotranslocation, and MMTV-SP function. These mutants further characterize intermediates in Rem trafficking and have implications for all proteins affected by ERAD.
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http://dx.doi.org/10.1128/JVI.06197-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3255906PMC
January 2012

Regulatory T-cell expansion during chronic viral infection is dependent on endogenous retroviral superantigens.

Proc Natl Acad Sci U S A 2011 Mar 14;108(9):3677-82. Epub 2011 Feb 14.

Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

Regulatory T cells (Treg) play critical roles in the modulation of immune responses to infectious agents. Further understanding of the factors that control Treg activation and expansion in response to pathogens is needed to manipulate Treg function in acute and chronic infections. Here we show that chronic, but not acute, infection of mice with lymphocytic choriomeningitis virus results in a marked expansion of Foxp3(+) Treg that is dependent on retroviral superantigen (sag) genes encoded in the mouse genome. Sag-dependent Treg expansion was MHC class II dependent, CD4 independent, and required dendritic cells. Thus, one unique mechanism by which certain infectious agents evade host immune responses may be mediated by endogenous Sag-dependent activation and expansion of Treg.
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http://dx.doi.org/10.1073/pnas.1100213108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3048095PMC
March 2011

Retroviral Rem protein requires processing by signal peptidase and retrotranslocation for nuclear function.

Proc Natl Acad Sci U S A 2010 Jul 21;107(27):12287-92. Epub 2010 Jun 21.

Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology, The University of Texas, Austin, TX 78712, USA.

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 N-terminal 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 cleavage-site 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.
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http://dx.doi.org/10.1073/pnas.1004303107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2901445PMC
July 2010

Mapping of the functional boundaries and secondary structure of the mouse mammary tumor virus Rem-responsive element.

J Biol Chem 2009 Sep 24;284(38):25642-52. Epub 2009 Jul 24.

Section of Molecular Genetics and Microbiology, The University of Texas, Austin, Texas 78712-0162, USA.

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.
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http://dx.doi.org/10.1074/jbc.M109.012476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2757966PMC
September 2009

Rev and Rex proteins of human complex retroviruses function with the MMTV Rem-responsive element.

Retrovirology 2009 Feb 3;6:10. Epub 2009 Feb 3.

Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, USA.

Background: Mouse mammary tumor virus (MMTV) encodes the Rem protein, an HIV Rev-like protein that enhances nuclear export of unspliced viral RNA in rodent cells. We have shown that Rem is expressed from a doubly spliced RNA, typical of complex retroviruses. Several recent reports indicate that MMTV can infect human cells, suggesting that MMTV might interact with human retroviruses, such as human immunodeficiency virus (HIV), human T-cell leukemia virus (HTLV), and human endogenous retrovirus type K (HERV-K). In this report, we test whether the export/regulatory proteins of human complex retroviruses will increase expression from vectors containing the Rem-responsive element (RmRE).

Results: MMTV Rem, HIV Rev, and HTLV Rex proteins, but not HERV-K Rec, enhanced expression from an MMTV-based reporter plasmid in human T cells, and this activity was dependent on the RmRE. No RmRE-dependent reporter gene expression was detectable using Rev, Rex, or Rec in HC11 mouse mammary cells. Cell fractionation and RNA quantitation experiments suggested that the regulatory proteins did not affect RNA stability or nuclear export in the MMTV reporter system. Rem had no demonstrable activity on export elements from HIV, HTLV, or HERV-K. Similar to the Rem-specific activity in rodent cells, the RmRE-dependent functions of Rem, Rev, or Rex in human cells were inhibited by a dominant-negative truncated nucleoporin that acts in the Crm1 pathway of RNA and protein export.

Conclusion: These data argue that many retroviral regulatory proteins recognize similar complex RNA structures, which may depend on the presence of cell-type specific proteins. Retroviral protein activity on the RmRE appears to affect a post-export function of the reporter RNA. Our results provide additional evidence that MMTV is a complex retrovirus with the potential for viral interactions in human cells.
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http://dx.doi.org/10.1186/1742-4690-6-10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2661877PMC
February 2009

BALB/Mtv-null mice responding to strong mouse mammary tumor virus superantigens restrict mammary tumorigenesis.

J Virol 2009 Jan 15;83(1):484-8. Epub 2008 Oct 15.

Section of Molecular Genetics and Microbiology, The University of Texas at Austin, One University Station, A5000, 2506 Speedway, NMS 2.104, Austin, TX 78712-0162, USA.

The absence of endogenous mouse mammary tumor viruses (MMTVs) in the congenic mouse strain, BALB/Mtv-null, restricts the early steps of exogenous C3H MMTV infection, preventing the superantigen (Sag) response and mammary tumorigenesis. Here we demonstrate that BALB/Mtv-null mice also resist tumor induction by FM MMTV, which encodes a stronger Sag compared to C3H MMTV. In contrast to infections with C3H MMTV, Mtv-null mice show FM-MMTV Sag-specific responses comparable to those observed in susceptible BALB/c mice. Neither virus shows significant replication in the spleen or mammary gland. Thus, Mtv-null mice restrict MMTV replication and mammary tumorigenesis even after a robust Sag response.
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http://dx.doi.org/10.1128/JVI.01374-08DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2612332PMC
January 2009

ALY is a common coactivator of RUNX1 and c-Myb on the type B leukemogenic virus enhancer.

J Virol 2007 Apr 17;81(7):3503-13. Epub 2007 Jan 17.

Section of Molecular Genetics and Microbiology, University of Texas at Austin, One University Station, A5000 24th Street and Speedway, ESB 226, Austin, TX 78712-0162, USA.

Type B leukemogenic virus (TBLV), a mouse mammary tumor virus (MMTV) variant, often induces T-cell leukemias and lymphomas by c-myc activation following viral DNA integration. Transfection assays using a c-myc reporter plasmid indicated that the TBLV long terminal repeat (LTR) enhancer is necessary for T-cell-specific increases in basal reporter activity. The sequence requirements for this effect were studied using mutations of the 62-bp enhancer region in an MMTV LTR reporter vector. Deletion of a nuclear factor A-binding site dramatically reduced reporter activity in Jurkat T cells. However, a 41-bp enhancer missing the RUNX1 site still retained minimal enhancer function. DNA affinity purification using a TBLV enhancer oligomer containing the RUNX1 binding site followed by mass spectrometry resulted in the identification of ALY. Subsequent experiments focused on the reconstitution of enhancer activity in epithelial cells. ALY overexpression synergized with RUNX1B on TBLV enhancer activity, and synergism required the RUNX1B-binding site. A predicted c-Myb binding site in the enhancer was confirmed after c-myb overexpression elevated TBLV LTR reporter activity, and overexpression of c-Myb and RUNX1B together showed additive effects on reporter gene levels. ALY also synergized with c-Myb, and coimmunoprecipitation experiments demonstrated an interaction between ALY and c-Myb. These experiments suggest a central role for ALY in T-cell enhancer function and oncogene activation.
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http://dx.doi.org/10.1128/JVI.02253-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1866045PMC
April 2007

Endogenous MMTV proviruses induce susceptibility to both viral and bacterial pathogens.

PLoS Pathog 2006 Dec;2(12):e128

Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, United States of America.

Most inbred mice carry germline proviruses of the retrovirus, mouse mammary tumor virus (MMTV) (called Mtvs), which have multiple replication defects. A BALB/c congenic mouse strain lacking all endogenous Mtvs (Mtv-null) was resistant to MMTV oral and intraperitoneal infection and tumorigenesis compared to wild-type BALB/c mice. Infection of Mtv-null mice with an MMTV-related retrovirus, type B leukemogenic virus, also resulted in severely reduced viral loads and failure to induce T-cell lymphomas, indicating that resistance is not dependent on expression of a superantigen (Sag) encoded by exogenous MMTV. Resistance to MMTV in Mtv-null animals was not due to neutralizing antibodies. Further, Mtv-null mice were resistant to rapid mortality induced by intragastric inoculation of the Gram-negative bacterium, Vibrio cholerae, but susceptibility to Salmonella typhimurium was not significantly different from BALB/c mice. Susceptibility to both MMTV and V. cholerae was reconstituted by the presence of any one of three endogenous Mtvs located on different chromosomes and was associated with increased pathogen load. One of these endogenous proviruses is known to encode only Sag. Therefore, Mtv-encoded Sag appears to provide a unique genetic susceptibility to specific viruses and bacteria. Since human endogenous retroviruses also encode Sags, these studies have broad implications for pathogen-induced responses in mice and humans.
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http://dx.doi.org/10.1371/journal.ppat.0020128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1665650PMC
December 2006

Differentiation-induced cleavage of Cutl1/CDP generates a novel dominant-negative isoform that regulates mammary gene expression.

Mol Cell Biol 2006 Oct 5;26(20):7466-78. Epub 2006 Aug 5.

Section of Molecular Genetics and Microbiology, The University of Texas at Austin, 24th and Speedway, ESB 226, Austin, TX 78712-0162, USA.

Cutl1/CCAAT displacement protein (CDP) is a transcriptional repressor of mouse mammary tumor virus (MMTV), a betaretrovirus that is a paradigm for mammary-specific gene regulation. Virgin mammary glands have high levels of full-length CDP (200 kDa) that binds to negative regulatory elements (NREs) to repress MMTV transcription. During late pregnancy, full-length CDP levels decline, and a 150-kDa form of CDP (CDP150) appears concomitantly with a decline in DNA-binding activity for the MMTV NREs and an increase in viral transcripts. Developmental regulation of CDP was recapitulated in the normal mammary epithelial line, SCp2. Western blotting of tissue and SCp2 nuclear extracts confirmed that CDP150 lacks the C terminus. Transfection of tagged full-length and mutant cDNAs into SCp2 cells and use of a cysteine protease inhibitor demonstrated that CDP is proteolytically processed within the homeodomain to remove the C terminus during differentiation. Mixing of virgin and lactating mammary extracts or transfection of mutant CDP cDNAs missing the homeodomain into cells containing full-length CDP also abrogated NRE binding. Loss of DNA binding correlated with increased expression of MMTV and other mammary-specific genes, indicating that CDP150 is a developmentally induced dominant-negative protein. Thus, a novel posttranslational process controls Cutl1/CDP activity and gene expression in the mammary gland.
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http://dx.doi.org/10.1128/MCB.01083-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1636867PMC
October 2006

Mouse mammary tumor virus encodes a self-regulatory RNA export protein and is a complex retrovirus.

J Virol 2005 Dec;79(23):14737-47

The University of Texas at Austin, Section of Molecular Genetics and Microbiology, One University Station, A5000, Austin, TX 78712, USA.

Mouse mammary tumor virus (MMTV) has been classified as a simple retrovirus with two accessory genes, dut and sag. Cloned MMTV proviruses carrying a trimethoprim (trim) cassette in the envelope gene were defective for Gag protein production and the nuclear export of unspliced gag-pol RNA. Complementation experiments indicated that a trans-acting product was responsible for the Gag defect of such mutants. Analysis of MMTV-infected cells revealed the presence of a novel, doubly spliced RNA that encodes a putative product of 301 amino acids. Overexpression of cDNA from this RNA increased Gag levels from env mutant proviruses or reporter gene expression from unspliced mRNAs and allowed detection of a 33-kDa protein product, which has been named regulator of export of MMTV mRNA, or Rem. The Rem N terminus has motifs similar to the Rev-like export proteins of complex retroviruses, and mutation of the nuclear localization signal (NLS) abolished RNA export and detection within the nucleus. The Rem C terminus has few identifiable features, but removal of this domain increased Rem-mediated export, suggesting an autoregulatory function. A reporter vector developed from the 3' end of the MMTV provirus was Rem responsive and required both the presence of the MMTV env-U3 junction and a functional Crm1 pathway. The identification of a third accessory protein from a doubly spliced transcript suggests that MMTV is the first murine complex retrovirus to be documented. Manipulation of the MMTV genome may provide mouse models for human retroviral diseases, such as AIDS.
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http://dx.doi.org/10.1128/JVI.79.23.14737-14747.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1287593PMC
December 2005

Conversion of mouse mammary tumor virus to a lymphomagenic virus.

J Virol 2005 Oct;79(19):12592-6

Section of Molecular Genetics and Microbiology, The University of Texas at Austin, Austin, TX 78712-0162, USA.

Type B leukemogenic virus is a variant of mouse mammary tumor virus (MMTV) that causes thymic lymphomas rather than mammary tumors in mice. We demonstrate that conversion of a mammotropic MMTV to a T-cell-tropic virus requires two alterations in the long terminal repeat: (i) acquisition of a T-cell-specific enhancer and (ii) loss of transcriptional repression through deletion of negative regulatory elements (NREs) or by suppression of NRE activity after appropriate positioning of the enhancer.
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http://dx.doi.org/10.1128/JVI.79.19.12592-12596.2005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1211542PMC
October 2005

Nuclear matrix binding regulates SATB1-mediated transcriptional repression.

J Biol Chem 2005 Jul 25;280(26):24600-9. Epub 2005 Apr 25.

Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas 78712, USA.

Special AT-rich binding protein 1 (SATB1) originally was identified as a protein that bound to the nuclear matrix attachment regions (MARs) of the immunoglobulin heavy chain intronic enhancer. Subsequently, SATB1 was shown to repress many genes expressed in the thymus, including interleukin-2 receptor alpha, c-myc, and those encoded by mouse mammary tumor virus (MMTV), a glucocorticoid-responsive retrovirus. SATB1 binds to MARs within the MMTV provirus to repress transcription. To address the role of the nuclear matrix in SATB1-mediated repression, a series of SATB1 deletion constructs was used to determine protein localization. Wild-type SATB1 localized to the soluble nuclear, chromatin, and nuclear matrix fractions. Mutants lacking amino acids 224-278 had a greatly diminished localization to the nuclear matrix, suggesting the presence of a nuclear matrix targeting sequence (NMTS). Transient transfection experiments showed that NMTS fusions to green fluorescent protein or LexA relocalized these proteins to the nuclear matrix. Difficulties with previous assay systems prompted us to develop retroviral vectors to assess effects of different SATB1 domains on expression of MMTV proviruses or integrated reporter genes. SATB1 overexpression repressed MMTV transcription in the presence and absence of functional glucocorticoid receptor. Repression was alleviated by deletion of the NMTS, which did not affect DNA binding, or by deletion of the MAR-binding domain. Our studies indicate that both nuclear matrix association and DNA binding are required for optimal SATB1-mediated repression of the integrated MMTV promoter and may allow insulation from cellular regulatory elements.
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http://dx.doi.org/10.1074/jbc.M414076200DOI Listing
July 2005

The homeodomain protein CDP regulates mammary-specific gene transcription and tumorigenesis.

Mol Cell Biol 2004 Jun;24(11):4810-23

Section of Molecular Genetics and Microbiology, The University of Texas at Austin, One University Station, A5000, 24th and Speedway, ESB 226, Austin, TX 78712-0162, USA.

The CCAAT-displacement protein (CDP) has been implicated in developmental and cell-type-specific regulation of many cellular and viral genes. We previously have shown that CDP represses mouse mammary tumor virus (MMTV) transcription in tissue culture cells. Since CDP-binding activity for the MMTV long terminal repeat declines during mammary development, we tested whether binding mutations could alter viral expression. Infection of mice with MMTV proviruses containing CDP binding site mutations elevated viral RNA levels in virgin mammary glands and shortened mammary tumor latency. To determine if CDP has direct effects on MMTV transcription rather than viral spread, virgin mammary glands of homozygous CDP-mutant mice lacking one of three Cut repeat DNA-binding domains (DeltaCR1) were examined by reverse transcription-PCR. RNA levels of endogenous MMTV as well as alpha-lactalbumin and whey acidic protein (WAP) were elevated. Heterozygous mice with a different CDP mutation that eliminated the entire C terminus and the homeodomain (DeltaC mice) showed increased levels of MMTV, beta-casein, WAP, and alpha-lactalbumin RNA in virgin mammary glands compared to those from wild-type animals. No differences in amounts of WDNM1, epsilon-casein, or glyceraldehyde-3-phosphate dehydrogenase RNA were observed between the undifferentiated mammary tissues from wild-type and mutant mice, indicating the specificity of this effect. These data show independent contributions of different CDP domains to negative regulation of differentiation-specific genes in the mammary gland.
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http://dx.doi.org/10.1128/MCB.24.11.4810-4823.2004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC416401PMC
June 2004

Rorgamma (Rorc) is a common integration site in type B leukemogenic virus-induced T-cell lymphomas.

J Virol 2004 May;78(9):4943-6

Section of Microbiology and Molecular Genetics and Institute of Cellular and Molecular Biology, Austin, Texas 78712, USA.

The retrovirus type B leukemogenic virus (TBLV) causes T-cell lymphomas in mice. We have identified the Rorgamma locus as an integration site in 19% of TBLV-induced tumors. Overexpression of one or more Rorgamma isoforms in >77% of the tumors tested may complement apoptotic effects of c-myc overexpression.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC387709PMC
http://dx.doi.org/10.1128/jvi.78.9.4943-4946.2004DOI Listing
May 2004

Binding of CCAAT displacement protein CDP to adenovirus packaging sequences.

J Virol 2003 Jun;77(11):6255-64

Department of Molecular Genetics and Microbiology, School of Medicine, Stony Brook University, New York 11794, USA.

Adenovirus (Ad) type 5 DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent upon the cis-acting packaging domain located between nucleotides 194 and 380. Seven A/T-rich repeats have been identified within this domain that direct packaging. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans-acting factor(s) that plays a role in packaging. Two cellular activities that bind to minimal packaging domains in vitro have been previously identified. These binding activities are P complex, an uncharacterized protein(s), and chicken ovalbumin upstream promoter transcription factor (COUP-TF). In this work, we report that a third cellular protein, octamer-1 protein (Oct-1), binds to minimal packaging domains. In vitro binding analyses and in vivo packaging assays were used to examine the relevance of these DNA binding activities to Ad DNA packaging. The results of these experiments reveal that COUP-TF and Oct-1 binding does not play a functional role in Ad packaging, whereas P-complex binding directly correlates with packaging function. We demonstrate that P complex contains the cellular protein CCAAT displacement protein (CDP) and that full-length CDP is found in purified virus particles. In addition to cellular factors, previous evidence indicates that viral factors play a role in the initiation of viral DNA packaging. We propose that CDP, in conjunction with one or more viral proteins, binds to the packaging sequences of Ad to initiate the encapsidation process.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC154998PMC
http://dx.doi.org/10.1128/jvi.77.11.6255-6264.2003DOI Listing
June 2003

Tag, you're hit: retroviral insertions identify genes involved in cancer.

Trends Mol Med 2003 Feb;9(2):43-5

Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, TX 78705, USA.

Retroviral integrations have been used for many years to identify genes involved in cancer. The recently published mouse genome sequence has allowed large-scale identification of potential human cancer genes and their classification into distinct signaling pathways.
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http://dx.doi.org/10.1016/s1471-4914(03)00003-0DOI Listing
February 2003

The type B leukemogenic virus truncated superantigen is dispensable for T-cell lymphomagenesis.

J Virol 2003 Mar;77(6):3866-70

Section of Molecular Genetics and Microbiology and Institute for Cellular and Molecular Biology, the University of Texas at Austin, Austin, Texas 78712, USA.

Type B leukemogenic virus (TBLV) is a variant of mouse mammary tumor virus (MMTV) that causes T-cell lymphomas in mice. We have constructed a TBLV-MMTV hybrid, pHYB-TBLV, in which 756 bp of the C3H MMTV long terminal repeat (LTR) was replaced with 438 bp of the TBLV LTR. Intraperitoneal injection of pHYB-TBLV transfectants consistently resulted in T-cell lymphomas in 50% of injected weanling BALB/c mice with an average latency period of 5.7 (+/- 1.5) months. Transfectants of pHYB-TBLV containing a double-frameshift mutation in the truncated superantigen gene (sag) induced T-cell lymphomas with similar incidences, latency periods, and phenotypes, suggesting that cis-acting elements in the TBLV LTR determine disease specificity.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC149533PMC
http://dx.doi.org/10.1128/jvi.77.6.3866-3870.2003DOI Listing
March 2003

CDP binding to multiple sites in the mouse mammary tumor virus long terminal repeat suppresses basal and glucocorticoid-induced transcription.

J Virol 2002 Mar;76(5):2168-79

Section of Molecular Genetics and Microbiology, Institute for Cellular and Molecular Biology, The University of Texas at Austin, 100 W. 24th St., Austin, TX 78705, USA.

Mouse mammary tumor virus (MMTV) is transcribed at high levels in the lactating mammary gland to ensure transmission of virus from the milk of infected female mice to susceptible offspring. We previously have shown that the transcription factor CCAAT displacement protein (CDP) is expressed in high amounts in virgin mammary gland, yet DNA-binding activity for the MMTV long terminal repeat (LTR) disappears as mammary tissue differentiates during lactation. CDP is a repressor of MMTV expression and, therefore, MMTV expression is suppressed during early mammary gland development. In this study, we have shown using DNase I footprinting and electrophoretic mobility shift assays that there are at least five CDP-binding sites in the MMTV LTR upstream of those previously described in the promoter-proximal negative regulatory element (NRE). Single mutations in two of these upstream sites (+691 or +692 and +735 relative to the first base of the LTR) reduced CDP binding to the cognate sites and elevated reporter gene expression from the full-length MMTV LTR. Combination of a mutation in the promoter-distal NRE with a mutation in the proximal NRE gave approximately additive increases in LTR-reporter gene activity, suggesting that these binding sites act independently. Mutations in several different CDP-binding sites allowed elevation of reporter gene activity from the MMTV promoter in the absence and presence of glucocorticoids, hormones that contribute to high levels of MMTV transcription during lactation by activation of hormone receptor binding to the LTR. In addition, overexpression of CDP in transient-transfection assays suppressed both basal and glucocorticoid-induced LTR-mediated transcription in a dose-dependent manner. These data suggest that multiple CDP-binding sites contribute independently to regulate binding of positive factors, including glucocorticoid receptor, to the MMTV LTR during mammary gland development.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC135928PMC
http://dx.doi.org/10.1128/jvi.76.5.2168-2179.2002DOI Listing
March 2002