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Application of representational difference analysis to genomic fragments of Marek's disease virus.

ABSTRACT: A rapid and simple method for isolation of DNA fragments of Marek's disease virus (MDV) based on representational difference analysis (RDA) was developed. Multiple viral DNA fragments, the sizes of which were restricted to 0.3 to 3.5 kbp, were simultaneously amplified after subtraction of chicken DNA from BamHI-, BglII-, EcoRI-, HindIII-, or XhoI-digested DNA fragments of MDV-infected cells. Nucleotide sequence of two RDA-derived fragments coincided with the sequence determined from direct sequencing of the MDV genome. We detected an interstrain difference in the size of restriction enzyme-digested fragments on agarose gel. This method was used on a single feather pulp to generate sufficient MDV DNA for cloning.

SUBMITTER: Endoh D

PROVIDER: S-EPMC87597 | biostudies-literature | 2000 Dec

REPOSITORIES: biostudies-literature

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Application of representational difference analysis to genomic fragments of Marek's disease virus.

Endoh D D Cho K O KO Tsukamoto K K Morimura T T Kon Y Y Hayashi M M

Journal of clinical microbiology 20001201 12

A rapid and simple method for isolation of DNA fragments of Marek's disease virus (MDV) based on representational difference analysis (RDA) was developed. Multiple viral DNA fragments, the sizes of which were restricted to 0.3 to 3.5 kbp, were simultaneously amplified after subtraction of chicken DNA from BamHI-, BglII-, EcoRI-, HindIII-, or XhoI-digested DNA fragments of MDV-infected cells. Nucleotide sequence of two RDA-derived fragments coincided with the sequence determined from direct seque ...[more]

PMID: 11101556

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Project description:The highly oncogenic alphaherpesvirus Marek's disease virus (MDV) causes immense economic losses in the poultry industry. MDV induces a variety of symptoms in infected chickens, including neurological disorders and immunosuppression. Most notably, MDV induces transformation of lymphocytes, leading to T cell lymphomas in visceral organs with a mortality of up to 100%. While several factors involved in MDV tumorigenesis have been identified, the transformation process and tumor composition remain poorly understood. Here we developed an imaging mass spectrometry (IMS) approach that allows sensitive visualization of MDV-induced lymphoma with a specific mass profile and precise differentiation from the surrounding tissue. To identify potential tumor markers in tumors derived from a very virulent wild-type virus and a telomerase RNA-deficient mutant, we performed laser capture microdissection (LCM) and thereby obtained tumor samples with no or minimal contamination from surrounding nontumor tissue. The proteomes of the LCM samples were subsequently analyzed by quantitative mass spectrometry based on stable isotope labeling. Several proteins, like interferon gamma-inducible protein 30 and a 70-kDa heat shock protein, were identified that are differentially expressed in tumor tissue compared to surrounding tissue and naive T cells. Taken together, our results demonstrate for the first time that MDV-induced tumors can be visualized using IMS, and we identified potential MDV tumor markers by analyzing the proteomes of virus-induced tumors.IMPORTANCE Marek's disease virus (MDV) is an oncogenic alphaherpesvirus that infects chickens and causes the most frequent clinically diagnosed cancer in the animal kingdom. Not only is MDV an important pathogen that threatens the poultry industry but it is also used as a natural virus-host model for herpesvirus-induced tumor formation. In order to visualize MDV-induced lymphoma and to identify potential biomarkers in an unbiased approach, we performed imaging mass spectrometry (IMS) and noncontact laser capture microdissection. This study provides a first description of the visualization of MDV-induced tumors by IMS that could be applied also for diagnostic purposes. In addition, we identified and validated potential biomarkers for MDV-induced tumors that could provide the basis for future research on pathogenesis and tumorigenesis of this malignancy.

Project description:UnlabelledMarek'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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Application of representational difference analysis to genomic fragments of Marek's disease virus.

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Application of representational difference analysis to genomic fragments of Marek's disease virus.

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