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.

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.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is the infectious cause of the highly vascularized tumor Kaposi’s sarcoma (KS), which is characterized by proliferating spindle cells of endothelial origin, extensive neo-angiogenesis and inflammatory infiltrates. The KSHV K15 protein contributes to the angiogenic and invasive properties of KSHV-infected endothelial cells. Here, we asked whether K15 could also play a role in KSHV lytic replication. Deletion of the K15 gene from the viral genome or its depletion by siRNA lead to reduced virus reactivation, as evidenced by the decreased expression levels of KSHV lytic proteins RTA, K-bZIP, ORF 45 and K8.1 as well as reduced release of infectious virus. Similar results were found for a K1 deletion virus. Deleting either K15 or K1 from the viral genome also compromised the ability of KSHV to activate PLCγ1, Erk1/2 and Akt1. In infected primary lymphatic endothelial (LEC-rKSHV) cells, which have previously been shown to spontaneously display a viral lytic transcription pattern, transfection of siRNA against K15, but not K1, abolished viral lytic replication as well as KSHV-induced spindle cell formation. Using a newly generated monoclonal antibody to K15, we found an abundant K15 protein expression in KS tumor biopsies obtained from HIV positive patients, emphasizing the physiological relevance of our findings. Finally, we used a dominant negative inhibitor of the K15-PLCγ1 interaction to establish proof of principle that pharmacological intervention with K15-dependent pathways may represent a novel approach to block KSHV reactivation and thereby its pathogenesis. Both the latent and lytic replication phases of the KSHV life cycle are thought to contribute to its persistence and pathogenesis. The non-structural signaling membrane protein K15 is involved in the angiogenic and invasive properties of KSHV-infected endothelial cells. Here we show that the K15 protein is required for virus replication, early viral gene expression and virus production through its activation of the cellular signaling pathways PLCγ1 and Erk 1/2. K15 is abundantly expressed in KSHV-infected lymphatic endothelial cells (LECs) and contributes to KSHV-induced endothelial spindle cell formation. The abundant K15 protein expression observed in LECs is also observed in KS tumors. We also show that it may be possible to target K15 in order to intervene therapeutically with KSHV lytic replication and pathogenesis.

Merkel cell polyomavirus (MCV or MCPyV) is an alphapolyomavirus causing human Merkel cell carcinoma and encodes four tumor (T) antigen proteins: large T (LT), small tumor (sT), 57 kT, and middle T (MT)/alternate LT open reading frame proteins. We show that MCV MT is generated as multiple isoforms through internal methionine translational initiation that insert into membrane lipid rafts. The membrane-localized MCV MT oligomerizes and promiscuously binds to lipid raft-associated Src family kinases (SFKs). MCV MT-SFK interaction is mediated by a Src homology (SH) 3 recognition motif as determined by surface plasmon resonance, coimmunoprecipitation, and bimolecular fluorescence complementation assays. SFK recruitment by MT leads to tyrosine phosphorylation at a SH2 recognition motif (pMT ^Y114 ), allowing interaction with phospholipase C gamma 1 (PLCγ1). The secondary recruitment of PLCγ1 to the SFK-MT membrane complex promotes PLCγ1 tyrosine phosphorylation on Y783 and activates the NF-κB inflammatory signaling pathway. Mutations at either the MCV MT SH2 or SH3 recognition sites abrogate PLCγ1-dependent activation of NF-κB signaling and increase viral replication after MCV genome transfection into 293 cells. These findings reveal a conserved viral targeting of the SFK-PLCγ1 pathway by both MCV and murine polyomavirus (MuPyV) MT proteins. The molecular steps in how SFK-PLCγ1 activation is achieved, however, differ between these two viruses.

Merkel cell polyomavirus (MCV) causes one of the most aggressive human skin cancers, but laboratory studies on MCV replication have proven technically difficult. We report the first recombinase-mediated MCV minicircle (MCVmc) system that generates high levels of circularized virus, allowing facile MCV genetic manipulation and characterization of viral gene expression kinetics during replication. Mutations to Fbw7, Skp2, β-TrCP and hVam6p interaction sites, or to the stem loop sequence for the MCV-encoded miRNA precursor, markedly increase viral replication, whereas point mutation to an origin-binding site eliminates active virus replication. To further increase the utility of this system, an mScarlet fusion protein was inserted into the VP1 c-terminus to generate a non-infectious reporter virus for studies on virus kinetics. When this reporter virus genome is heterologously expressed together with MCV VP1 and VP2, virus-like particles are generated. The reporter virus genome is encapsidated and can be used at lower biosafety levels for one-round infection studies. Our findings reveal that MCV has multiple, self-encoded viral restriction mechanisms to promote viral latency over lytic replication, and these mechanisms are now amenable to examination using a recombinase technology.

Merkel cell polyomavirus (MCV) establishes a near-ubiquitous, asymptomatic infection in humans but on rare occasions drives Merkel cell carcinoma, an aggressive skin cancer. How MCV replication is coordinated with host cell-cycle progression, and how this coordination shapes infected-cell fate, has remained unresolved. Here we combine reporter virus live-cell imaging, pharmacologic cell-cycle perturbation, flow cytometry, and single-cell transcriptomics to examine MCV replication dynamics. VP1 late gene expression occurs almost exclusively during late S or G2, is associated with prolonged G2 arrest, and frequently culminates in cell death. Strikingly, cells that divide before virus replication give rise to daughter cells that synchronously initiate viral replication and VP1 expression, revealing mitotic inheritance of silent viral genomes, a pattern seen for viruses with a known latency lifecycle. Single-cell RNA sequencing further identifies a transient antiviral and inflammatory response that is subsequently suppressed in surviving, cell cycle arrested cells. These findings define the cell-cycle logic, inheritance, and host consequences of MCV replication at single-cell resolution.