The Experts below are selected from a list of 255 Experts worldwide ranked by ideXlab platform
Stephen P Goff - One of the best experts on this subject based on the ideXlab platform.
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transcriptional silencing of moloney Murine Leukemia virus in human embryonic carcinoma cells
Journal of Virology, 2017Co-Authors: Gary Z Wang, Stephen P GoffAbstract:ABSTRACT Embryonic carcinoma (EC) cells are malignant counterparts of embryonic stem (ES) cells and serve as useful models for investigating cellular differentiation and human embryogenesis. Though the susceptibility of Murine EC cells to retroviral infection has been extensively analyzed, few studies of retrovirus infection of human EC cells have been performed. We tested the susceptibility of human EC cells to transduction by retroviral vectors derived from three different retroviral genera. We show that human EC cells efficiently express reporter genes delivered by vectors based on human immunodeficiency virus type 1 (HIV-1) and Mason-Pfizer monkey virus (M-PMV) but not Moloney Murine Leukemia virus (MLV). In human EC cells, MLV integration occurs normally, but no viral gene expression is observed. The block to MLV expression of MLV genomes is relieved upon cellular differentiation. The lack of gene expression is correlated with transcriptional silencing of the MLV promoter through the deposition of repressive histone marks as well as DNA methylation. Moreover, depletion of SETDB1, a histone methyltransferase, resulted in a loss of transcriptional silencing and upregulation of MLV gene expression. Finally, we provide evidence showing that the lack of MLV gene expression may be attributed in part to the lack of MLV enhancer function in human EC cells. IMPORTANCE Human embryonic carcinoma (EC) cells are shown to restrict the expression of Murine Leukemia virus genomes but not retroviral genomes of the lentiviral or betaretroviral families. The block occurs at the level of transcription and is accompanied by the deposition of repressive histone marks and methylation of the integrated proviral DNA. The host machinery required for silencing in human EC cells is distinct from that in Murine EC cell lines: the histone methyltransferase SETDB1 is required, but the widely utilized corepressor TRIM28/Kap1 is not. A transcriptional enhancer element from the Mason-Pfizer monkey virus can override the silencing and promote transcription of chimeric proviral DNAs. The findings reveal novel features of human EC gene regulation not present in their Murine counterparts.
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structural basis of suppression of host translation termination by moloney Murine Leukemia virus
Nature Communications, 2016Co-Authors: Xuhua Tang, Stephen P Goff, Yiping Zhu, Stacey L Baker, Matthew W Bowler, Benjamin Jieming Chen, Chen Chen, Robert J HoggAbstract:Retroviral reverse transcriptase (RT) of Moloney Murine Leukemia virus (MoMLV) is expressed in the form of a large Gag-Pol precursor protein by suppression of translational termination in which the maximal efficiency of stop codon read-through depends on the interaction between MoMLV RT and peptidyl release factor 1 (eRF1). Here, we report the crystal structure of MoMLV RT in complex with eRF1. The MoMLV RT interacts with the C-terminal domain of eRF1 via its RNase H domain to sterically occlude the binding of peptidyl release factor 3 (eRF3) to eRF1. Promotion of read-through by MoMLV RNase H prevents nonsense-mediated mRNA decay (NMD) of mRNAs. Comparison of our structure with that of HIV RT explains why HIV RT cannot interact with eRF1. Our results provide a mechanistic view of how MoMLV manipulates the host translation termination machinery for the synthesis of its own proteins.
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ebp1 a novel host factor involved in primer binding site dependent restriction of moloney Murine Leukemia virus in embryonic cells
Journal of Virology, 2014Co-Authors: Gary Z Wang, Daniel De Wolf, Stephen P GoffAbstract:ABSTRACT Mouse embryonic cells are unable to support the replication of Moloney Murine Leukemia virus (MLV). The integrated viral DNA is transcriptionally silenced, largely due to binding of host transcriptional repressors to the primer binding site (PBS) of the provirus. We have previously shown that a PBS DNA-binding repressor complex contains ZFP809 and TRIM28. Here, we identified ErbB3-binding protein 1 (EBP1) to be a novel component of the ZFP809-TRIM28 silencing complex and show that EBP1 depletion reduces PBS-mediated retroviral silencing.
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xenotropic Murine Leukemia virus related virus establishes an efficient spreading infection and exhibits enhanced transcriptional activity in prostate carcinoma cells
Journal of Virology, 2010Co-Authors: Jason J. Rodriguez, Stephen P GoffAbstract:Xenotropic Murine Leukemia virus-related virus (XMRV) is a novel human gammaretrovirus discovered in association with human prostate tumors. XMRV was first identified in prostate stromal cells surrounding the tumors of patients carrying a mutation in the HPC1 gene locus. To determine the tropism of XMRV in cell culture, we tested the ability of XMRV to spread and replicate in various prostate and nonprostate cell lines. We found that although the expression of XMRV viral proteins and the spread of infectious virus were minimal in a variety of cell lines, XMRV displayed robust expression and infection in LNCaP prostate tumor cells. The transcriptional activity of the XMRV long terminal repeat (LTR) was found to be higher than the Moloney Murine Leukemia virus LTRs in both LNCaP and WPMY-1 (simian virus 40-transformed prostate stromal cells). The U3 promoter of XMRV and a glucocorticoid response element (GRE) within the U3 were required for the transcriptional activity in LNCaP cells. Coexpression of the androgen receptor and stimulation with dihydrotestosterone stimulated XMRV-LTR-dependent transcription in 293T cells, and the GRE was required for this activity. These data suggest that XMRV may replicate more efficiently in LNCaP cells in part due to the transcriptional environment in LNCaP cells.
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primer binding site dependent restriction of Murine Leukemia virus requires hp1 binding by trim28
Journal of Virology, 2008Co-Authors: Daniel H. Wolf, Florence Cammas, Regine Losson, Stephen P GoffAbstract:TRIM28 is a transcriptional corepressor which is required for primer binding site (PBS)-dependent restriction of Murine Leukemia virus (MLV) replication in embryonic stem and embryonic carcinoma (EC) cells. PBS-dependent restriction of MLV leads to transcriptional silencing of the integrated provirus and has been shown to correlate with TRIM28-mediated recruitment of HP1 to the silenced loci. Here we show, using a cell line with a point mutation in the HP1 binding domain of TRIM28, that interaction with HP1 is absolutely required for the PBS-dependent restriction of MLV in the F9 EC cell line.
Sriram Aiyer - One of the best experts on this subject based on the ideXlab platform.
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bet proteins target Murine Leukemia virus integration to transcription start sites
Retrovirology, 2013Co-Authors: Amit Sharma, Matthew R Plumb, Frances Male, Nikoloz Shkriabai, Alison Slaughter, Jacques J. Kessl, Ross C. Larue, Nirav Malani, Elizabeth K. Coward, Sriram AiyerAbstract:Background The selection of chromosomal targets for retroviral integration varies markedly, tracking with the genus of the retrovirus, suggestive of specific targeting by cellular factors. Gamma-retroviral Murine Leukemia virus (MLV) DNA integration into the host genome is favored at transcription start sites, but the underlying mechanism for this preference is unknown. The molecular mechanisms of MLV integration are of particular significance due to the fact that MLV-based vectors are used for human gene-therapy. In clinical trials the use of MLV-based vectors to correct primary immunodeficiencies has been curative, but adverse events have occurred that are associated with the insertional activation of proto-oncogenes. Therefore the identification of cellular factors that control MLV integration may provide mechanistic clues to facilitate the development of safer gene-therapy vectors.
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bet proteins promote efficient Murine Leukemia virus integration at transcription start sites
Proceedings of the National Academy of Sciences of the United States of America, 2013Co-Authors: Amit Sharma, Matthew R Plumb, Frances Male, Nikoloz Shkriabai, Alison Slaughter, Jacques J. Kessl, Ross C. Larue, Nirav Malani, Elizabeth K. Coward, Sriram AiyerAbstract:The selection of chromosomal targets for retroviral integration varies markedly, tracking with the genus of the retrovirus, suggestive of targeting by binding to cellular factors. γ-Retroviral Murine Leukemia virus (MLV) DNA integration into the host genome is favored at transcription start sites, but the underlying mechanism for this preference is unknown. Here, we have identified bromodomain and extraterminal domain (BET) proteins (Brd2, -3, -4) as cellular-binding partners of MLV integrase. We show that purified recombinant Brd4(1-720) binds with high affinity to MLV integrase and stimulates correct concerted integration in vitro. JQ-1, a small molecule that selectively inhibits interactions of BET proteins with modified histone sites impaired MLV but not HIV-1 integration in infected cells. Comparison of the distribution of BET protein-binding sites analyzed using ChIP-Seq data and MLV-integration sites revealed significant positive correlations. Antagonism of BET proteins, via JQ-1 treatment or RNA interference, reduced MLV-integration frequencies at transcription start sites. These findings elucidate the importance of BET proteins for MLV integration efficiency and targeting and provide a route to developing safer MLV-based vectors for human gene therapy.
Henrik Garoff - One of the best experts on this subject based on the ideXlab platform.
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sequential activation of the three protomers in the moloney Murine Leukemia virus env
Proceedings of the National Academy of Sciences of the United States of America, 2017Co-Authors: Mathilda Sjoberg, Robin Loving, Birgitta Lindqvist, Henrik GaroffAbstract:Viral membrane fusion proteins of class I are trimers in which the protomeric unit is a complex of a surface subunit (SU) and a fusion active transmembrane subunit (TM). Here we have studied how the protomeric units of Moloney Murine Leukemia virus envelope protein (Env) are activated in relation to each other, sequentially or simultaneously. We followed the isomerization of the SU-TM disulfide and subsequent SU release from Env with biochemical methods and found that this early activation step occurred sequentially in the three protomers, generating two asymmetric oligomer intermediates according to the scheme (SU-TM)3 → (SU-TM)2TM → (SU-TM)TM2 → TM3. This was the case both when activation was triggered in vitro by depleting stabilizing Ca2+ from solubilized Env and when viral Env was receptor triggered on rat XC cells. In the latter case, the activation reaction was too fast for direct observation of the intermediates, but they could be caught by alkylation of the isomerization active thiol.
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maturation cleavage of the Murine Leukemia virus env precursor separates the transmembrane subunits to prime it for receptor triggering
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Robin Loving, Shang Rung Wu, Mathilda Sjoberg, Birgitta Lindqvist, Henrik GaroffAbstract:The Env protein of Murine Leukemia virus matures by two cleavage events. First, cellular furin separates the receptor binding surface (SU) subunit from the fusion-active transmembrane (TM) subunit and then, in the newly assembled particle, the viral protease removes a 16-residue peptide, the R-peptide from the endodomain of the TM. Both cleavage events are required to prime the Env for receptor-triggered activation. Cryoelectron microscopy (cryo-EM) analyses have shown that the mature Env forms an open cage-like structure composed of three SU–TM complexes, where the TM subunits formed separated Env legs. Here we have studied the structure of the R-peptide precursor Env by cryo-EM. TM cleavage in Moloney Murine Leukemia virus was inhibited by amprenavir, and the Envs were solubilized in Triton X-100 and isolated by sedimentation in a sucrose gradient. We found that the legs of the R-peptide Env were held together by trimeric interactions at the very bottom of the Env. This suggested that the R-peptide ties the TM legs together and that this prevents the activation of the TM for fusion. The model was supported by further cryo-EM studies using an R-peptide Env mutant that was fusion-competent despite an uncleaved R-peptide. The Env legs of this mutant were found to be separated, like in the mature Env. This shows that it is the TM leg separation, normally caused by R-peptide cleavage, that primes the Env for receptor triggering.
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production of infectious recombinant moloney Murine Leukemia virus particles in bhk cells using semliki forest virus derived rna expression vectors
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: Henrik GaroffAbstract:We describe a heterologous, Semliki Forest virus (SFV)-driven packaging system for the production of infectious recombinant Moloney Murine Leukemia virus particles. The gag-pol and env genes, as well as a recombinant retrovirus genome (LTR-psi (+)-neoR-LTR), were inserted into individual SFV1 expression plasmids. Replication-competent RNAs were transcribed in vitro and introduced into the cytoplasm of BHK-21 cells using electroporation. The expressed Moloney Murine Leukemia virus structural proteins produced extracellular virus-like particles. In these particles the gag precursor was processed into mature products, indicating that the particles contained an active protease. The protease of the gag-pol fusion protein was also shown to be active in a trans-complementation assay using a large excess of Pr65gag. Moreover, the particles possessed reverse transcriptase (RT) activity as measured in an in vitro assay. Cotransfection of BHK-21 cells by all three SFV1 constructs resulted in the production of transduction-competent particles at 4 x 10(6) colony-forming units (cfu)/ml during a 5-hr incubation period. Altogether, 2.9 x 10(7) transduction-competent particles were obtained from about 4 x 10(6) transfected cells. Thus, this system represents the first RNA-based packaging system for the production of infectious retroviral particles. The facts that no helper virus could be detected in the virus stocks and that particles carrying the amphotropic envelope could be produced with similar efficiency as those that carry the ecotropic envelope make the system very interesting for gene therapy.
Robert H Silverman - One of the best experts on this subject based on the ideXlab platform.
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xenotropic Murine Leukemia virus related virus is susceptible to azt
Virology, 2010Co-Authors: Ryuta Sakuma, Seiga Ohmine, Toshie Sakuma, Robert H Silverman, Yasuhiro H IkedaAbstract:The xenotropic Murine Leukemia virus-related virus (XMRV) is a human retrovirus, recently isolated from tissues of prostate cancer patients with impaired RNase L activity. In this study, we evaluated 10 licensed anti-HIV-1 compounds for their activity against XMRV, including protease inhibitors (PI), nucleoside reverse transcriptase (RT) inhibitors (NRTI), non-nucleoside RT inhibitors (NNRTI) and an integrase inhibitor. No PI affected XMRV production; even high concentrations of Ritonavir failed to inhibit the maturation of XMRV Gag polyproteins. Among the NRTI, NNRTI and integrase inhibitors used in this study, only AZT blocked XMRV infection and replication through inhibition of viral reverse transcription. This sensitivity of XMRV to AZT may be explained by the modest homology in the motif D sequences of HIV-1 and XMRV reverse transcriptases. If XMRV becomes established as an etiological agent for prostate cancer or other diseases, AZT may be useful for preventing or treating XMRV infections in humans.
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androgen stimulates transcription and replication of xenotropic Murine Leukemia virus related virus
Journal of Virology, 2010Co-Authors: Beihua Dong, Robert H SilvermanAbstract:Xenotropic Murine Leukemia virus-related virus (XMRV) is a gammaretrovirus originally identified in a subset of prostate cancer patients. Because androgens stimulate prostate tumors and some retroviruses, we investigated the effects of dihydrotestosterone (DHT) on XMRV transcription and replication. Transcription from the XMRV U3 region was stimulated up to 2-fold by DHT, but only in cells containing a functional androgen receptor. Mutations in the glucocorticoid response element (GRE) of XMRV impaired basal transcription and androgen responsiveness. Furthermore, DHT stimulated XMRV replication 3-fold, whereas androgen inhibitors (casodex and flutamide) suppressed viral growth up to 3-fold. Findings suggest that integration of the XMRV long terminal repeat (LTR) into host DNA could impart androgen stimulation on cellular genes.
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integration site preference of xenotropic Murine Leukemia virus related virus a new human retrovirus associated with prostate cancer
Journal of Virology, 2008Co-Authors: Beihua Dong, Robert H Silverman, David Boren, Jaydip Das Gupta, Christina Gaughan, Eric A Klein, Samson A ChowAbstract:Xenotropic Murine Leukemia virus-related virus (XMRV) is a new human gammaretrovirus identified in prostate cancer tissue from patients homozygous for a reduced-activity variant of the antiviral enzyme RNase L. Neither a casual relationship between XMRV infection and prostate cancer nor a mechanism of tumorigenesis has been established. To determine the integration site preferences of XMRV and the potential risk of proviral insertional mutagenesis, we carried out a genome-wide analysis of viral integration sites in the prostate cell line DU145 after an acute XMRV infection and compared the integration site pattern of XMRV with those found for Murine Leukemia virus and two human retroviruses, human immunodeficiency virus type 1 and human T-cell Leukemia virus type 1. Among all retroviruses analyzed, XMRV has the strongest preference for transcription start sites, CpG islands, DNase-hypersensitive sites, and gene-dense regions; all are features frequently associated with structurally open transcription regulatory regions of a chromosome. Analyses of XMRV integration sites in tissues from prostate cancer patients found a similar preference for the aforementioned chromosomal features. Additionally, XMRV integration sites in cancer tissues were associated with cancer breakpoints, common fragile sites, microRNA, and cancer-related genes, suggesting a selection process that favors certain chromosomal integration sites. In both acutely infected cells and cancer tissues, no common integration site was detected within or near proto-oncogenes or tumor suppressor genes. These results are consistent with a model in which XMRV may contribute to tumorigenicity via a paracrine mechanism.
Timothy J Henrich - One of the best experts on this subject based on the ideXlab platform.
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xenotropic Murine Leukemia virus related virus prevalence in patients with chronic fatigue syndrome or chronic immunomodulatory conditions
The Journal of Infectious Diseases, 2010Co-Authors: Jonathan Z Li, Timothy J Henrich, Camille N. Kotton, Donna FelsensteinAbstract:We investigated the prevalence of xenotropic Murine Leukemia virus-related virus (XMRV) among 293 participants seen at academic hospitals in Boston, Massachusetts. Participants were recruited from the following 5 groups of patients: chronic fatigue syndrome (n = 32), human immunodeficiency virus infection (n = 43), rheumatoid arthritis (n = 97), hematopoietic stem-cell or solid organ transplant (n = 26), or a general cohort of patients presenting for medical care (n = 95). XMRV DNA was not detected in any participant samples. We found no association between XMRV and patients with chronic fatigue syndrome or chronic immunomodulatory conditions.
