Joseph M. Ziegelbauer, Ph.D.

     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 human 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. 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. In addition, we have mapped functional microRNA target sites in multiple human genes using site-directed mutagenesis. Furthermore, using KS biopsies from patient enrolled in clinical trials, 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 are currently investigating the functional roles of a couple of selected miRNA targets identified in these approaches. Below are some of the selected miRNA targets and their functional significance. 

     From various expression screens to identify miRNA targets, we found that Kaposi's sarcoma-associated herpesvirus (KSHV) viral microRNAs (miRNAs) target several enzymes in the mevalonate/cholesterol pathway. 3-Hydroxy-3-methylglutaryl-coenzyme A (CoA) synthase 1 (HMGCS1), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR [a rate-limiting step in the mevalonate pathway]), and farnesyl-diphosphate farnesyltransferase 1 (FDFT1 [a committed step in the cholesterol branch]) are repressed by multiple KSHV miRNAs. Transfection of viral miRNA mimics in primary endothelial cells (human umbilical vein endothelial cells [HUVECs]) reduced intracellular cholesterol levels. We also found that cholesterol levels were decreased in de novo-infected HUVECs after seven days. This reduction is at least partially due to viral miRNAs, since the mutant form of KSHV lacking 10 of the 12 miRNA genes had increased cholesterol compared to wild-type KSHV infections. We hypothesized that KSHV is down-regulating cholesterol to suppress the antiviral response by a modified form of cholesterol, 25-hydroxycholesterol (25HC). We found that the cholesterol 25-hydroxylase (CH25H) gene, which is responsible for generating 25HC, had increased expression in de novo-infected HUVECs, but was strongly suppressed in long-term latently infected cell lines. We found that 25HC inhibits KSHV infection when added exogenously prior to de novo infection. In conclusion, we found that multiple KSHV viral miRNAs target enzymes in the mevalonate pathway to modulate cholesterol in infected cells during latency. This repression of cholesterol levels could potentially be beneficial to viral infection by decreasing the levels of antiviral 25HC. These results suggest a new virus-host relationship and indicate a previously unidentified viral strategy to lower cholesterol levels.

    We were interested in identifying cellular networks that were targeted by KSHV-miRNAs and employed network building strategies using validated KSHV miRNA targets and gene-gene associations based on published literature. Here, we report the identification of a network of genes repressed by KSHV miRNAs that center on the transcription factor- signal transducer and activator of transcription 3 (STAT3) that is also targeted by KSHV miRNAs. KSHV miRNAs suppressed STAT3 and STAT5 activation and inhibited STAT3-dependent transcriptional activity upon IL6-treatment. KSHV miRNAs also repressed the induction of antiviral interferon-stimulated genes upon IFNα- treatment. Finally, we observed increased lytic reactivation of KSHV from latently infected cells upon STAT3 repression with siRNAs or a small molecule inhibitor of STAT3. Our data suggest that treatment of infected cells with a STAT3 inhibitor and a viral replication inhibitor, ganciclovir, represents a possible strategy to target latently infected cells without increasing virion production. Together, we show that KSHV miRNAs suppress a network of targets associated with STAT3, deregulate cytokine-mediated gene activation, suppress an interferon response, and influence the transition into the lytic phase of viral replication.

    In patient-derived cell lines, certain KSHV miRNAs are extremely abundantly expressed and can be expressed at a much higher level than cellular miRNAs. This raises the question of how the virus can manipulate the miRNA biogenesis pathway to promote expression of viral miRNAs. MCP-1-induced protein-1 (MCPIP1), a critical regulator of immune homeostasis, has been shown to suppress miRNA biosynthesis via cleavage of precursor miRNAs through its RNase domain. Since MCPIP1 in strongly unregulated by inflammatory cytokines, including IL-1, we hypothesized that MCPIP1 could be used as an antiviral strategy to inhibit biogenesis of viral miRNAs at the time of infection. We demonstrate that MCPIP1 can directly cleave KSHV and EBV precursor miRNAs and that MCPIP1 expression is repressed following de novo KSHV infection. In addition, repression with siRNAs to MCPIP1 in KSHV-infected cells increased IL-6 and KSHV miRNA expression, supporting a role for MCPIP1 in IL-6 and KSHV miRNA regulation. We also provide evidence that KSHV miRNAs repress MCPIP1 expression by targeting the 3'UTR of MCPIP1. Conversely, expression of essential miRNA biogenesis components Dicer and TRBP is increased following latent KSHV infection. We propose that KSHV infection inhibits a negative regulator of miRNA biogenesis (MCPIP1) and up-regulates critical miRNA processing components to evade host mechanisms that inhibit expression of viral miRNAs. KSHV-mediated alterations in miRNA biogenesis represent a novel mechanism by which KSHV interacts with its host and a new mechanism for the regulation of viral miRNA expression.

    Circular RNAs have recently been shown inhibit specific miRNAs and RNA-binding proteins (RBPs). We have used novel techniques to discover human circular RNAs that increased in expression with KSHV infection and contain predicted binding sites for KSHV miRNAs. We recently found a human circular RNA that inhibits KSHV infection by an unknown mechanism. In addition, we have discovered that KSHV encodes and expresses viral circular RNAs. These are some of the first examples of sub-genomic viral circular RNAs. We have also found these viral circular RNAs in patient samples and are working to understand the functions of these human and viral circular RNAs. 

    In conclusion, we have been successful in meeting our goals of identifying human targets of viral miRNAs and investigating the roles of these genes in viral infection. In our efforts to identify miRNA targets, our combined approach of multiple methods have identified valid miRNA targets that are missed using a single approach. This combined method could be directly applied by others studying viral and cellular miRNA functions. This research has led to finding new potential strategies for treating viral infections. Some examples include targeting latent infections in combination with a lytic inhibitory drug and another discovery that a modified form of cholesterol has potent antiviral activities.  We also have discovered new virus-host interactions (including circular RNAs), which has opened the possibility of disrupting specific cellular pathways to inhibit viral infection. We also reported the first example of a cellular factor that degrade viral miRNAs. This has led to new discoveries about viral miRNA biogenesis regulation and how cytokines influence this miRNA biogenesis pathway.