scholarly journals A Cell Culture System to Investigate Marek’s Disease Virus Integration into Host Chromosomes

2021 ◽  
Vol 9 (12) ◽  
pp. 2489
Author(s):  
Yu You ◽  
Tereza Vychodil ◽  
Giulia Aimola ◽  
Renato L. Previdelli ◽  
Thomas W. Göbel ◽  
...  
Keyword(s):  
Disease Virus ◽  
Marek's Disease Virus ◽  
Telomeric Repeat ◽  
Marek's Disease ◽  
Target Cells ◽  
Neoplastic Disease ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
Integration Mechanism

Marek’s disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes a devastating neoplastic disease in chickens. MDV has been shown to integrate its genome into the telomeres of latently infected and tumor cells, which is crucial for efficient tumor formation. Telomeric repeat arrays present at the ends of the MDV genome facilitate this integration into host telomeres; however, the integration mechanism remains poorly understood. Until now, MDV integration could only be investigated qualitatively upon infection of chickens. To shed further light on the integration mechanism, we established a quantitative integration assay using chicken T cell lines, the target cells for MDV latency and transformation. We optimized the infection conditions and assessed the establishment of latency in these T cells. The MDV genome was efficiently maintained over time, and integration was confirmed in these cells by fluorescence in situ hybridization (FISH). To assess the role of the two distinct viral telomeric repeat arrays in the integration process, we tested various knockout mutants in our in vitro integration assay. Efficient genome maintenance and integration was thereby dependent on the presence of the telomeric repeat arrays in the virus genome. Taken together, we developed and validated a novel in vitro integration assay that will shed light on the integration mechanism of this highly oncogenic virus into host telomeres.

scholarly journals Efficient Mutagenesis of Marek’s Disease Virus-Encoded microRNAs Using a CRISPR/Cas9-Based Gene Editing System

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 466 ◽  
Author(s):  
Jun Luo ◽  
Man Teng ◽  
Xusheng Zai ◽  
Na Tang ◽  
Yaoyao Zhang ◽  
...  
Keyword(s):  
Disease Virus ◽  
Gene Editing ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
Bac Clone ◽  
Virus Growth ◽  
Virus Serotype

The virus-encoded microRNAs (miRNAs) have been demonstrated to have important regulatory roles in herpesvirus biology, including virus replication, latency, pathogenesis and/or tumorigenesis. As an emerging efficient tool for gene editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has been successfully applied in manipulating the genomes of large DNA viruses. Herein, utilizing the CRISPR/Cas9 system with a double-guide RNAs transfection/virus infection strategy, we have established a new platform for mutagenesis of viral miRNAs encoded by the Marek’s disease virus serotype 1 (MDV-1), an oncogenic alphaherpesvirus that can induce rapid-onset T-cell lymphomas in chickens. A series of miRNA-knocked out (miR-KO) mutants with deletions of the Meq- or the mid-clustered miRNAs, namely RB-1B∆Meq-miRs, RB-1B∆M9-M2, RB-1B∆M4, RB-1B∆M9 and RB-1B∆M11, were generated from vvMDV strain RB-1B virus. Interestingly, mutagenesis of the targeted miRNAs showed changes in the in vitro virus growth kinetics, which is consistent with that of the in vivo proliferation curves of our previously reported GX0101 mutants produced by the bacterial artificial chromosome (BAC) clone and Rec E/T homologous recombination techniques. Our data demonstrate that the CRISPR/Cas9-based gene editing is a simple, efficient and relatively nondisruptive approach for manipulating the small non-coding genes from the genome of herpesvirus and will undoubtedly contribute significantly to the future progress in herpesvirus biology.

scholarly journals MicroRNAs 221 and 222 target p27Kip1 in Marek's disease virus-transformed tumour cell line MSB-1

2009 ◽  
Vol 90 (5) ◽  
pp. 1164-1171 ◽  
Author(s):  
Luke S. Lambeth ◽  
Yongxiu Yao ◽  
Lorraine P. Smith ◽  
Yuguang Zhao ◽  
Venugopal Nair
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Disease Virus ◽  
Expression Profiles ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Tumour Cell Line ◽  
Neoplastic Disease ◽  
Marek’S Disease Virus ◽  
Marek’S Disease

MicroRNAs (miRNAs) are a class of short RNAs that function as post-transcriptional suppressors of protein expression and are involved in a variety of biological processes, including oncogenesis. Several recent studies have implicated the involvement of miR-221 and miR-222 in tumorigenesis as these miRNAs are upregulated in a number of cancers and affect the expression of cell cycle regulatory proteins such as the cyclin-dependent kinase (cdk) inhibitor p27Kip1. Marek's disease virus (MDV) is a highly oncogenic herpesvirus that affects poultry, causing acute neoplastic disease with lymphomatous lesions in several organs. MDV-encoded oncogenes such as Meq are directly implicated in the neoplastic transformation of T cells and have been well studied. More recently, however, the involvement of both host and virus-encoded miRNAs in the induction of MD lymphomas is being increasingly recognized. We analysed the miRNA expression profiles in the MDV-transformed lymphoblastoid cell line MSB-1 and found that endogenous miRNAs miR-221 and miR-222 were significantly upregulated. Demonstration of the conserved binding sites for these miRNAs in the chicken p27Kip1 3′-untranslated region sequence and the repression of luciferase activity of reporter constructs indicated that miR-221 and miR-222 target p27Kip1 in these cells. We also found that overexpression of miR-221 and miR-222 decreased p27Kip1 levels and that treatment with retrovirally expressed antagomiRs partially alleviated this suppression. These data show that an oncogenic herpesvirus, as in the case of many cancers, can exploit the miRNA machinery for suppressing cell cycle regulatory molecules such as p27Kip1 in the induction and progression of T-cell lymphomas.

scholarly journals MicroRNA Profile of Marek's Disease Virus-Transformed T-Cell Line MSB-1: Predominance of Virus-Encoded MicroRNAs

2008 ◽  
Vol 82 (8) ◽  
pp. 4007-4015 ◽  
Author(s):  
Yongxiu Yao ◽  
Yuguang Zhao ◽  
Hongtao Xu ◽  
Lorraine P. Smith ◽  
Charles H. Lawrie ◽  
...  
Keyword(s):  
T Cell ◽  
Cell Line ◽  
Disease Virus ◽  
Northern Blotting ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Neoplastic Disease ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
Microrna Profile

ABSTRACT Research over the last few years has demonstrated the increasing role of microRNAs (miRNAs) as major regulators of gene expression in diverse cellular processes and diseases. Several viruses, particularly herpesviruses, also use the miRNA pathway of gene regulation by encoding their own miRNAs. Marek's disease (MD) is a widespread lymphomatous neoplastic disease of poultry caused by the highly contagious Marek's disease virus type 1 (MDV-1). Recent studies using virus-infected chicken embryo fibroblasts have identified at least eight miRNAs that map to the RL/RS region of the MDV genome. Since MDV is a lymphotropic virus that induces T-cell lymphomas, analysis of the miRNA profile in T-cell lymphoma would be more relevant for examining their role in oncogenesis. We determined the viral and host miRNAs expressed in MSB-1, a lymphoblastoid cell line established from an MDV-induced lymphoma of the spleen. In this paper, we report the identification of 13 MDV-1-encoded miRNAs (12 by direct cloning and 1 by Northern blotting) from MSB-1 cells. These miRNAs, five of which are novel MDV-1 miRNAs, map to the Meq and latency-associated transcript regions of the MDV genome. Furthermore, we show that miRNAs encoded by MDV-1 and the coinfected MDV-2 accounted for >60% of the 5,099 sequences of the MSB-1 “miRNAome.” Several chicken miRNAs, some of which are known to be associated with cancer, were also cloned from MSB-1 cells. High levels of expression of MDV-1-encoded miRNAs and potentially oncogenic host miRNAs suggest that miRNAs may have major roles in MDV pathogenesis and neoplastic transformation.

scholarly journals Transcriptome-wide N6-methyladenosine modification profiling of long non-coding RNAs during replication of Marek’s disease virus in vitro

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Aijun Sun ◽  
Xiaojing Zhu ◽  
Ying Liu ◽  
Rui Wang ◽  
Shuaikang Yang ◽  
...  
Keyword(s):  
Disease Virus ◽  
Chicken Embryo Fibroblast ◽  
Uninfected Cell ◽  
Marek's Disease Virus ◽  
Marek's Disease ◽  
Marek’S Disease Virus ◽  
Marek’S Disease ◽  
Rna Immunoprecipitation ◽  
Non Coding Rnas

Abstract Background The newly discovered reversible N6-methyladenosine (m6A) modification plays an important regulatory role in gene expression. Long non-coding RNAs (lncRNAs) participate in Marek’s disease virus (MDV) replication but how m6A modifications in lncRNAs are affected during MDV infection is currently unknown. Herein, we profiled the transcriptome-wide m6A modification in lncRNAs in MDV-infected chicken embryo fibroblast (CEF) cells. Results Methylated RNA immunoprecipitation sequencing results revealed that the lncRNA m6A modification is highly conserved with MDV infection increasing the expression of lncRNA m6A modified sites compared to uninfected cell controls. Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that lncRNA m6A modifications were highly associated with signaling pathways associated with MDV infection. Conclusions In this study, the alterations seen in transcriptome-wide m6A occurring in lncRNAs following MDV-infection suggest this process plays important regulatory roles during MDV replication. We report for the first time profiling of the alterations in transcriptome-wide m6A modification in lncRNAs of MDV-infected CEF cells.

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