Merkel Cell Polyomavirus Encodes Circular RNAs (circRNAs) Enabling a Dynamic circRNA/microRNA/mRNA Regulatory Network

Abstract

Viral noncoding RNAs have acquired increasing prominence as important regulators of infection and mediators of pathogenesis. Circular RNAs (circRNAs) generated by backsplicing events have been identified in several oncogenic human DNA viruses. Here, we show that Merkel cell polyomavirus (MCV), the etiologic cause of ∼80% of Merkel cell carcinomas (MCCs), also expresses circular RNAs. By RNase R-resistant RNA sequencing, four putative circRNA backsplice junctions (BSJs) were identified from the MCV early region (ER). The most abundantly expressed MCV circRNA, designated circMCV-T, is generated through backsplicing of all of ER exon II to form a 762-nucleotide (nt) circular RNA molecule. Curiously, circMCV-T, as well as two other less abundantly expressed putative MCV circRNAs, overlaps in a complementary fashion with the MCV microRNA (miRNA) locus that encodes MCV-miR-M1. circMCV-T is consistently detected in concert with linear T antigen transcripts throughout infection, suggesting a crucial role for this RNA molecule in the regulatory functions of the early region, known to be vital for viral replication. Knocking out the hairpin structure of MCV-miR-M1 in genomic early region expression constructs and using a new high-efficiency, recombinase-mediated, recircularized MCV molecular clone demonstrates that circMCV-T levels decrease in the presence of MCV-miR-M1, underscoring the interplay between MCV circRNA and miRNA. Furthermore, circMCV-T partially reverses the known inhibitory effect of MCV-miR-M1 on early gene expression. RNase R-resistant RNA sequencing of lytic rat polyomavirus 2 (RatPyV2) identified an analogously located circRNA, stipulating a crucial, conserved regulatory function of this class of RNA molecules in the family of polyomaviruses.IMPORTANCE Covalently closed circular RNAs were recently described in the human DNA tumor viruses Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus (KSHV), and human papillomavirus (HPV). Here, we show that MCV, another DNA tumor virus, generates circRNAs from its early regulatory region in concert with T antigen linear transcripts. MCV circMCV-T interacts with another MCV noncoding RNA, miR-M1, to functionally modulate early region transcript expression important for viral replication and long-term episomal persistence. This work describes a dynamic regulatory network integrating circRNA/miRNA/mRNA biomolecules and underscores the intricate functional modulation between several classes of polyomavirus-encoded RNAs in the control of viral replication.

Characterization of ALTO-encoding circular RNAs expressed by Merkel cell polyomavirus and trichodysplasia spinulosa polyomavirus

“…Moreover, mutation of in the MCPyV circALTO backsplicing sites suppresses expression of the ALTO protein isoform. In contrast to our findings, Abere et al were not able to find convincing evidence that circMCV-T/ALTO could be translated [ 56 ]. However, when using a mutant circMCV-T/ALTO1 that does not generate the MCPyV miRNAs, they were able to detect the circMCV-T/ALTO1 on polysomes, though ALTO expression from this construct was still not detected.…”

Characterizing Expression and Regulation of Gamma-Herpesviral Circular RNAs

“…Viral non-coding RNAs have a wide range of functions and are often crucial for successful infection. Novel circRNAs were recently identified in various DNA viruses including KSHV, EBV, HPV (human papillomavirus), and MCPyV (Merkel cell polyomavirus) ( Tagawa et al, 2018 ; Toptan et al, 2018 ; Ungerleider et al, 2018 ; Huang et al, 2019 ; Zhao et al, 2019 ; Abere et al, 2020b ). Circular RNAs have gene regulatory functions in human cells and viral circRNAs can be additional regulators of host-virus interactions ( Kristensen et al, 2019 ).…”

“…Currently no KSHV circRNAs are found to be associated with polysomes ( Toptan et al, 2018 ; Abere et al, 2020b ), but it is possible that these viral circRNAs regulate genes by interacting with other RNA species or RNA-binding proteins. Though a magnitude of transcriptomic change of single viral circRNA may be mild as seen in our volcano plots ( Figure 6A ), there can be significant change when all viral circRNAs are expressed simultaneously or in combination with their natural viral cofactors.…”

“…New bioinformatics tools have allowed researchers to look for these back-spliced junctions, a unique marker of circRNAs ( Hansen, 2018 ). Much like the beginning of the miRNA field, initial studies have focused on the expression profiling of cellular and viral circular RNAs in different diseases, cell types, and infection ( Tagawa et al, 2018 ; Toptan et al, 2018 ; Ungerleider et al, 2018 ; Huang et al, 2019 ; Zhao et al, 2019 ; Abere et al, 2020b ).…”

“…Early work on cellular and viral circRNAs in the context of infection have found differential expression patterns and some limited examples of functions, including a cellular circRNA that inhibits infection and viral circRNAs that alter cell growth ( Tagawa et al, 2018 ; Toptan et al, 2018 ; Huang et al, 2019 ; Liu et al, 2019 ; Ungerleider et al, 2019 , 2018 ; Zhao et al, 2019 ; Abere et al, 2020a , b ). Here we focus on circRNAs in the context of Kaposi sarcoma herpesvirus (KSHV) and Epstein–Barr virus (EBV).…”