Functions of KSHV microRNAs and circular RNAs

Division Of Basic Sciences - Nci

Investigators

Linked publications, trials & patents

Abstract

Previously, we have discovered and published around 40 validated cellular mRNA targets of Kaposi's Sarcoma Herpesvirus (KSHV) miRNAs. Recently, we have started studying circular RNAs, which are formed by back-splicing events, lack poly-A tails, can regulate gene expression, and are more stable than linear 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 (circ_0001400, circRELL1), is induced by various pathogenic viruses, namely KSHV, Epstein-Barr virus, and human cytomegalovirus. Our findings that circ_0001400 levels increase upon infection with multiple human herpesviruses suggests a common mechanism shared across diverse viral infections. However, how these viruses regulate circRNA transcript levels remains elusive. We observed that while circ_0001400 increases during infection, the mRNA from the same gene locus, RELL1, does not show similar regulation. This discrepancy led us to postulate that infection changes of circRNAs occur co-/post-transcriptionally. Using 4SU RNA-Seq, we examined whether circRNAs, including circ_0001400, are regulated transcriptionally, co-transcriptionally, or post-transcriptionally. Our data suggested a majority of human circRNAs were differentially regulated in a co-transcriptional manner. We further identified proteins interacting with circ_0001400 using a CRISPR-assisted RNA-protein interaction detection method, CARPID. Among various proteins identified, we confirmed the interaction between circ_0001400 and a splicing factor, PNISR. This finding, together with 4SU RNA-Seq analysis, proposes that KSHV mainly regulates human circRNAs, including circ_0001400, in a co-transcriptional manner. We investigated the effects of circ_0001400 after KSHV infection and found that it suppresses KSHV lytic gene expression and virus production. Ectopic expression of circ_0001400 reduced KSHV gene expression, while knock-down of circ_0001400 led to an increase in viral transcript levels. Notably, we found that manipulation of circ_0001400 expression levels did not affect viral entry, indicating that viral gene expression is regulated at a post-entry step. Furthermore, we observed that circ_0001400 suppresses lytic infection more significantly than latent infection in primary endothelial cells. We also explored the broader influence of circ_0001400 on cell physiology, focusing on cell growth, immune responses, and miRNA machinery. We observed that manipulation of circ_0001400 leads to differential expression of various genes related to these processes. Circ_0001400 seems to promote cell growth and inhibit apoptosis, which was confirmed by our phenotypic analyses. We also found that circ_0001400 expression correlates with the activation of immunity-related pathways. In addition, our study revealed that circ_0001400 interacts with a component of the mTOR complex, a critical regulator of cell growth, thus offering new insights into its cellular function. We also characterized the circRNAome of HSV-1, KSHV, and MHV68 infections. We used cell culture and mouse models (with our collaborators) to thoroughly profile circRNAs expressed during lytic or latent phases. To facilitate high-throughput analysis of circular transcripts, we developed a custom bioinformatic pipeline called circRNA DAQ (Detection, Annotation, Quantitation). To confirm that predicted transcripts were truly circular, we complemented sequencing approaches with RNase R digestion assays and divergent primer amplification. Through these approaches, we identified thousands of viral circRNAs, some of which approach the abundance of the housekeeping gene GAPDH. It is exciting to note that 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 that promote circRNA biogenesis. We found that viral circRNA synthesis was resistant to major spliceosome inhibition and not reliant on canonical splice donor-acceptor sites. During lytic infection, viral circRNAs tiled the entire genome, with the late phase of lytic transcription promoting rampant back-splicing. Additionally, using eCLIP and Nascent (4SU) RNA-Seq, we determined that the KSHV RNA binding protein (ORF57) enhanced viral circRNA accumulation post-transcriptionally. Our work has identified thousands of novel herpesvirus transcripts and elucidated a unique splicing mechanism driven by lytic replication. As viral circRNAs were completely unstudied until the last six years, our work lays the foundation for a novel class of molecules that may contribute to herpesvirus replication, persistence, and tumorigenesis. Importantly, since circRNAs appear to be ubiquitously expressed across assorted virus families, our findings have implications for other viral pathogens and associated diseases.