T-cell transformation by Marek's disease virus

ABSTRACTMarek’s disease virus 1 (MDV-1), an oncogenic α-herpesvirus that induces T-cell lymphomas in chickens, serves as model system to study transformation by lymphotropic herpesviruses. Like the oncogenic human γ-herpesviruses Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), MDV-1 encodes several viral microRNAs (miRNAs). One MDV-1 miRNA, miR-M4, shares the same “seed” targeting sequence with both a KSHV miRNA, miR-K11, and cellular miR-155. Importantly, miR-M4 plays a critical role in T-cell transformation by MDV-1, while miR-K11 and cellular miR-155 are thought to play key roles in B-cell transformation by KSHV and EBV, respectively. Here, we present an analysis of the mRNAs targeted by viral miRNAs expressed in the chicken T-cell line MSB1, which is naturally coinfected with MDV-1 and the related nonpathogenic virus MDV-2. Our analysis identified >1,000 endogenous mRNAs targeted by miRNAs encoded by each virus, many of which are targeted by both MDV-1 and MDV-2 miRNAs. We present a functional analysis of an MDV-1 gene, RLORF8, targeted by four MDV-1 miRNAs and a cellular gene, encoding interleukin-18 (IL-18) and targeted by both MDV-1 and MDV-2 miRNAs, and show that ectopic expression of either protein in a form resistant to miRNA inhibition results in inhibition of cell proliferation. Finally, we present a restricted list of 9 genes targeted by not only MDV-1 miR-M4 but also KSHV miR-K11 and human miR-155. Given the critical role played by miR-155 seed family members in lymphomagenesis in humans and chickens, these mRNA targets may contain genes whose inhibition plays a conserved role in herpesvirus transformation.IMPORTANCEHerpesviruses cause lymphomas in both humans and chickens, and in both cases, evidence indicates that virally encoded miRNAs, or virally subverted cellular miRNAs, belonging to the miR-155 seed family, play a critical role in this process. However, because each miRNA regulates numerous cellular mRNAs species, it has been difficult to elucidate which miRNA targets are important. Given the evolutionary distance between chickens and humans and the observation that miR-155 is nevertheless highly conserved in both species, we reasoned that the identification of shared miR-155 targets might shed light on this process. Here, we present an analysis of the mRNAs targeted by miRNAs encoded by the oncogenic avian herpesvirus MDV-1 in transformed chicken T cells, including a short list of mRNAs that are also targeted by miR-155 seed family miRNAs in EBV- or KSHV-transformed human B cells, and present an initial functional analysis of some of these miRNA targets.

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Marek's Disease Virus-Encoded MicroRNA 155 Ortholog Critical for the Induction of Lymphomas Is Not Essential for the Proliferation of Transformed Cell Lines

Journal of Virology ◽

10.1128/jvi.00713-19 ◽

2019 ◽

Vol 93 (17) ◽

Cited By ~ 8

Author(s):

Yaoyao Zhang ◽

Na Tang ◽

Jun Luo ◽

Man Teng ◽

Katy Moffat ◽

...

Keyword(s):

T Cell ◽

Disease Virus ◽

Cell Lines ◽

Critical Role ◽

Marek's Disease Virus ◽

Marek's Disease ◽

Marek’S Disease Virus ◽

Marek’S Disease ◽

T Cell Lymphomas

ABSTRACT MicroRNAs (miRNAs) are small noncoding RNAs with profound regulatory roles in many areas of biology, including cancer. MicroRNA 155 (miR-155), one of the extensively studied multifunctional miRNAs, is important in several human malignancies such as diffuse large B cell lymphoma and chronic lymphocytic leukemia. Moreover, miR-155 orthologs KSHV-miR-K12-11 and MDV-miR-M4, encoded by Kaposi’s sarcoma-associated herpesvirus (KSHV) and Marek’s disease virus (MDV), respectively, are also involved in oncogenesis. In MDV-induced T-cell lymphomas and in lymphoblastoid cell lines derived from them, MDV-miR-M4 is highly expressed. Using excellent disease models of infection in natural avian hosts, we showed previously that MDV-miR-M4 is critical for the induction of T-cell lymphomas as mutant viruses with precise deletions were significantly compromised in their oncogenicity. However, those studies did not elucidate whether continued expression of MDV-miR-M4 is essential for maintaining the transformed phenotype of tumor cells. Here using an in situ CRISPR/Cas9 editing approach, we deleted MDV-miR-M4 from the MDV-induced lymphoma-derived lymphoblastoid cell line MDCC-HP8. Precise deletion of MDV-miR-M4 was confirmed by PCR, sequencing, quantitative reverse transcription-PCR (qRT-PCR), and functional analysis. Continued proliferation of the MDV-miR-M4-deleted cell lines demonstrated that MDV-miR-M4 expression is not essential for maintaining the transformed phenotype, despite its initial critical role in the induction of lymphomas. Ability to examine the direct role of oncogenic miRNAs in situ in tumor cell lines is valuable in delineating distinct determinants and pathways associated with the induction or maintenance of transformation in cancer cells and will also contribute significantly to gaining further insights into the biology of oncogenic herpesviruses. IMPORTANCE Marek’s disease virus (MDV) is an alphaherpesvirus associated with Marek’s disease (MD), a highly contagious neoplastic disease of chickens. MD serves as an excellent model for studying virus-induced T-cell lymphomas in the natural chicken hosts. Among the limited set of genes associated with MD oncogenicity, MDV-miR-M4, a highly expressed viral ortholog of the oncogenic miR-155, has received extensive attention due to its direct role in the induction of lymphomas. Using a targeted CRISPR-Cas9-based gene editing approach in MDV-transformed lymphoblastoid cell lines, we show that MDV-miR-M4, despite its critical role in the induction of tumors, is not essential for maintaining the transformed phenotype and continuous proliferation. As far as we know, this was the first study in which precise editing of an oncogenic miRNA was carried out in situ in MD lymphoma-derived cell lines to demonstrate that it is not essential in maintaining the transformed phenotype.

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Marek’s disease virus may interfere with T cell immunity by TLR3 signals

Veterinary Research Communications ◽

10.1007/s11259-014-9598-x ◽

2014 ◽

Vol 38 (2) ◽

pp. 149-156 ◽

Cited By ~ 9

Author(s):

Xuming Hu ◽

Wencai Xu ◽

Aijian Qin ◽

Genghua Wu ◽

Kun Qian ◽

...

Keyword(s):

T Cell ◽

Disease Virus ◽

Marek's Disease Virus ◽

Marek's Disease ◽

T Cell Immunity ◽

Marek’S Disease Virus ◽

Marek’S Disease ◽

Cell Immunity

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Differential Virus-Specific IFN-Gamma Producing T Cell Responses to Marek’s Disease Virus in Chickens With B19 and B21 MHC Haplotypes

Frontiers in Immunology ◽

10.3389/fimmu.2021.784359 ◽

2022 ◽

Vol 12 ◽

Author(s):

Nitish Boodhoo ◽

Shahriar Behboudi

Keyword(s):

T Cell ◽

Disease Virus ◽

Ex Vivo ◽

T Cell Responses ◽

Marek's Disease Virus ◽

Marek's Disease ◽

Marek’S Disease Virus ◽

Marek’S Disease ◽

Ifn Gamma ◽

Cell Responses

Marek’s disease virus (MDV), the etiologic agent for Marek’s disease (MD), causes a deadly lymphoproliferative disease in chickens. Causes of the well-documented association between genetically defined lines of chicken and resistance to MD remain unknown. Here, the frequencies of IFN-gamma producing pp38 and MEQ-specific T cell responses were determined in line N (B21 haplotype; MD-resistant) and line P2a (B19 haplotype, MD-susceptible) chickens after infection with vaccine and/or virulent (RB1B) strains of MDV using both standard ex vivo and cultured chIFN-gamma ELISPOT assays. Notably, MDV infection of naïve and vaccinated MD-resistant chickens induced higher frequencies of IFN-gamma producing MDV-specific T cell responses using the cultured and ex vivo ELISPOT assay, respectively. Remarkably, vaccination did not induce or boost MEQ-specific effector T cells in the susceptible chickens, while it boosted both pp38-and MEQ-specific response in resistant line. Taken together, our results revealed that there is a direct association between the magnitude of T cell responses to pp38 and MEQ of MDV antigens and resistance to the disease.

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

Journal of Virology ◽

10.1128/jvi.02659-07 ◽

2008 ◽

Vol 82 (8) ◽

pp. 4007-4015 ◽

Cited By ~ 100

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.

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Homodimerization of the Meq Viral Oncoprotein Is Necessary for Induction of T-Cell Lymphoma by Marek's Disease Virus

Journal of Virology ◽

10.1128/jvi.01393-09 ◽

2009 ◽

Vol 83 (21) ◽

pp. 11142-11151 ◽

Cited By ~ 38

Author(s):

Andrew C. Brown ◽

Lorraine P. Smith ◽

Lydia Kgosana ◽

Susan J. Baigent ◽

Venugopal Nair ◽

...

Keyword(s):

T Cell ◽

Disease Virus ◽

Leucine Zipper ◽

Target Genes ◽

Structural Features ◽

Marek's Disease Virus ◽

Marek's Disease ◽

Marek’S Disease Virus ◽

Marek’S Disease ◽

Viral Oncoprotein

ABSTRACT Marek's disease virus (MDV) is a lymphotropic alphaherpesvirus that induces fatal rapid-onset T-cell lymphomas in chickens, its natural host. The MDV-encoded nuclear oncoprotein Meq is essential for lymphomagenesis and acts as a regulator of transcription. Meq has structural features, including a basic domain adjacent to a leucine zipper motif (B-ZIP), that suggest it is related to the Jun/Fos family of transcription factors. Via the leucine zipper, Meq can form homodimers or heterodimerize with c-Jun. Meq/Meq homodimers are associated with transrepression, and Meq/Jun heterodimers can transactivate target genes carrying an AP-1-like binding site. In order to determine the role of the leucine zipper and of Meq dimerization in T lymphomagenesis, specific point mutations were engineered into the highly oncogenic RB-1B strain of MDV to produce virus completely lacking a functional Meq leucine zipper (RB-1B MeqBZIP/BZIP) or virus encoding Meq that cannot homodimerize but can still bind to c-Jun and an AP-1-like site on DNA (RB-1B MeqHom/Hom). Both of these mutant viruses were capable of replication in cultured chicken embryo fibroblasts. However both mutations resulted in a complete loss of oncogenicity, since no lymphomas were produced up to 90 days postinfection in experimentally infected chicks. We conclude that the leucine zipper is necessary for the oncogenic activity of Meq and/or the efficient establishment of long-term MDV latency in T cells. Moreover, it appears that the ability to form homodimers is an absolute requirement and the ability to bind c-Jun alone is insufficient for the T-cell lymphomagenesis associated with virulent MDV.

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Cytogenetic stability of chicken T-cell line transformed with Marek's disease virus: atomic force microscope, a new tool for investigation

Journal of Molecular Recognition ◽

10.1002/jmr.1094 ◽

2010 ◽

Vol 24 (4) ◽

pp. 608-618 ◽

Cited By ~ 4

Author(s):

Sebastiano Di Bucchianico ◽

Maria Federica Giardi ◽

Patrizia De Marco ◽

Luca Ottaviano ◽

Dario Botti

Keyword(s):

T Cell ◽

Atomic Force Microscope ◽

Cell Line ◽

Disease Virus ◽

Marek's Disease Virus ◽

Marek's Disease ◽

Marek’S Disease Virus ◽

Marek’S Disease ◽

Atomic Force

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An Anti-Tumor Vaccine Against Marek's Disease Virus Induces Differential Activation and Memory Response of γδ T Cells and CD8 T Cells in Chickens

Frontiers in Immunology ◽

10.3389/fimmu.2021.645426 ◽

2021 ◽

Vol 12 ◽

Author(s):

Xiaoli Hao ◽

Shuai Li ◽

Jiaqi Li ◽

Yi Yang ◽

Aijian Qin ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Disease Virus ◽

Γδ T Cells ◽

Marek's Disease Virus ◽

Receptor Expression ◽

Marek's Disease ◽

Marek’S Disease Virus ◽

Immune Protection ◽

Marek’S Disease

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes deadly T-cell lymphomas and serves as a natural virus-induced tumor model in chickens. The most efficacious vaccine, CVI988/Rispens (CVI988), against MD has been used for several decades. However, the mechanisms leading to protective immunity following vaccination are not fully understood. In this study, employing multi-parameter flow cytometry, we performed a comprehensive analysis of T cell responses in CVI988-vaccinated chickens. CVI988 vaccination induced significant expansion of γδ T cells and CD8α+ T cells but not CD4+ T cells in spleen, lung and blood at early time-points. The expansion of these cells was CVI988-specific as infection with very virulent MDV RB1B did not elicit expansion of either γδ or CD8α+ T cells. Phenotypic analysis showed that CVI988 vaccination elicited preferential proliferation of CD8α+ γδ T cells and CD8αα co-receptor expression was upregulated on γδ T cells and CD8α+ T cells after immunization. Additionally, cell sorting and quantitative RT-PCR showed that CVI988 vaccination activated γδ T cells and CD8α+ T cells which exhibited differential expression of cytotoxic and T cell-related cytokines. Lastly, secondary immunization with CVI988 induced the expansion of CD8+ T cells but not γδ T cells at higher magnitude, compared to primary immunization, suggesting CVI988 did induce memory CD8+ T cells but not γδ T cells in chickens. Our results, for the first time, reveal a potential role of γδ T cells in CVI988-induced immune protection and provide new insights into the mechanism of immune protection against oncogenic MDV.

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