The Experts below are selected from a list of 420 Experts worldwide ranked by ideXlab platform

Louis M. Mansky - One of the best experts on this subject based on the ideXlab platform.

  • human t cell leukemia virus type 1 gag domains have distinct rna binding specificities with implications for rna packaging and dimerization
    Journal of Biological Chemistry, 2018
    Co-Authors: Joshua Hatterschide, Louis M. Mansky, William A. Cantara, Yu Ci Syu, Ruth J Blower, Heather M Hanson, Karin Musierforsyth
    Abstract:

    Human T-cell leukemia virus type 1 (HTLV-1) is the first retrovirus that has conclusively been shown to cause human diseases. In HIV-1, specific interactions between the nucleocapsid (NC) domain of the Gag protein and genomic RNA (gRNA) mediate gRNA dimerization and selective packaging; however, the mechanism for gRNA packaging in HTLV-1, a Deltaretrovirus, is unclear. In other Deltaretroviruses, the matrix (MA) and NC domains of Gag are both involved in gRNA packaging, but MA binds nucleic acids with higher affinity and has more robust chaperone activity, suggesting that this domain may play a primary role. Here, we show that the MA domain of HTLV-1, but not the NC domain, binds short hairpin RNAs derived from the putative gRNA packaging signal. RNA probing of the HTLV-1 5' leader and cross-linking studies revealed that the primer-binding site and a region within the putative packaging signal form stable hairpins that interact with MA. In addition to a previously identified palindromic dimerization initiation site (DIS), we identified a new DIS in HTLV-1 gRNA and found that both palindromic sequences bind specifically the NC domain. Surprisingly, a mutant partially defective in dimer formation in vitro exhibited a significant increase in RNA packaging into HTLV-1-like particles, suggesting that efficient RNA dimerization may not be strictly required for RNA packaging in HTLV-1. Moreover, the lifecycle of HTLV-1 and other Deltaretroviruses may be characterized by NC and MA functions that are distinct from those of the corresponding HIV-1 proteins, but together provide the functions required for viral replication.

  • construction and characterization of Deltaretrovirus indicator cell lines
    Journal of Virological Methods, 2005
    Co-Authors: Nancy A Jewell, Louis M. Mansky
    Abstract:

    The Deltaretroviruses, which include bovine leukemia virus (BLV) and human T-cell leukemia virus types 1 and 2 (HTLV-1 and HTLV-2), replicate poorly in culture and the molecular details of their life cycles are limited. To facilitate the analysis of virus replication, mammalian cell lines were created with the long terminal repeats (LTRs) of each virus driving expression of the enhanced green fluorescent protein gene (egfp). The BLGFP, H1GFP and H2GFP cell lines detect virus infection by the expression of GFP via the transactivation of the LTR via the Tax protein of BLV, HTLV-1 or HTLV-2, respectively. GFP expression was measured by flow cytometry, yielding sensitive and rapid detection of virus infectivity. Interestingly, we observed that the Tax proteins of HTLV-1 and HTLV-2 could transactivate the BLV LTR at levels that were comparable to that of BLV Tax. In contrast, the BLV Tax showed low levels of transactivation in H1GFP and H2GFP cells. HTLV-1 and HTLV-2 Tax proteins efficiently transactivated both the HTLV-1 and HTLV-2 LTRs. Finally, spinoculation of BLV resulted in only a two-fold increase in viral titer.

  • Analysis of Bovine Leukemia Virus Gag Membrane Targeting and Late Domain Function
    Journal of Virology, 2002
    Co-Authors: Huating Wang, Kendra M. Norris, Louis M. Mansky
    Abstract:

    Assembly of retrovirus-like particles only requires the expression of the Gag polyprotein precursor. We have exploited this in the development of a model system for studying the virus particle assembly pathway for bovine leukemia virus (BLV). BLV is closely related to the human T-cell leukemia viruses (HTLVs), and all are members of the Deltaretrovirus genus of the Retroviridae family. Overexpression of a BLV Gag polyprotein containing a carboxy-terminal influenza virus hemagglutinin (HA) epitope tag in mammalian cells led to the robust production of virus-like particles (VLPs). Site-directed mutations were introduced into HA-tagged Gag to test the usefulness of this model system for studying certain aspects of the virus assembly pathway. First, mutations that disrupted the amino-terminal glycine residue that is important for Gag myristylation led to a drastic reduction in VLP production. Predictably, the nature of the VLP production defect was correlated to Gag membrane localization. Second, mutation of the PPPY motif (located in the MA domain) greatly reduced VLP production in the absence of the viral protease. This reduction in VLP production was more severe in the presence of an active viral protease. Examination of particles by electron microscopy revealed an abundance of particles that began to pinch off from the plasma membrane but were not completely released from the cell surface, indicating that the PPPY motif functions as a late domain (L domain).

  • Analysis of bovine leukemia virus Gag membrane targeting and late domain function
    2002
    Co-Authors: Huating Wang, Kendra M. Norris, Louis M. Mansky
    Abstract:

    Assembly of retrovirus-like particles only requires the expression of the Gag polyprotein precursor. We have exploited this in the development of a model system for studying the virus particle assembly pathway for bovine leukemia virus (BLV). BLV is closely related to the human T-cell leukemia viruses (HTLVs), and all are members of the Deltaretrovirus genus of the Retroviridae family. Overexpression of a BLV Gag polyprotein containing a carboxy-terminal influenza virus hemagglutinin (HA) epitope tag in mammalian cells led to the robust production of virus-like particles (VLPs). Site-directed mutations were introduced into HA-tagged Gag to test the usefulness of this model system for studying certain aspects of the virus assembly pathway. First, mutations that disrupted the amino-terminal glycine residue that is important for Gag myristylation led to a drastic reduction in VLP production. Predictably, the nature of the VLP production defect was correlated to Gag membrane localization. Second, mutation of the PPPY motif (located in the MA domain) greatly reduced VLP production in the absence of the viral protease. This reduction in VLP production was more severe in the presence of an active viral protease. Examination of particles by electron microscopy revealed an abundance of particles that began to pinch off from the plasma membrane but were not completely released from the cell surface, indicating that the PPPY motif functions as a late domain (L domain). The assembly of retrovirus particles requires the expressio

Karin Musierforsyth - One of the best experts on this subject based on the ideXlab platform.

  • solution conformation of bovine leukemia virus gag suggests an elongated structure
    Journal of Molecular Biology, 2019
    Co-Authors: Dominic F Qualley, William A. Cantara, Sarah Cooper, James Ross, Erik D Olson, Karin Musierforsyth
    Abstract:

    Abstract Bovine leukemia virus (BLV) is a Deltaretrovirus that infects domestic cattle. The structural protein Gag, found in all retroviruses, is a polyprotein comprising three major functional domains: matrix (MA), capsid (CA), and nucleocapsid (NC). Previous studies have shown that both mature BLV MA and NC are able to bind to nucleic acids; however, the viral assembly process and packaging of viral genomic RNA requires full-length Gag to produce infectious particles. Compared to lentiviruses, little is known about the structure of the Gag polyprotein of Deltaretroviruses. In this work, structural models of full-length BLV Gag and Gag lacking the MA domain were generated based on previous structural data of individual domains, homology modeling, and flexible fitting to SAXS data using molecular dynamics. The models were used in molecular dynamic simulations to determine the relative mobility of the protein backbone. Functional annealing assays revealed the role of MA in the nucleic acid chaperone activity of BLV Gag. Our results show that full-length BLV Gag has an elongated rod-shaped structure that is relatively rigid, with the exception of the linker between the MA and CA domains. Deletion of the MA domain maintains the elongated structure but alters the rate of BLV Gag-facilitated annealing of two complementary nucleic acids. These data are consistent with a role for the MA domain of retroviral Gag proteins in modulating nucleic acid binding and chaperone activity. Importance BLV is a retrovirus that is found worldwide in domestic cattle. Since BLV infection has serious implications for agriculture, and given its similarities to human retroviruses such as HTLV-1, the development of an effective treatment would have numerous benefits. The Gag polyprotein exists in all retroviruses and is a key player in viral assembly. However, the full-length structure of Gag from any virus has yet to be elucidated at high resolution. This study provides structural data for BLV Gag and could be a starting point for modeling Gag–small molecule interactions with the ultimate goal of developing of a new class of pharmaceuticals.

  • human t cell leukemia virus type 1 gag domains have distinct rna binding specificities with implications for rna packaging and dimerization
    Journal of Biological Chemistry, 2018
    Co-Authors: Joshua Hatterschide, Louis M. Mansky, William A. Cantara, Yu Ci Syu, Ruth J Blower, Heather M Hanson, Karin Musierforsyth
    Abstract:

    Human T-cell leukemia virus type 1 (HTLV-1) is the first retrovirus that has conclusively been shown to cause human diseases. In HIV-1, specific interactions between the nucleocapsid (NC) domain of the Gag protein and genomic RNA (gRNA) mediate gRNA dimerization and selective packaging; however, the mechanism for gRNA packaging in HTLV-1, a Deltaretrovirus, is unclear. In other Deltaretroviruses, the matrix (MA) and NC domains of Gag are both involved in gRNA packaging, but MA binds nucleic acids with higher affinity and has more robust chaperone activity, suggesting that this domain may play a primary role. Here, we show that the MA domain of HTLV-1, but not the NC domain, binds short hairpin RNAs derived from the putative gRNA packaging signal. RNA probing of the HTLV-1 5' leader and cross-linking studies revealed that the primer-binding site and a region within the putative packaging signal form stable hairpins that interact with MA. In addition to a previously identified palindromic dimerization initiation site (DIS), we identified a new DIS in HTLV-1 gRNA and found that both palindromic sequences bind specifically the NC domain. Surprisingly, a mutant partially defective in dimer formation in vitro exhibited a significant increase in RNA packaging into HTLV-1-like particles, suggesting that efficient RNA dimerization may not be strictly required for RNA packaging in HTLV-1. Moreover, the lifecycle of HTLV-1 and other Deltaretroviruses may be characterized by NC and MA functions that are distinct from those of the corresponding HIV-1 proteins, but together provide the functions required for viral replication.

Nicolas Rosewick - One of the best experts on this subject based on the ideXlab platform.

  • characterization of novel bovine leukemia virus blv antisense transcripts by deep sequencing reveals constitutive expression in tumors and transcriptional interaction with viral micrornas
    Retrovirology, 2016
    Co-Authors: Keith Durkin, Nicolas Rosewick, Vincent Hahaut, Maria Artesi, Arsène Burny, Natasa Arsic, Philip J Griebel, Michel Georges
    Abstract:

    Bovine Leukemia Virus (BLV) is a Deltaretrovirus closely related to the Human T cell leukemia virus-1 (HTLV-1). Cattle are the natural host of BLV where it integrates into B-cells, producing a lifelong infection. Most infected animals remain asymptomatic but following a protracted latency period about 5 % develop an aggressive leukemia/lymphoma, mirroring the disease trajectory of HTLV-1. The mechanisms by which these viruses provoke cellular transformation remain opaque. In both viruses little or no transcription is observed from the 5′LTR in tumors, however the proviruses are not transcriptionally silent. In the case of BLV a cluster of RNA polymerase III transcribed microRNAs are highly expressed, while the HTLV-1 antisense transcript HBZ is consistently found in all tumors examined. Here, using RNA-seq, we demonstrate that the BLV provirus also constitutively expresses antisense transcripts in all leukemic and asymptomatic samples examined. The first transcript (AS1) can be alternately polyadenylated, generating a transcript of ~600 bp (AS1-S) and a less abundant transcript of ~2200 bp (AS1-L). Alternative splicing creates a second transcript of ~400 bp (AS2). The coding potential of AS1-S/L is ambiguous, with a small open reading frame of 264 bp, however the transcripts are primarily retained in the nucleus, hinting at a lncRNA-like role. The AS1-L transcript overlaps the BLV microRNAs and using high throughput sequencing of RNA-ligase-mediated (RLM) 5′RACE, we show that the RNA-induced silencing complex (RISC) cleaves AS1-L. Furthermore, experiments using altered BLV proviruses with the microRNAs either deleted or inverted point to additional transcriptional interference between the two viral RNA species. The identification of novel viral antisense transcripts shows the BLV provirus to be far from silent in tumors. Furthermore, the consistent expression of these transcripts in both leukemic and nonmalignant clones points to a vital role in the life cycle of the virus and its tumorigenic potential. Additionally, the cleavage of the AS1-L transcript by the BLV encoded microRNAs and the transcriptional interference between the two viral RNA species suggest a shared role in the regulation of BLV.

  • Identification and characterization of novel Bovine Leukemia Virus ( BLV ) antisense transcripts reveals their constitutive expression in leukemic and pre-leukemic clones
    n/a, 2016
    Co-Authors: Keith Durkin, Nicolas Rosewick, Vincent Hahaut, Philip Griebel, Arsène Burny, Michel Georges, Maria Artesi, Anne Van Den Broeke
    Abstract:

    Bovine Leukemia Virus (BLV) is a Deltaretrovirus closely related to the Human T-cell leukemia virus-1 (HTLV-1). Cattle are the natural host of BLV where it integrates into B-cells and produces a lifelong infection. Most infected animals remain asymptomatic but following a protracted latency period about ~5% develop an aggressive leukemia/lymphoma, mirroring the disease trajectory of HTLV-1. The 5’LTRs of both the BLV and HTLV-1 proviruses are transcriptionally silent in tumors, however they are not entirely quiescent, with the HLTV-1 antisense transcript HBZ and the BLV microRNAs constitutively expressed in tumors. Here, using RNA-seq, we demonstrate that in addition to microRNAs, the BLV provirus also constitutively expresses two antisense transcripts in all BLV infected samples examined. The first transcript (AS1) has alternate potential polyadenylation sites generating a short transcript of ~600bp (AS1-S) and a less abundant longer transcript of ~2200bp (AS1-L). Alternative splicing also creates a second transcript of ~400bp (AS2) utilizing the first exon of AS1. Production of AS transcripts from the 3’LTR was supported by reporter assays demonstrating that the BLV LTR has substantial and Tax-independent antisense promoter activity. BLV AS transcripts predominantly localize in the nucleus. Examination of protein coding potential showed AS2 to be non-coding, while the AS1-S/L transcripts coding potential is ambiguous, with a small potential open reading frame (ORF) of 264bp present. The AS1-L transcript overlaps the BLV microRNAs transcribed in the sense direction. Using high throughput sequencing of RNA-ligase-mediated (RLM) 5' RACE products, we show that the perfect complementary between the transcripts leads to RNA-induced silencing complex (RISC) mediated cleavage of AS1-L. Furthermore, experiments using BLV proviruses where the microRNAs were removed or inverted point to additional transcriptional interactions between the two viral RNA species. Knock down of AS1-S/L using locked nucleic acids (LNAs) showed no obvious effect on the cells phenotype. While a detailed elucidation of the BLV antisense transcripts function remains in the future, the constitutive expression in all samples examined, points to a vital role for the transcripts in the life cycle and oncogenic potential of BLV.

  • next generation transcriptome analysis of Deltaretrovirus induced leukemia from micrornas to macrornas
    2015
    Co-Authors: Nicolas Rosewick, Philippe Martiat, Anne Van Den Broeke
    Abstract:

    Plus de 20 million de personnes a travers le monde sont infectees par le virus T-lymphotrope humain de type 1 (HTLV-1), causant des leucemies a cellules T dans ~5 % des individus infectes. Le virus de la leucemie bovine (BLV), structurellement et fonctionnellement proche de HTLV-1, induit des leucemies a cellules B dans des modeles animaux suite a une infection naturelle (bovin) ou experimentale (mouton). Les mecanismes moleculaires responsables du potentiel oncogene de ces deux virus restent largement incompris. Dans les deux maladies, leucemies T chez l’homme, leucemies B chez l’animal, le site integration du virus dans les cellules leucemiques est tres variable. Il est donc generalement admis que le potentiel oncogene du provirus est principalement lie a l’activite de l’oncoproteine virale Tax. De maniere paradoxale cependant, ni HTLV-1 ni BLV n’expriment de proteines virales au stade tumoral. Dans ce travail, nous avons etudie le transcriptome non codant des leucemies induites par BLV et HTLV-1 par sequencage a haut debit. Dans la premiere partie, nous demontrons que le provirus BLV n’est en fait pas silencieux dans les cellules tumorales. BLV produit un ensemble de dix microARNs (miRNAs) tres abondants et extremement conserves dans toutes les tumeurs. Cette observation constitue la premiere description de miRNAs encodes par un retrovirus. Les microARNs encodes par BLV sont transcrits par la RNA Polymerase III, strategie qui permet leur production de facon independante de celle des messagers viraux ainsi que leur expression abondante dans le contexte tumoral caracterise par l’absence d’activite RNA Polymerase II. Nous avons ensuite montre que, comme HTLV-1, BLV produit des transcrits encodes par le brin negatif du provirus. L’analyse par sequencage ARN a haut debit (RNA-Seq) de tumeurs induites par BLV montre l’absence d’expression virale a partir du promoteur viral situe dans le LTR 5’. Cependant, elle revele la presence de deux transcrits viraux anti-sens non codants (AS1 et AS2) produits par le LTR 3’. Nous avons identifie ces transcrits dans toutes les tumeurs BLV analysees. Enfin, l’analyse RNA-Seq de tumeurs induites par HTLV-1 et BLV a revele la presence d’interactions transcriptionnelles virus-hote. Les genes hotes affectes sont significativement enrichis en genes lies au cancer. Ces resultats suggerent que les transcrits HTLV hbz et BLV AS1 jouent un role essentiel dans la tumorigenese en interagissant avec le genome de l’hote. Nous avons egalement detecte ce type de perturbation a des temps precoces dans le modele experimental BLV chez le mouton. Ces observations suggerent que ces interactions virus-hote constituent des evenements precoces qui procurent un avantage selectif aux clones associes, mais que d’autres alterations -genetiques et/ou epigenetiques- sont necessaires a l’etablissement de la tumeur. En conclusion, nos travaux vont permettre de mieux comprendre le role des interactions virus-genome hote dans l’oncogenese ainsi que la fonction de transcrits non codants dans le developpement des cancers qu’ils soient ou non d’etiologie virale.More than 20 million people are infected by Human T-cell Lymphotropic Virus type 1 (HTLV-1) worldwide and this will cause T-cell leukemia in 5% of them. Yet the molecular mechanisms that underlie the oncogenic potential of this virus remain largely unknown. Bovine Leukemia Virus (BLV) is closely related to HTLV1 and causes a very similar B-cell leukemia in cattle and sheep. As for HTLV1, the oncogenic mechanisms underlying BLV-induced leukemia remain poorly understood. In both diseases, leukemic cells harbor mainly one integrated provirus, yet the integration sites are very variable. As a consequence, it is generally assumed that the oncogenic effect of the provirus is largely mediated by the virally encoded Tax protein. Paradoxically, however, both HTLV1 and BLV proviruses are found to be epigenetically silenced in tumor cells. Thus Tax, as any other virally encoded protein, is not expressed in leukemic cells suggesting that other factors are involved in tumorigenesis. In this study we made three observations that might dramatically change the prevalent dogma of HTLV1 and BLV-induced leukemia. First, we demonstrated that the BLV provirus is not silent at all in tumor cells. A cluster of BLV-encoded microRNAs (miRNAs) is highly expressed, accounting for 40% of the miRNAs present in leukemic cells. This finding is the first description of retroviral-encoded miRNAs. BLV miRNAs are transcribed from five independent RNA Pol III units and are exceedingly conserved across BLV isolates (more than the protein coding genes), strongly supporting an essential yet still unknown function. Next we showed that – as HTLV1 – BLV strongly expresses antisense RNAs. High-throughput sequencing of RNA libraries (RNA-seq) from BLV associated tumors, as expected, showed no expression of viral mRNA from the 5’ LTR. However, it did reveal the presence of two novel non-coding antisense transcripts originating in the 3’ LTR of BLV. Finally, RNA-Seq analysis of HTLV-1 and BLV-induced tumors revealed that the viral 3’ LTR-driven antisense RNAs produced by both viruses interact with host genes localized in the vicinity of proviral integration. Enrichment analysis of affected host genes suggests a significant bias towards cancer-related genes. Host gene perturbations were also found at early stages post-infection in the BLV experimental model in sheep, suggesting that provirus-dependent cancer driver gene perturbations trigger initial amplification of the corresponding clones, requiring additional genetic and/or epigenetic changes to develop full blown leukemia. Overall, our findings reveal an unexpected role for BLV and HTLV antisense transcripts and contribute to the understanding of non-coding RNA-mediated mechanisms in leukemogenesis.

Van Den Broeke Anne - One of the best experts on this subject based on the ideXlab platform.

  • Improving the bioinformatics analysis of HTS clonality data in virus-induced leukemia
    2018
    Co-Authors: Hahaut Vincent, Burny Arsene, Rosewick Nicolas, Artesi Maria, Durkin Keith, Bron Dominique, Georges Michel, Van Den Broeke Anne
    Abstract:

    Proviral integration into the host genome is one of the main hallmarks of infection by oncogenic retroviruses. This event creates a life-long signature, each infected cell being characterized by a specific integration site (IS). Monitoring of the clonal architecture over time (clone: population of cells sharing an identical IS) has significantly contributed to a better understanding of HIV persistence, gene therapy vector mediated treatment and Deltaretrovirus-induced leukemia. Our lab recently developed an optimized high-throughput sequencing (HTS) based clonality method. It enables the identification of proviral integration sites genome-wide while simultaneously quantifying the abundance of the corresponding clones. The method is superior to any of the previously available protocols, mainly in terms of sensitivity, cost-effectiveness and hands-on time, making it suitable for routine clinical observation of infected individuals. Using this method, we recently showed that longitudinal monitoring of the dominant leukemic clone in patients infected by Human T-cell Leukemia Virus-1 (HTLV-1) better predicts therapeutic response (Artesi et al, Leukemia, 2017). We applied the method to biological samples isolated from HTLV-1 infected patients and Bovine Leukemia Virus (BLV) infected animals (bovine and sheep). This resulted in the generation of an unprecedented volume of raw sequence data. In this study we developed a novel clonality analysis pipeline that better exploits the potential of the method, improving previously published protocols

  • Investigating non-coding viral transcripts in Bovine Leukemia Virus induced leukemia
    2017
    Co-Authors: Hahaut Vincent, Burny Arsene, Rosewick Nicolas, Artesi Maria, Durkin Keith, Georges Michel, Griebel Philip, Natasa Arsic, Van Den Broeke Anne
    Abstract:

    Bovine Leukemia Virus (BLV) is a Deltaretrovirus closely related to the Human T-cell leukemia virus-1 (HTLV-1). The natural host of BLV is cattle and much like the case of HTLV-1 in humans, about ~5% of infected individuals develop leukemia/lymphoma following a long period of asymptomatic infection (~7 years in cattle, several decades in human). Experimental infection of sheep with BLV results in a reduced latency period (2 years on average), making for an attractive cancer model. A further advantage of the BLV system is that it is possible to infect sheep via injection of a cloned provirus, facilitating the mutation of specific parts of the viral genome to examine the function of viral products in vivo. Like HTLV-1, the BLV mRNAs/proteins are transcribed from the viral 5’ long terminal repeat (LTR), a region rich in regulatory elements. It was previously believed that the BLV provirus was transcriptionally silent in tumors, however we identified a cluster of five abundantly expressed non-canonical RNA polymerase III dependent microRNAs (miRNAs) encoded by BLV (Rosewick et al., PNAS 2013). In addition, using RNA sequencing we recently discovered viral antisense transcripts originating in the 3' Long Terminal Repeat (LTR) of the BLV provirus (Durkin et al., Retrovirology 2016) . While 5'LTR dependent transcription is absent in malignant cells, both the viral miRNAs and the antisense transcripts are expressed in all BLV induced leukemic and pre-leukemic samples examined to date, pointing to a vital role in the life cycle of the virus and a critical function in cellular transformation

  • Somatic Structural And Numerical Aberrations In Bovine Leukemia Virus Induced Tumors
    2017
    Co-Authors: Durkin Keith, Hahaut Vincent, Artesi Maria, Georges Michel, Griebel Philip, Rosewick Rosewick, Arsic Natasa, Burny Arsène, Van Den Broeke Anne
    Abstract:

    peer reviewedaudience: researcherBovine Leukemia Virus (BLV) is a Deltaretrovirus that integrates into B-cells producing a lifelong infection in cattle. Like its close relative Human T-cell leukemia virus-1 (HTLV-1), BLV induces an aggressive leukemia/lymphoma in about ~5% of infected individuals. While not a natural host it is possible to infect sheep with BLV and in contrast to cattle, all infected sheep develop tumors at an accelerated rate (~18 months). Historically research into both viruses has primarily focused on their transcripts/proteins. However secondary events are likely to be important as only a subset of infected individuals, following many decades of infection, develop a tumor. At the current time little is known about the landscape of somatic changes in BLV induced tumors. To examine gross numerical and structural variants (SVs) we assayed 12 bovine tumors on the BovineSNP50 Illumina BeadChip as well as 22 ovine tumors on the OvineSNP50 Illumina BeadChip. We also carried out whole genome sequencing (~30X) on 4 ovine tumors with matched normal tissue. Initial examination of the tumors revealed frequent aneuploidy, with orthologous regions of the genome involved in both species. Focal SVs identified included an amplification (>4 copies) of the terminus of BTA16 in three tumors (contains PTPRC & miR-181), while the tumor suppressor CDKN2A on OAR2 was deleted in multiple ovine tumors. For the 4 sequenced tumors multiple time points over the course of infection were available allowing us to determine when these SVs arose via nested PCR. Interestingly we observed that the SVs involving well know cancer driver genes generally appear many months prior to tumor development. These preliminary results indicate that tumors induced by HTLV-1 and BLV display somatic structural changes that impinge on overlapping sets of genes and point to the emergence of SVs affecting cancer driver genes in the preleukemic clone, well before the clone undergoes rapid expansion

  • The Landscape And Evolution Of Somatic Mutations In Bovine Leukemia Virus Induced Tumors
    2017
    Co-Authors: Durkin Keith, Hahaut Vincent, Rosewick Nicolas, Artesi Maria, Georges Michel, Griebel Philip, Arsic Natasa, Burny Arsène, Van Den Broeke Anne
    Abstract:

    peer reviewedaudience: researcherBovine Leukemia Virus (BLV) is a Deltaretrovirus that integrates into B-cells producing a lifelong infection in cattle. Like its close relative Human T-cell leukemia virus-1 (HTLV-1), BLV induces an aggressive leukemia/lymphoma in about ~5% of infected individuals. While not a natural host it is possible to infect sheep with BLV and in contrast to cattle, all infected sheep develop tumors at an accelerated rate (~18 months). Historically research into both viruses has primarily focused on their transcripts/proteins. However secondary somatic events are likely to be important as only a subset of infected individuals, following many decades of infection, develop a tumor. At the current time little is known about the landscape of somatic changes in BLV induced tumors and the timing of their occurrence. To address this we have carried out whole genome sequencing of BLV induced tumors from two cattle, and from five sheep with matched normal tissue. This revealed frequent aneuploidy, with orthologous regions of the genome involved in both species and elevated mitochondria DNA copy numbers in tumors. Recurrent structural variants (SVs) were seen affecting the tumor suppressors CDKN2A and ARID1A, both on OAR2. On average ~1400 somatic SNVs were observed in each ovine tumor, with high/moderate impact variants in known cancer drivers genes such as KMT2A, ATRX, RPL22 and KRAS. The five sheep were also sampled at regular time points, prior to leukemia onset, allowing us to examine tumor clone evolution. High throughput sequencing of proviral integration sites showed that the tumor clone represents only a small fraction of the infected cells for the majority of the disease, only expanding rapidly in the terminal stages. Low coverage sequencing of samples prior to tumor development indicates that aneuploidy of OAR9 is a feature of the majority of BLV infected clones. Preliminary nested PCR also showed that many SVs were present prior to tumor development. High throughput approaches are being developed to track both SVs and SNV in the preleukemic stages of the disease

  • Structural And Numerical Somatic Changes In BLV Induced Tumors
    2017
    Co-Authors: Durkin Keith, Hahaut Vincent, Rosewick Nicolas, Artesi Maria, Georges Michel, Griebel Philip, Arsic Natasa, Burny Arsène, Van Den Broeke Anne
    Abstract:

    peer reviewedaudience: researcherBackground Bovine Leukemia Virus (BLV) is a Deltaretrovirus that integrates into B-cells producing a lifelong infection in cattle. Like its close relative Human T-cell leukemia virus-1 (HTLV-1) BLV induces an aggressive leukemia/lymphoma in about ~5% of infected individuals. While not a natural host it is possible to infect sheep with BLV and in contrast to cattle, all infected sheep develop tumors at an accelerated rate (~18 months). Historically research into both viruses has primarily focused on their transcripts/proteins. However secondary events are likely to be important as only a subset of infected individuals, following many decades of infection, develop a neoplasm. Recent work in HTLV-1 induced adult T cell leukemia/lymphoma (ATL) identified a large number of somatic changes associated with malignancy. At the current time little is known about the landscape of somatic changes in BLV induced tumors. Methods To examine gross numerical and structural aberrations in BLV induced tumors we assayed 12 bovine tumors on the BovineSNP50 Illumina BeadChip as well as 22 ovine tumors on the OvineSNP50 Illumina BeadChip. The resultant data was examined with penCNV in combination with visual inspection of the Log R ratios and B allele frequencies. Results The tumors from both species showed frequent aneuploidy with the whole or a large part of chromosomes BTA5, BTA10, BTA14 and BTA24 duplicated in >50% of the bovine tumors. In the ovine tumors chromosomes OAR5, OAR7, OAR9 and OAR16 were frequently duplicated. It is interesting to note that BTA14 is orthologous to OAR9 and both are orthologous to HSA8q, a part of the human genome frequently duplicated in ATLs and other leukemias. In addition a number of focal structural variants were observed. In cattle the terminus of BTA16, which includes the CD45 gene and miR-181 was amplified (>4 copies) in three tumors. In sheep, mirroring observations in ATL, the CDKN2A gene was deleted in multiple tumors. Conclusion These preliminary results indicate that tumors induced by HTLV-1 and BLV display somatic structural changes that impinge on overlapping sets of genes. Secondarily, it appears that in the case of BLV despite the much shorter incubation periods in sheep, the resultant tumors in both the natural and the experimental host display evidence of substantial genome instability

Burny Arsene - One of the best experts on this subject based on the ideXlab platform.

  • Improving the bioinformatics analysis of HTS clonality data in virus-induced leukemia
    2018
    Co-Authors: Hahaut Vincent, Burny Arsene, Rosewick Nicolas, Artesi Maria, Durkin Keith, Bron Dominique, Georges Michel, Van Den Broeke Anne
    Abstract:

    Proviral integration into the host genome is one of the main hallmarks of infection by oncogenic retroviruses. This event creates a life-long signature, each infected cell being characterized by a specific integration site (IS). Monitoring of the clonal architecture over time (clone: population of cells sharing an identical IS) has significantly contributed to a better understanding of HIV persistence, gene therapy vector mediated treatment and Deltaretrovirus-induced leukemia. Our lab recently developed an optimized high-throughput sequencing (HTS) based clonality method. It enables the identification of proviral integration sites genome-wide while simultaneously quantifying the abundance of the corresponding clones. The method is superior to any of the previously available protocols, mainly in terms of sensitivity, cost-effectiveness and hands-on time, making it suitable for routine clinical observation of infected individuals. Using this method, we recently showed that longitudinal monitoring of the dominant leukemic clone in patients infected by Human T-cell Leukemia Virus-1 (HTLV-1) better predicts therapeutic response (Artesi et al, Leukemia, 2017). We applied the method to biological samples isolated from HTLV-1 infected patients and Bovine Leukemia Virus (BLV) infected animals (bovine and sheep). This resulted in the generation of an unprecedented volume of raw sequence data. In this study we developed a novel clonality analysis pipeline that better exploits the potential of the method, improving previously published protocols

  • New insights into Bovine Leukemia Virus (BLV) transcriptional regulation
    2018
    Co-Authors: Bellefroid Maxime, Burny Arsene, Plant Estelle, Rodari Anthony, Van Driessche Benoît, Fauquenoy Sylvain, Vanhulle Caroline, Nestola Lorena, Van Lint Carine
    Abstract:

    Bovine leukemia virus (BLV) is a B-lymphotropic oncogenic Deltaretrovirus infecting cattle and closely related to human T-cell leukemia viruses I and II (HTLV-I and II). Despite the well-established repression of the 5'LTR-driven viral gene expression, we and others have discovered and characterized two alternative viral promoters[1][2][3][4], allowing a high expression of viral miRNAs[2][3] and antisense viral transcripts[4], potentially contributing to tumor progression and to escape from the host immune system. In addition, our data have suggested a collision phenomenon between the RNAPIII transcribing the miRNA cluster and the RNAPII coming in an antisense orientation from the 3’LTR[1] with, as a result, a stalling of both RNA polymerase complexes. These latter results have indicated that transcriptional interference could be seen as a new mechanism used by BLV to regulate its three transcriptional activities.In this work, we investigated the interplay and self-regulation between the three BLV promoter activities and showed putative critical functions of the transcriptional interference to drive or repress BLV transcriptional activities. In addition, we highlighted the implication of new transcription factors in BLV transcriptional and epigenetic regulations but also in BLV-mediated pathogenesis. Overall in this study, we further investigated new alternative ways used by BLV to regulate its transcriptional and epigenetic status and provided new fundamental insights into BLV transcriptional and epigenetic regulations which could explain the escape from the host immune system and/or the BLV-induced pathogenesis.info:eu-repo/semantics/publishe

  • Investigating non-coding viral transcripts in Bovine Leukemia Virus induced leukemia
    2017
    Co-Authors: Hahaut Vincent, Burny Arsene, Rosewick Nicolas, Artesi Maria, Durkin Keith, Georges Michel, Griebel Philip, Natasa Arsic, Van Den Broeke Anne
    Abstract:

    Bovine Leukemia Virus (BLV) is a Deltaretrovirus closely related to the Human T-cell leukemia virus-1 (HTLV-1). The natural host of BLV is cattle and much like the case of HTLV-1 in humans, about ~5% of infected individuals develop leukemia/lymphoma following a long period of asymptomatic infection (~7 years in cattle, several decades in human). Experimental infection of sheep with BLV results in a reduced latency period (2 years on average), making for an attractive cancer model. A further advantage of the BLV system is that it is possible to infect sheep via injection of a cloned provirus, facilitating the mutation of specific parts of the viral genome to examine the function of viral products in vivo. Like HTLV-1, the BLV mRNAs/proteins are transcribed from the viral 5’ long terminal repeat (LTR), a region rich in regulatory elements. It was previously believed that the BLV provirus was transcriptionally silent in tumors, however we identified a cluster of five abundantly expressed non-canonical RNA polymerase III dependent microRNAs (miRNAs) encoded by BLV (Rosewick et al., PNAS 2013). In addition, using RNA sequencing we recently discovered viral antisense transcripts originating in the 3' Long Terminal Repeat (LTR) of the BLV provirus (Durkin et al., Retrovirology 2016) . While 5'LTR dependent transcription is absent in malignant cells, both the viral miRNAs and the antisense transcripts are expressed in all BLV induced leukemic and pre-leukemic samples examined to date, pointing to a vital role in the life cycle of the virus and a critical function in cellular transformation

  • Characterization of novel Bovine Leukemia Virus (BLV) antisense transcripts by deep sequencing reveals constitutive expression in tumors and transcriptional interaction with viral microRNAs
    'Springer Science and Business Media LLC', 2016
    Co-Authors: Durkin Keith, Burny Arsene, Hahaut Vincent, Rosewick Nicolas, Artesi Maria, Georges Michel, Griebel Philip, Arsic Natasa, Van Den Broeke Anne
    Abstract:

    Background: Bovine Leukemia Virus (BLV) is a Deltaretrovirus closely related to the Human T cell leukemia virus-1 (HTLV-1). Cattle are the natural host of BLV where it integrates into B-cells, producing a lifelong infection. Most infected animals remain asymptomatic but following a protracted latency period about 5% develop an aggressive leukemia/lymphoma, mirroring the disease trajectory of HTLV-1. The mechanisms by which these viruses provoke cellular transformation remain opaque. In both viruses little or no transcription is observed from the 5'LTR in tumors, however the proviruses are not transcriptionally silent. In the case of BLV a cluster of RNA polymerase III transcribed microRNAs are highly expressed, while the HTLV-1 antisense transcript HBZ is consistently found in all tumors examined. Results: Here, using RNA-seq, we demonstrate that the BLV provirus also constitutively expresses antisense transcripts in all leukemic and asymptomatic samples examined. The first transcript (AS1) can be alternately polyadenylated, generating a transcript of ~600bp (AS1-S) and a less abundant transcript of ~2200bp (AS1-L). Alternative splicing creates a second transcript of ~400bp (AS2). The coding potential of AS1-S/L is ambiguous, with a small open reading frame of 264bp, however the transcripts are primarily retained in the nucleus, hinting at a lncRNA-like role. The AS1-L transcript overlaps the BLV microRNAs and using high throughput sequencing of RNA-ligase-mediated (RLM) 5'RACE, we show that the RNA-induced silencing complex (RISC) cleaves AS1-L. Furthermore, experiments using altered BLV proviruses with the microRNAs either deleted or inverted point to additional transcriptional interference between the two viral RNA species. Conclusions: The identification of novel viral antisense transcripts shows the BLV provirus to be far from silent in tumors. Furthermore, the consistent expression of these transcripts in both leukemic and nonmalignant clones points to a vital role in the life cycle of the virus and its tumorigenic potential. Additionally, the cleavage of the AS1-L transcript by the BLV encoded microRNAs and the transcriptional interference between the two viral RNA species suggest a shared role in the regulation of BLV.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

  • Identification and characterization of novel bovine leukemia virus (BLV) antisense transcripts in leukemic and pre-leukemic clones
    2016
    Co-Authors: Durkin Keith, Burny Arsene, Hahaut Vincent, Rosewick Nicolas, Artesi Maria, Georges Michel, Griebel Philip, Arsic Natasa, Van Den Broeke Anne
    Abstract:

    The Deltaretrovirus Bovine Leukemia Virus (BLV) is closely related to the Human T-cell leukemia virus-1 (HTLV-1). Cattle are the natural host of BLV where it integrates into B-cells, produces a lifelong infection. Most infected animals remain asymptomatic but following a protracted latency period about ~5% develop an aggressive leukemia/lymphoma, mirroring the disease trajectory of HTLV-1. Like the case in HTLV-1 the 5’LTR BLV provirus is transcriptionally silent in tumors, however the provirus is not entirely quiescent, constitutively express the BLV microRNAs in tumors. Using RNA-seq, we found that in addition to microRNAs, the BLV provirus also constitutively expresses two antisense transcripts in all BLV infected samples examined. The first transcript (AS1) has alternate potential polyadenylation sites generating a short transcript of ~600bp (AS1-S) and a less abundant longer transcript of ~2200bp (AS1-L). Alternative splicing also creates a second transcript of ~400bp (AS2) utilizing the first exon of AS1. Production of AS transcripts from the 3’LTR was supported by reporter assays demonstrating that the BLV LTR has substantial and Tax-independent antisense promoter activity. BLV AS transcripts predominantly localize in the nucleus. Examination of protein coding potential showed AS2 to be non-coding, while the AS1-S/L transcripts coding potential is ambiguous, with a small potential open reading frame (ORF) of 264bp present. The AS1-L transcript overlaps the BLV microRNAs transcribed in the sense direction. Using high throughput sequencing of RNA-ligase-mediated (RLM) 5' RACE products, we show that the perfect complementary between the transcripts leads to RNA-induced silencing complex (RISC) mediated cleavage of AS1-L. Furthermore, experiments using BLV proviruses where the microRNAs were removed or inverted point to additional transcriptional interactions between the two viral RNA species. Knock down of AS1-S/L using locked nucleic acids (LNAs) showed no obvious effect on the cells phenotype. While a detailed elucidation of the BLV antisense transcripts function remains in the future, the constitutive expression in all samples examined, points to a vital role for the transcripts in the life cycle and oncogenic potential of BLV.Peer reviewe