Functions of KSHV microRNAs and circular RNAs

Division Of Basic Sciences - Nci

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Abstract

Herpesviruses possess linear double-stranded DNA genomes that replicate in the host nucleus, relying on host transcriptional machinery to produce more than 100 viral RNAs. Like their hosts, these viruses generate diverse RNA classes, including mRNAs, long non-coding RNAs, microRNAs, and the recently recognized circular RNAs (circRNAs). CircRNAs are single-stranded molecules in which the 5-prime and 3-prime ends are covalently linked. Since the first reports of virus-encoded circRNAs in 2018, their presence has been experimentally confirmed in numerous DNA and RNA viruses, including members of the herpesvirus, papillomavirus, polyomavirus, hepatitis B virus, respiratory syncytial virus, and coronavirus families. Bioinformatic studies suggest that additional viruses also produce circRNAs. In host cells, circRNAs can act as microRNA sponges, protein scaffolds, transcriptional enhancers, or translation templates. Although the functions of viral circRNAs are less clear, some are translated and can be packaged into virions or extracellular vesicles. CircRNAs from oncogenic gammaherpesviruses have been shown to influence cell growth and apoptosis, and cancer-specific circRNA expression profiles have prompted interest in these molecules as potential biomarkers. Because of their low immunogenicity, long half-life, and capacity for cap-independent translation, circRNAs are also being investigated as vehicles for mRNA-based vaccines. Eukaryotic circRNAs are typically generated by spliceosome-mediated back splicing, a process facilitated by RNA-binding proteins or tandem repeat sequences that bring flanking regions of the back-splice junction together. A spliceosome-independent pathway, involving tRNA splicing endonuclease and the RNA ligase RTCB, produces circularized tRNA introns in both archaea and metazoans. Additional chemical and enzymatic strategies (such as those using T4 DNA or RNA ligases) have been employed for in-vitro circRNA production. Some herpesvirus circRNAs (for example, those derived from multi-exon genes with canonical splice sites) may follow the spliceosome-dependent route; however, most viral genes are short, single-exon units that overlap extensively, and herpes simplex virus 1 actively suppresses spliceosome activity during lytic replication. Moreover, circRNAs have been detected in cytoplasmic RNA viruses, challenging the notion that the spliceosome is always required. Thus, whether viral circRNAs arise through canonical or alternative mechanisms remains unresolved. Because circRNAs share almost the entire sequence of their linear counterparts (with the back-splice junction representing the only unique region) high-throughput sequencing coupled with chimeric-read analysis is essential for global detection and quantification. Using these approaches, circRNAs have been shown to be widespread across organisms and tissues. In our present work, we profiled the circRNA repertoire of human herpesviruses HSV-1 and KSHV, as well as murine gammaherpesvirus 68 (MHV68), across lytic, latent, and reactivation phases in both cell culture and mouse models. To support this effort, we developed CHARLIE (Circular RNAs in Host And Viruses Analysis Pipeline), a custom workflow for high-throughput viral circRNA analysis. More than 90 percent of identified viral circRNAs lacked canonical splice donor-acceptor motifs, and key back-splicing hotspots proved resistant to spliceosome or lariat debranching enzyme inhibition. Depletion of RNA ligases (RLIG1 in HSV-1; RLIG1 or RTCB in KSHV) significantly reduced viral circRNA abundance, implicating these enzymes as alternative trans-acting factors with locus-specific roles. Furthermore, enhanced crosslinking immunoprecipitation and nascent RNA sequencing revealed that the KSHV RNA-binding protein ORF57 promotes circRNA synthesis for select viral and host transcripts. Collectively, these findings uncover dozens of previously unrecognized herpesvirus circRNAs and highlight distinct biogenesis pathways distinguishing viral circRNAs from those of the host.