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

ABSTRACT: Circular RNAs (circRNAs) are a conserved class of RNAs with diverse functions, including serving as messenger RNAs that are translated into peptides. Here we describe circular RNAs generated by human polyomaviruses (HPyVs), some of which encode variants of the previously described alternative large T antigen open reading frame (ALTO) protein. Circular ALTO RNAs (circALTOs) can be detected in virus positive Merkel cell carcinoma (VP-MCC) cell lines and tumor samples. CircALTOs are stable, predominantly located in the cytoplasm, and N6-methyladenosine (m6A) modified. The translation of MCPyV circALTOs into ALTO protein is negatively regulated by MCPyV-generated miRNAs in cultured cells. MCPyV ALTO expression increases transcription from some recombinant promoters in vitro and upregulates the expression of multiple genes previously implicated in MCPyV pathogenesis. MCPyV circALTOs are enriched in exosomes derived from VP-MCC lines and circALTO-transfected 293T cells, and purified exosomes can mediate ALTO expression and transcriptional activation in MCPyV-negative cells. The related trichodysplasia spinulosa polyomavirus (TSPyV) also expresses a circALTO that can be detected in infected tissues and produces ALTO protein in cultured cells. Thus, human polyomavirus circRNAs are expressed in human tumors and infected tissues and express proteins that have the potential to modulate the infectious and tumorigenic properties of these viruses.

SUBMITTER: Yang R

PROVIDER: S-EPMC8158866 | biostudies-literature | 2021 May

REPOSITORIES: biostudies-literature

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

Yang Rong R Lee Eunice E EE Kim Jiwoong J Choi Joon H JH Kolitz Elysha E Chen Yating Y Crewe Clair C Salisbury Nicholas J H NJH Scherer Philipp E PE Cockerell Clay C Smith Taylor R TR Rosen Leslie L Verlinden Louisa L Galloway Denise A DA Buck Christopher B CB Feltkamp Mariet C MC Sullivan Christopher S CS Wang Richard C RC

PLoS pathogens 20210517 5

Circular RNAs (circRNAs) are a conserved class of RNAs with diverse functions, including serving as messenger RNAs that are translated into peptides. Here we describe circular RNAs generated by human polyomaviruses (HPyVs), some of which encode variants of the previously described alternative large T antigen open reading frame (ALTO) protein. Circular ALTO RNAs (circALTOs) can be detected in virus positive Merkel cell carcinoma (VP-MCC) cell lines and tumor samples. CircALTOs are stable, predomi ...[more]

PMID: 33999949

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Project description:UnlabelledThe polyomavirus tumor (T) antigens play crucial roles in viral replication, transcription, and cellular transformation. They are encoded by partially overlapping open reading frames (ORFs) located in the early region through alternative mRNA splicing. The T expression pattern of the trichodysplasia spinulosa-associated polyomavirus (TSPyV) has not been established yet, hampering further study of its pathogenic mechanisms and taxonomic relationship. Here, we characterized TSPyV T antigen expression in human cell lines transfected with the TSPyV early region. Sequencing of T antigen-encoded reverse transcription-PCR (RT-PCR) products revealed three splice donor and acceptor sites creating six mRNA splice products that potentially encode the antigens small T (ST), middle T (MT), large T (LT), tiny T, 21kT, and alternative T (ALTO). Except for 21kT, these splice products were also detected in skin of TSPyV-infected patients. At least three splice products were confirmed by Northern blotting, likely encoding LT, MT, ST, 21kT, and ALTO. Protein expression was demonstrated for LT, ALTO, and possibly MT, with LT detected in the nucleus and ALTO in the cytoplasm of transfected cells. Splice site and start codon mutations indicated that ALTO is encoded by the same splice product that encodes LT and uses internal start codons for initiation. The genuineness of ALTO was indicated by the identification of acetylated N-terminal ALTO peptides by mass spectrometry. Summarizing, TSPyV exhibits an expression pattern characterized by both MT and ALTO expression, combining features of rodent and human polyomaviruses. This unique expression pattern provides important leads for further study of polyomavirus-related disease and for an understanding of polyomavirus evolution.ImportanceThe human trichodysplasia spinulosa-associated polyomavirus (TSPyV) is distinguished among polyomaviruses for combining productive infection with cell-transforming properties. In the research presented here, we further substantiate this unique position by indicating expression of both middle T antigen (MT) and alternative T antigen (ALTO) in TSPyV. So far, none of the human polyomaviruses was shown to express MT, which is considered the most important viral oncoprotein of rodent polyomaviruses. Coexpression of ALTO and MT, which involves a conserved, recently recognized overlapping ORF subject to positive selection, has not been observed before for any polyomavirus. As a result of our findings, this study provides valuable new insights into polyomavirus T gene use and expression. Obviously, these insights will be instrumental in further study and gaining an understanding of TSPyV pathogenicity. More importantly, however, they provide important leads with regard to the interrelationship, functionality, and evolution of polyomaviruses as a whole, indicating that TSPyV is a suitable model virus to study these entities further.

Project description:The Polyomaviridae constitute a family of small DNA viruses infecting a variety of hosts. In humans, polyomaviruses can cause infections of the central nervous system, urinary tract, skin, and possibly the respiratory tract. Here we report the identification of a new human polyomavirus in plucked facial spines of a heart transplant patient with trichodysplasia spinulosa, a rare skin disease exclusively seen in immunocompromized patients. The trichodysplasia spinulosa-associated polyomavirus (TSV) genome was amplified through rolling-circle amplification and consists of a 5232-nucleotide circular DNA organized similarly to known polyomaviruses. Two putative "early" (small and large T antigen) and three putative "late" (VP1, VP2, VP3) genes were identified. The TSV large T antigen contains several domains (e.g. J-domain) and motifs (e.g. HPDKGG, pRb family-binding, zinc finger) described for other polyomaviruses and potentially involved in cellular transformation. Phylogenetic analysis revealed a close relationship of TSV with the Bornean orangutan polyomavirus and, more distantly, the Merkel cell polyomavirus that is found integrated in Merkel cell carcinomas of the skin. The presence of TSV in the affected patient's skin was confirmed by newly designed quantitative TSV-specific PCR, indicative of a viral load of 10(5) copies per cell. After topical cidofovir treatment, the lesions largely resolved coinciding with a reduction in TSV load. PCR screening demonstrated a 4% prevalence of TSV in an unrelated group of immunosuppressed transplant recipients without apparent disease. In conclusion, a new human polyomavirus was discovered and identified as the possible cause of trichodysplasia spinulosa in immunocompromized patients. The presence of TSV also in clinically unaffected individuals suggests frequent virus transmission causing subclinical, probably latent infections. Further studies have to reveal the impact of TSV infection in relation to other populations and diseases.

Project description: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.

Dataset Information

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

Publications

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

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