Hansen, A.; (2010) Kaposi sarcoma herpesvirus microRNA function in lymphatic endothelial cells. Doctoral thesis , UCL (University College London).

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Abstract

Kaposi’s sarcoma herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma (KS), the most common AIDS-associated malignancy. KS lesions are comprised of poorly differentiated, spindle-shaped endothelial cells, the precursors of which are lymphatic endothelial cells (LEC). KSHV infection of LEC induces transcriptional reprogramming towards a phenotype more analogous to blood vessel endothelial cells BEC. KSHV encodes 17 mature miRNAs (miRNA), 14 of which are co-expressed as a cluster. miRNAs are small non-coding RNA molecules that act posttranscriptionally to negatively regulate gene expression. It was the aim of this thesis to investigate the function of the KSHV miRNAs through identifying viral miRNA targets in LEC. miRNAs silence their target genes by either blocking translation or inducing mRNA degradation. Gene expression microarray (GEM) analysis was used to quantify changes in mRNA abundance of LEC transcripts induced by the KSHV miRNA cluster. MAF, a leucine zipper transcription factor, was significantly down-regulated in the presence of the viral miRNAs. miRNA expression analysis of KS lesions identified those KSHV miRNAs which are expressed and therefore relevant to KS pathogenesis. In silico prediction analysis identified multiple KSHV miRNAs binding sites in the MAF 3’UTR, MAF silencing was mediated through specific interactions with these sites. In particular, KSHV miRNAs miR-K12-6 and miR-K12-11 were identified as individual miRNAs responsible for MAF down-regulation. MAF was silenced specifically by these miRNAs during primary KSHV infection of LEC. MAF has been previously characterised as a LEC-specific transcription factor, although its function and target genes were unknown. GEM profiling of LEC in which MAF was silenced by siRNA revealed an increase in expression of BEC markers. Therefore, in LEC, MAF acts a transcriptional repressor of BEC transcripts helping to maintain lymphatic identity. Gene set enrichment analysis of three GEM data sets revealed a significant concordant increase in BEC genes during primary KSHV infection, expression of the KSHV miRNA cluster and MAF silencing. The miRNA cluster-induced up-regulation of BEC markers was shown to be by way of MAF silencing. In this study I have identified and validated MAF as a LEC-specific KSHV miRNA target and have shown MAF to have a role in maintaining LEC identity through repression of BEC markers. Consequently, down-regulation of MAF following KSHV miRNA expression contributes to the mechanism behind the transcription reprogramming of LEC observed upon KSHV infection.

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