Joseph M. Ziegelbauer, Ph.D.
HIV and AIDS Malignancy Branch
Building 10, Room 6N110
Bethesda, MD 20892
Multiple groups recently discovered that Kaposi's sarcoma-associated Herpesvirus (KSHV or human herpesvirus 8), which is associated with Kaposi's sarcoma, multicentric Castleman's disease and primary effusion lymphoma, expresses multiple microRNAs. Small non-coding RNAs called microRNAs have become newly appreciated molecules for gene regulation with each microRNA potentially targeting hundreds of target genes for repression. KSHV microRNAs are expressed during latency and represent an alternative method to alter host gene expression without generating viral proteins that could be detected by the host immune system. However, a current challenge is identifying the targets of microRNAs in order to determine the functions of microRNAs.
To address this challenge, we developed a novel system to identify host targets of virally encoded microRNAs using a combination of gain and loss of microRNA function experiments, followed by expression profiling and sequence analysis. This dataset represents one of the largest microRNA target databases known since we have individually inhibited and expressed all seventeen viral microRNAs in multiple cell types. The first validated microRNA target from this approach was the human gene BCL2-Associated Transcription Factor 1 (BCLAF1). Note this target may have been missed using other common microRNA target prediction methods. Further studies revealed BCLAF1 may inhibit viral replication. Using a variety of expression profiling data (including SILAC), 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 and help us 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.
Ramalingam D, Ziegelbauer JM. Viral microRNAs Target a Gene Network, Inhibit STAT Activation, and Suppress Interferon Responses. Sci Rep. 2017;7:40813.
Serquiña AKP, Kambach DM, Sarker O, Ziegelbauer JM. Viral MicroRNAs Repress the Cholesterol Pathway, and 25-Hydroxycholesterol Inhibits Infection. MBio. 2017;8(4).
Happel C, Ramalingam D, Ziegelbauer JM. Virus-Mediated Alterations in miRNA Factors and Degradation of Viral miRNAs by MCPIP1. PLoS Biol. 2016;14(11):e2000998.
Gallaher AM, Das S, Xiao Z, Andresson T, Kieffer-Kwon P, Happel C, Ziegelbauer J. Proteomic screening of human targets of viral microRNAs reveals functions associated with immune evasion and angiogenesis. PLoS Pathog. 2013;9(9):e1003584.
Abend JR, Ramalingam D, Kieffer-Kwon P, Uldrick TS, Yarchoan R, Ziegelbauer JM. 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. 2012;86(21):11663-74.
Related Scientific Focus Areas
Microbiology and Infectious Diseases
This page was last updated on June 15th, 2017