Discovery of Kaposi's sarcoma herpesvirus-encoded circular RNAs and a human antiviral circular RNA

Abstract

Noncoding RNAs have substantial effects in host-virus interactions. Circular RNAs (circRNAs) are novel single-stranded noncoding RNAs which can decoy other RNAs or RNA-binding proteins to inhibit their functions. The role of circRNAs is largely unknown in the context of Kaposi's sarcoma herpesvirus (KSHV). We hypothesized that circRNAs influence viral infection by inhibiting host and/or viral factors. Transcriptome analysis of KSHV-infected primary endothelial cells and a B cell line identified human circRNAs that are differentially regulated upon infection. We confirmed the expression changes with divergent PCR primers and RNase R treatment of specific circRNAs. Ectopic expression of hsa_circ_0001400, a circRNA induced by infection, suppressed expression of key viral latent gene LANA and lytic gene RTA in KSHV de novo infections. Since human herpesviruses express noncoding RNAs like microRNAs, we searched for viral circRNAs encoded in the KSHV genome. We performed circRNA-Seq analysis with RNase R-treated, circRNA-enriched RNA from KSHV-infected cells. We identified multiple circRNAs encoded by the KSHV genome that are expressed in KSHV-infected endothelial cells and primary effusion lymphoma (PEL) cells. The KSHV circRNAs are located within ORFs of viral lytic genes, are up-regulated upon the induction of the lytic cycle, and alter cell growth. Viral circRNAs were also detected in lymph nodes from patients of KSHV-driven diseases such as PEL, Kaposi's sarcoma, and multicentric Castleman's disease. We revealed new host-virus interactions of circRNAs: human antiviral circRNAs are activated in response to KSHV infection, and viral circRNA expression is induced in the lytic phase of infection.

Keywords: KSHV; circRNA-Seq; circular RNA; herpesvirus; noncoding RNA.

Fig. 1.

KSHV induces certain human circular RNAs upon infection. (A) Human circRNA expression levels in KSHV-infected cells were measured by microarrays. Signals for each human circRNA-specific probe from infected cells were normalized with signals from uninfected cells to calculate fold changes. The P values were determined by t tests. Data are shown as mean values of experiments with three independent experiments. (B) Human circRNA expression levels in RNase R-treated infected HUVECs are shown. circRNAs that were most strongly and consistently up-regulated according to microarray analysis and abundant in circRNA-Seq data were selected. Sequenced reads mapped to back-spliced junctions of known human circRNAs were counted and shown as counts per million reads. Raw values of two independent experiments are shown. (C and D) Expression levels of human circRNAs were assessed with RT-qPCR in KSHV-infected HUVECs (C), 293T cells (D), and MC116 cells (E). Various MOI conditions were used; MOIs were 30 and 100 for HUVECs and 7.5 and 15 for 293T cells. Transcript levels were normalized to GAPDH or ACTB and uninfected controls. Data are shown as mean values and SD of three independent experiments. *P < 0.05.

Fig. 2.

KSHV lytic genes encode circular RNAs. (A) Schematic overview of the circRNA discovery analysis. Numbers of back-spliced junctions of viral origins are shown. (B) KSHV circRNAs identified by circRNA-Seq are shown. All back-spliced junctions detected in KSHV-infected HUVECs (with RNase R, n = 2; without RNase R, n = 4) were accumulated in 200 nucleotide bins and plotted. Viral coding sequences and PAN RNA are shown to describe genomic loci. Amplicons of divergent primers are shown. (Right inset) A partial genomic map of identified KSHV circRNAs. Positions of coding sequences (CDSs) of viral genes, ncRNAs, and divergent primers are shown. The genomic positions are according to the KSHV reference genome, NC_009333. (C) Viral circRNA levels were assessed with RT-qPCR using divergent primers in doxycycline-treated TREx-BCBL1 and TREx-BCBL1-RTA cells. Transcript levels were normalized to GAPDH and TREx-BCBL1 controls. Samples with a Ct value of more than 35 were determined to be under the detection limit and designated as nondetected (n.d.). (D) Viral circRNAs were tested for RNase R resistance. Total RNA from doxycycline-treated TREx-BCBL1-RTA cells was treated with RNase R and was subjected to RT-qPCR. Transcript levels of RNase R-treated samples were normalized to mock-treated controls and are shown as percentages. Data are shown as raw and mean values and SD of three independent experiments. (E) Viral transcripts and circRNAs were assessed from lymph node samples from five KSHV-positive donors (Patients 1 to 5). Diagnosis (lymph nodes and patients) and LANA detection (lymph nodes) results are shown below each donor. Transcript levels were normalized to ACTB and donor 5. Samples with a Ct value of more than 35 were determined to be under the detection limit and designated as not detected (n.d.). FH, follicular hyperplasia; KS, Kaposi’s sarcoma; MCD, multicentric Castleman’s disease; PEL, primary effusion lymphoma.

Fig. 3.

Circular RNAs regulate viral gene expression and cell growth. (A) Transcript levels of hsa_circ_0001400 were assessed in SLK cells stably expressing hsa_circ_0001400 with RT-dPCR (digital PCR). Data were normalized to ACTB and are shown as mean values and SD of three independent experiments. (B) Transcript levels of viral genes were assessed in SLK cells stably expressing hsa_circ_0001400 with RT-qPCR 3 d after KSHV de novo infection. Data were normalized to ACTB and shown as mean values and SD of four independent experiments. *P < 0.05. (C) Transcript levels of human and viral genes were assessed in BAC16 KSHV-infected SLK (SLK-K) cells transfected with siRNAs for 48 h with RT-qPCR. Transcript levels were normalized to ACTB and to siNT (nontargeting negative control) transfected SLK-K cells. Data are shown as mean values and SD of three independent experiments. *P < 0.05. siNT, nontargeting siRNA; siCirc1400-1/2, hsa_circ_0001400 targeting siRNA 1 and 2. (D) Transcript levels of human and viral genes were assessed in RTA-induced BCBL1 cells and in de novo infected SLK cells stably expressing circRNAs with RT-dPCR. Data were normalized to ACTB and are shown as mean values and SD of three independent experiments. n.d., not detected. (E) Viability of SLK cells stably expressing circRNAs was assessed after BAC16 KSHV infections by measuring reducing potentials of living cells. Cell viabilities were normalized to circGFP-expressing SLK cells. Data are shown as mean values and SD of three experiments.