Functions of KSHV microRNAs and circular RNAs

Division Of Basic Sciences - Nci

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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 to understand the regulation of the cholesterol biosynthesis pathway by viral microRNAs to discover the functions and regulations of circular RNAs. Kaposi's sarcoma-associated herpesvirus (KSHV) expresses miRNAs during latency. 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 are more stable than mRNAs. Circular RNAs have recently been shown to inhibit specific miRNAs and have other activities. Our goals have been: 1. To identify circular RNAs that change in expression with viral infection. 2. To discover new viral circular RNAs and understand their biogenesis. 3. To determine the functions and mechanisms of human and viral circular RNAs. We previously reported that Kaposi's sarcoma herpesvirus (KSHV) infection alters the expression of hundreds of human circular RNAs. We showed that a human circRNA, hsa_circ_0001400, is induced by various pathogenic viruses, namely KSHV, Epstein-Barr virus, and human cytomegalovirus. The induction of circRNAs including circ_0001400 by KSHV is co-transcriptionally regulated, likely at splicing. Consistently, screening for circ_0001400-interacting proteins identified a splicing factor, PNISR. Functional studies using infected primary endothelial cells revealed that circ_0001400 inhibits KSHV lytic transcription and virus production. Simultaneously, the circRNA promoted cell cycle, inhibited apoptosis, and induced immune genes. RNA-pulldown assays identified transcripts interacting with circ_0001400, including TTI1, which is a component of the pro-growth mTOR complexes. We thus identified a circRNA that is pro-growth and anti-lytic replication. These results support a model in which KSHV induces circ_0001400 expression to maintain latency. Since circ_0001400 is induced by multiple viruses, this novel viral strategy may be widely employed by other viruses. We also characterized the circRNAome of HSV-1, KSHV, and MHV68 infections. We used cell culture and mouse models to thoroughly profile circRNAs expressed during lytic or latent phases. A custom bioinformatic pipeline, called circRNA DAQ (Detection, Annotation, Quantitation) was developed to facilitate high-throughput analysis of circular transcripts. Sequencing approaches were complemented with RNase R digestion assays and divergent primer amplification to confirm that predicted transcripts were truly circular. Using these approaches, we identified thousands of viral circRNAs, some of which approach abundance of the housekeeping gene GAPDH. Excitingly, we are the first to identify HSV-1 encoded circRNAs, including circular transcripts derived from the latency-associated transcript (LAT). Next, we characterized cis- and trans-acting factors which promote circRNA biogenesis. We found viral circRNA synthesis was resistant to major spliceosome inhibition and not reliant on canonical splice donor-acceptor sites. Viral circRNAs tiled the entire genome during lytic infection, with the late phase of lytic transcription promoting rampant back-splicing. Finally, using eCLIP and Nascent (4SU) RNA-Seq, we determined that the KSHV RNA binding protein (ORF57) enhanced viral circRNA accumulation post-transcriptionally. Our work identifies thousands of novel herpesvirus transcripts and elucidates a unique splicing mechanism driven by lytic replication. As viral cirRNAs have been completely unstudied until the last five years, our work lays the foundation for a novel class of molecules which may contribute to herpesvirus replication, persistence, and tumorigenesis. Since circRNAs appear to be ubiquitously expressed across assorted virus families, our work has implications for other viral pathogens and associated diseases.