Unknown,Transcriptomics,Genomics,Proteomics

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Custom microchip analysis identifies differentially expressed non-coding RNAs in Epstein-Barr virus-infected B cells

ABSTRACT: Upon Epstein-Barr virus (EBV) infection of human B lymphocytes non-coding RNAs (ncRNAs) regulate expression of viral and cellular genes. In this study, we generated a specialized cDNA library from EBV-immortalized cells and subjected it to deep sequencing. We identified 631 unique ncRNA genes, comprised of 321 potential novel differentially expressed ncRNA candidates. Subsequently, we investigated differential expression of known and potential novel ncRNA candidates by custom-designed microchips by comparing expression of ncRNA genes of EBV-immortalized versus non-infected control cells. Among the differentially expressed candidates from chip analysis, differential expression of six novel ncRNA candidates was verified by northern blot analysis. In addition, microchip analysis resulted in observation of increased expression levels of a significant number of potential ncRNA candidates that were preferentially derived from genomic loci annotated as Alu repetitive elements. Alu elements are members of the repeat subfamily of short interspersed nuclear elements (SINE) and were reported to be transcribed upon stress stimulation. While EBV infection significantly up-regulated expression of Alu-derived RNA transcripts, no significant increase in expression of these transcripts was observed under additional tested stress conditions. By employing deep sequencing followed by custom microchip analysis, we identified six novel differentially expressed ncRNAs as well as significantly increased expression levels of Alu-derived RNA transcripts. These transcripts might be involved in crucial functions upon infection by EBV. 5 biological replica of non-infected BL2 samples were compared to 5 biological replica of LCL 197/2 EBV immortalized samples

ORGANISM(S): Homo sapiens

SUBMITTER: Sonja Vorwerk

PROVIDER: E-GEOD-20441 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The oral cavity is the persistent reservoir for EBV with lifelong infection of resident epithelial and B cells. Infection of these cell types results in distinct EBV gene expression patterns that are regulated by epigenetic modifications involving DNA methylation and chromatin structure. Such regulation of EBV gene expression relies on viral manipulation of the host epigenetic machinery that may inadvertently result in long-lasting, oncogenic host epigenetic reprogramming. To test this hypothesis in the context of EBV infection of epithelial cells, we established a transient infection model to identify the epigenetic consequences after EBV infection of immortalized normal oral keratinocytes and subsequent viral loss. With mounting evidence that EBV can induce epigenetic alterations, we developed a transient infection model where a clonal derivative (designated cl1) from a human telomerase immortalized normal oral keratinocyte (NOK) cell line was infected with a recombinant Akata EBV carrying neomycin resistance and GfP cassette in place of the BXLF1 open reading frame. Infected cells were passaged ten times, and then selection pressure was removed for an additional ten passages to allow for viral loss. Three transiently-infected EBV-negative clones were identified by single cell cloning (designated cl1, cl3, cl4). Uninfected parental clone and cells transfected with PTRUF5 plasmid were passaged alongside the transiently-infected clones. The transcription profiles were analyzed using Affymetrix microarray U133Plus2.0 arrays in duplicate. NOK cells were single cell cloned (designated as cl1) and EBV infected by co-culture with a recombinant Akata EBV strain carrying neomcycin resistance and GFP expression cassette in place of EBV BXLF1. Cells were selected with 350 ug/ml G418. As a selection control, cells were transfected a plasmid carrying a neomycin resistance cassette. Cells were passaged 10 times with G418, followed by removal of selection pressure. After an additional 10 passages, cells were single cell cloned and the presence of EBV was determined by various methods. Three clones were identified as being EBV negative and said to be transiently infected. Uninfected parental and vector transfected cells were passaged in the same manner and also single cell cloned.

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Custom microchip analysis identifies differentially expressed non-coding RNAs in Epstein-Barr virus-infected B cells

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