Functions of KSHV microRNAs and circular RNAs

Division Of Basic Sciences - Nci

Investigators

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Abstract

Previously, we have discovered and published around 40 validated cellular mRNA targets of Kaposi's Sarcoma Herpesvirus (KSHV) miRNAs. Importantly, many of these targets have not previously been studied in the context of KSHV infection. We have been continuing aims (1) to understand how viral infection influences miRNA biosynthesis and degradation (2) to understand the regulation of the cholesterol biosynthesis pathway by viral microRNAs (3) to discover the functions and regulations of circular RNAs. Kaposi's sarcoma-associated herpesvirus (KSHV) expresses miRNAs during latency. However, regulation of viral miRNAs remains largely unknown. Our prior studies demonstrated that MCPIP1 regulates KSHV miRNA biogenesis by degrading most KSHV pre-miRNAs through its RNase activity. Some viral pre-miRNAs are partially resistant to degradation by MCPIP1. We further characterized MCPIP1 substrate specificity and its antiviral potential against KSHV infection. In vitro cleavage assays and binding assays showed that MCPIP1 cleavage efficiency is related to binding affinity. Motif-based sequence analysis identified that KSHV pre-miRNAs that are well degraded by MCPIP1 have a 5-base motif (M5 base motif) within their terminal loops and this motif region consists of multiple pyrimidine-purine-pyrimidine (YRY) motifs. We further demonstrated that mutation of this M5 base motif within terminal loop of pre-miRNAs inhibited MCPIP1-mediated RNA degradation. We also revealed that MCPIP1 has an antiviral effect against KSHV infection. MCPIP1 can reduce the expression of Dicer, which in turn restricts KSHV infection. Our findings demonstrated that MCPIP1 inhibited KSHV infection and suppressed viral miRNA biogenesis by directly degrading KSHV pre-miRNAs and altering the expression of miRNA biogenesis factors. Taken together, these results demonstrated that MCPIP1 inhibited KSHV infection and suppressed viral miRNA biogenesis by directly degrading KSHV pre-miRNAs and altering expression of miRNA biogenesis factors. Increasing MCPIP1 expression might be a potential therapeutic strategy to inhibit spreading of infection. KSHV modulates host pathways partly through its miRNAs, short noncoding RNAs. Using various screens, we identified and validated host targets of these viral miRNAs involved in metabolism, particularly in the mevalonate/cholesterol pathway. We found that KSHV down regulates total cholesterol during de novo infection of primary endothelial cells. We hypothesized that KSHV modulates the mevalonate pathway with viral miRNAs to decrease amount of cholesterol that can be converted to antiviral 25HC (25-hydroxycholesterol). 25HC blocks KSHV de novo infection of primary endothelial cells at a post-entry step and decreases viral gene expression of LANA and RTA. Herein we expanded on this observation by determining transcriptomic changes associated with 25HC treatment of primary endothelial cells using RNA sequencing. We found that 25HC treatment inhibited KSHV gene expression and induced a type I interferon (IFN) response, including interferon-stimulated genes (ISGs) and several inflammatory cytokines (CXCL8, IL1A). Some 25HC-induced genes were partially responsible for the broadly antiviral effect of 25HC against several viruses. Additionally, we found that 25HC inhibited infection of primary B cells by a related oncogenic virus, Epstein-Barr Virus (EBV/ Human Herpesvirus-4) and investigated how 25HC inhibits EBV infection. We also found that the gene encoding cholesterol 25-hydroxylase (CH25H), which converts cholesterol to 25HC, can be induced by type I IFN in human peripheral blood mononuclear cells (PBMCs). We propose a model wherein viral miRNAs target the cholesterol pathway to prevent 25HC production and subsequent induction of antiviral ISGs. Together, these results answer some important questions about a widely acting antiviral (25HC), with implications for multiple viral and bacterial infections. Circular RNAs are formed by back-splicing events, lack poly-A tails, can regulate gene expression, and more stable than mRNAs. We previously reported that Kaposi's sarcoma herpesvirus (KSHV) infection alters the expression of hundreds of human circular RNAs. A human circular RNA, hsa_circ_0001400, is upregulated upon infection and inhibits KSHV gene expression. Still, functions and regulation of these human and viral circular RNAs are largely unknown in the context of KSHV infection. Multiple herpesviruses have been recently found to encode viral circular RNAs. Like cellular circular RNAs, these RNAs lack poly-A tails and their 5' and 3' ends have been joined, which confers protection from RNA exonucleases. We examined the expression patterns of circular RNAs from Kaposi's sarcoma herpesvirus (KSHV) in various environments. We performed deep sequencing of circRNA-enriched total RNA from a KSHV-positive patient lymph node for comparison with previous circRNA-Seq results. We found that circvIRF4 is highly expressed in the KSHV-positive patient sample relative to both B cell lines and de novo infected primary vascular and lymphatic endothelial cells (LECs). Overall, this patient sample showed a viral circRNA expression pattern more similar to the pattern from B cell lines, but we also discovered new back-spliced junctions and additional viral circular RNAs in this patient sample. We validated some of these back-spliced junctions as circular RNAs with standard assays. Differential expression patterns of circular RNAs in different cell types led us to investigate what cellular factors might be influencing the ratio of viral linear mRNAs to circular RNAs. We found that repression of certain RNA-binding proteins shifted the balance between viral linear mRNAs and circular RNAs. Taken together, examining viral circular RNA expression patterns may become useful tools for discovering their functions, the regulators of their expression, and determining the stage and cell types of infection in humans.