Circular DNA tumor viruses make circular RNAs

Significance Circular RNAs (circRNAs) play critical physiologic functions, but it is not known whether human DNA viruses express circRNAs. We surveyed Epstein−Barr virus (EBV) and Kaposi’s sarcoma herpesvirus (KSHV) tumors and cell lines, and found specific circRNAs expressed from both viruses. EBV circular BamHI A rightward transcripts (circBARTs) were expressed in all EBV tumor latency forms, including all EBV-infected posttransplant lymphoproliferative disease tumors tested, whereas EBV circBHLF1 and circLMP2 were more variably expressed. KSHV expressed circvIRF4 constitutively in primary effusion lymphoma cell lines, while the polyadenylated nuclear locus promiscuously generated variable, inducible, and bidirectional circRNAs. Tumor virus circRNAs can be long-lived, unique tumor biomarkers that may also open new research opportunities into understanding how these viruses cause cancer. Epstein−Barr virus (EBV) and Kaposi’s sarcoma herpesvirus (KSHV) cause ∼2% of all human cancers. RNase R-resistant RNA sequencing revealed that both gammaherpesviruses encode multiple, uniquely stable, circular RNAs (circRNA). EBV abundantly expressed both exon-only and exon−intron circRNAs from the BamHI A rightward transcript (BART) locus (circBARTs) formed from a spliced BART transcript and excluding the EBV miRNA region. The circBARTs were expressed in all verified EBV latency types, including EBV-positive posttransplant lymphoproliferative disease, Burkitt lymphoma, nasopharyngeal carcinoma, and AIDS-associated lymphoma tissues and cell lines. Only cells infected with the B95-8 EBV strain, with a 12-kb BART locus deletion, were negative for EBV circBARTs. Less abundant levels of EBV circRNAs originating from LMP2- and BHLF1-encoding genes were also identified. The circRNA sequencing of KSHV-infected primary effusion lymphoma cells revealed a KSHV-encoded circRNA from the vIRF4 locus (circvIRF4) that was constitutively expressed. In addition, KSHV polyadenylated nuclear (PAN) RNA locus generated a swarm (>100) of multiply backspliced, low-abundance RNase R-resistant circRNAs originating in both sense and antisense directions consistent with a novel hyperbacksplicing mechanism. In EBV and KSHV coinfected cells, exon-only EBV circBARTs were located more in the cytoplasm, whereas the intron-retaining circBARTs were found in the nuclear fraction. KSHV circvIRF4 and circPANs were detected in both nuclear and cytoplasmic fractions. Among viral circRNAs tested, none were found in polysome fractions from KSHV−EBV coinfected BC1 cells, although low-abundance protein translation from viral circRNAs could not be excluded. The circRNAs are a new class of viral transcripts expressed in gammaherpesvirus-related tumors that might contribute to viral oncogenesis.