Kaposi's sarcoma-associated herpesvirus (KSHV) is the responsible agent for Kaposi's sarcoma (KS), primary effusion lymphoma and multicentric Castleman's disease. KSHV expresses multiple microRNAs that modulate host gene expression. Most microRNAs repress target gene expression by destabilizing the mRNA transcript and decreasing translational efficiency. A goal of the project is to determine targets of viral microRNAs and understand why the virus has selected specific human target genes for inhibition. We hope to discover new functions of human genes as they relate to viral infection and cancer. Using a variety of expression profiling data, we constructed a dataset to integrate the expression data from multiple gain and loss of microRNA function experiments. We have tested over fifty predicted target genes and over thirty microRNA target genes were significantly inhibited by viral microRNAs using a variety of validation methods. In addition, we identified multiple examples of individual target genes being inhibited by multiple KSHV microRNAs. It is noteworthy to state approximately half of these microRNA:target interactions are not detected using common bioinformatic methods. A subset of these target genes has been further validated by looking at protein expression of endogenous target genes in response to viral microRNA expression, microRNA inhibition in infected cells and KSHV infection. To assess changes in protein expression, we utilize a near-infrared scanner to perform simultaneous two-color quantitative western blotting assays. In addition, we have mapped functional microRNA target sites in multiple human genes using site-directed mutagenesis. Furthermore, using KS biopsies we have determined multiple microRNA target genes that are inhibited in our cell culture systems are also inhibited at sites of KSHV infection in patients. We have used multiple methods to analyze the functional significance of the validated target genes. Many targets are involved with regulation of the cell cycle, apoptosis and other cancer-related functions. Our progress studying the functional significance of KSHV microRNAs has revealed a cytokine receptor (tumor necrosis factor receptor superfamily, member 12A) is targeted by a KSHV microRNA. We demonstrated KSHV microRNA-10A can inhibit TWEAK-induced apoptosis using multiple assays. Also KSHV microRNA-10A can suppress induction of pro-inflammatory cytokine IL-6 and IL-8 in response to the TWEAK cytokine. This represents a mechanism for KSHV to avoid death of an infected cell during latency and to inhibit immune responses by the host. We are actively studying the functional consequences of at least five other human genes that are targeted by KSHV microRNAs. Finally, we are using network analysis to understand how newly identified microRNA target genes could be interacting with each other. This analysis has revealed multiple examples of how microRNA target genes are in the same signaling pathway. These examples highlight significant pathways targeted by KSHV microRNAs.
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