Characterizing Expression and Regulation of Gamma-Herpesviral Circular RNAs.

Takanobu Tagawa,Daniel Oh,Joseph M Ziegelbauer,Guruswamy Mahesh,Thomas S Uldrick,Robert Yarchoan,Jerico Santos,Vishal N Kopardé,Sarah Dremel

doi:10.3389/fmicb.2021.670542

Abstract

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.

Highlights

Viruses have been known to express their own non-coding RNAs
Viral circRNA Expression Profiles in Infected Cells in vitro and in vivo Relevant cell types for Kaposi’s sarcoma herpesvirus (KSHV) infection include B cells and endothelial cells, and infections can lead to pathology of endothelial origin (Kaposi’s sarcoma) or B cell origin (PEL and multicentric Castleman disease) (Yarchoan and Uldrick, 2018)
Through circRNA-Seq, we and others have screened for KSHVencoded circRNAs in latently infected endothelial cells as well as B cell lines reactivated for lytic cycle in vitro (Tagawa et al, 2018; Toptan et al, 2018)

Summary

Introduction

Viruses have been known to express their own non-coding RNAs. Some of these earlier discoveries included HSV’s (Herpes simplex virus) latency-associated transcript (LAT), EBV (Epstein–Barr virus) -encoded small RNAs (EBERs), and KSHV’s (Kaposi’s sarcoma herpesvirus) polyadenylated nuclear (PAN) RNA (Withers et al, 2019; Tagawa et al, 2020), which would Herpesvirus Circular RNAs be called lncRNAs (long non-coding RNAs). Expression profiling of cellular and viral miRNAs was the main focus. The viral miRNAs function largely similar to cellular miRNAs and repress target gene expression. Hundreds of target genes of these viral miRNAs have been discovered (Ramalingam et al, 2012; Vojtechova and Tachezy, 2018; Židovec Lepej et al, 2020). Some of these viral miRNAs target cellular and viral transcripts and serve to regulate apoptosis, cytokine responses, and immunogenicity (Skalsky and Cullen, 2010; Tagawa et al, 2020). A recent addition to this field of non-coding RNAs is circular RNAs (circRNAs)

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