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 (miRNAs) repress target gene expression by destabilizing the mRNA transcript and decreasing translational efficiency. A goal of the project is to determine targets of viral miRNAs 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 miRNAs using a variety of validation methods. In addition, we identified multiple examples of individual target genes being inhibited by multiple KSHV miRNAs. 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 identified interleukin-1 receptor-associated kinase 1 (IRAK1) and myeloid differentiation primary-response protein 88 (MYD88) as potential KSHV microRNA targets from microarray expression data previously described (Nat Genet. 2009 Jan;41(1):130-4. Epub 2008 Dec 21.). We demonstrated that microRNAs expressed by Kaposi's sarcoma-associated herpesvirus (KSHV, HHV8) inhibit expression of two components in the toll-like receptor/interleukin-1 receptor pathway. In addition to predicting and validating two viral microRNA targets (which has been a challenge for the microRNA field), we show inhibition of IRAK1 and MYD88 inhibits activation of NF-kB and secretion of proinflammatory cytokines in response to TLR agonists and IL-1alpha. This represents a strategy for the virus to inhibit an immune response by using viral microRNAs during latency (which would not be antigenic). These results were published in November 2012. We have also completed a proteomic screen for miRNA targets, which identified novel validated and previously reported miRNA targets. These discoveries have led to novel discoveries about immune evasion and transcription factor regulation. Our analysis found that KSHV microRNAs can inhibit activation of intercellular adhesion molecule 1 (ICAM1) protein expression, by inhibiting rho-associated, coiled-coil containing protein kinase 2 (ROCK2) and signal transducer and activator of transcription 3 (STAT3) activation pathways of ICAM1. Analysis of the proteins that were upregulated in the presence of KSHV microRNAs, led to the discovery that KSHV microRNAs can upregulate hypoxia-inducible factor 1 alpha. These findings and the proteomic expression data are currently in press. We are also interested in the functions of microRNA targets in B cells, a relevant cell type for KSHV infection. We have developed methods to deliver small RNAs to silence specific genes in B cell lines with high efficiency and minimal toxicity without using viral transduction methods. Both EBV and KSHV express their own unique viral microRNAs and we sought the investigate validated and predicted human targets of these viral microRNAs. Using predicted miRNA targets from cross-linking and immunoprecipitation assays published by multiple research groups, we found a significant enrichment of targets involved in cell cycle regulation, apotosis, cytoskeleton remodeling, and immune responses. These findings were published in September 2012. 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 miRNAs and focusing our efforts on pathways with multiple miRNA targets.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Investigator-Initiated Intramural Research Projects (ZIA)
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National Cancer Institute Division of Basic Sciences
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Ramalingam, Dhivya; Happel, Christine; Ziegelbauer, Joseph M (2015) Kaposi's sarcoma-associated herpesvirus microRNAs repress breakpoint cluster region protein expression, enhance Rac1 activity, and increase in vitro angiogenesis. J Virol 89:4249-61
Viollet, Coralie; Davis, David A; Reczko, Martin et al. (2015) Next-Generation Sequencing Analysis Reveals Differential Expression Profiles of MiRNA-mRNA Target Pairs in KSHV-Infected Cells. PLoS One 10:e0126439
Gallaher, Amelia M; Das, Sudipto; Xiao, Zhen et al. (2013) Proteomic screening of human targets of viral microRNAs reveals functions associated with immune evasion and angiogenesis. PLoS Pathog 9:e1003584
Abend, Johanna R; Ramalingam, Dhivya; Kieffer-Kwon, Philippe et al. (2012) Kaposi's sarcoma-associated herpesvirus microRNAs target IRAK1 and MYD88, two components of the toll-like receptor/interleukin-1R signaling cascade, to reduce inflammatory-cytokine expression. J Virol 86:11663-74
Ramalingam, Dhivya; Kieffer-Kwon, Philippe; Ziegelbauer, Joseph M (2012) Emerging themes from EBV and KSHV microRNA targets. Viruses 4:1687-710
Ziegelbauer, Joseph M (2011) Functions of Kaposi's sarcoma-associated herpesvirus microRNAs. Biochim Biophys Acta 1809:623-30
Abend, Johanna R; Uldrick, Thomas; Ziegelbauer, Joseph M (2010) Regulation of tumor necrosis factor-like weak inducer of apoptosis receptor protein (TWEAKR) expression by Kaposi's sarcoma-associated herpesvirus microRNA prevents TWEAK-induced apoptosis and inflammatory cytokine expression. J Virol 84:12139-51