Research Article: Ago HITS-CLIP Expands Understanding of Kaposi’s Sarcoma-associated Herpesvirus miRNA Function in Primary Effusion Lymphomas

Date Published: August 23, 2012

Publisher: Public Library of Science

Author(s): Irina Haecker, Lauren A. Gay, Yajie Yang, Jianhong Hu, Alison M. Morse, Lauren M. McIntyre, Rolf Renne, Blossom Damania.


KSHV is the etiological agent of Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and a subset of multicentricCastleman’s disease (MCD). The fact that KSHV-encoded miRNAs are readily detectable in all KSHV-associated tumors suggests a potential role in viral pathogenesis and tumorigenesis. MiRNA-mediated regulation of gene expression is a complex network with each miRNA having many potential targets, and to date only few KSHV miRNA targets have been experimentally determined. A detailed understanding of KSHV miRNA functions requires high-through putribonomics to globally analyze putative miRNA targets in a cell type-specific manner. We performed Ago HITS-CLIP to identify viral and cellular miRNAs and their cognate targets in two latently KSHV-infected PEL cell lines. Ago HITS-CLIP recovered 1170 and 950 cellular KSHVmiRNA targets from BCBL-1 and BC-3, respectively. Importantly, enriched clusters contained KSHV miRNA seed matches in the 3′UTRs of numerous well characterized targets, among them THBS1, BACH1, and C/EBPβ. KSHV miRNA targets were strongly enriched for genes involved in multiple pathways central for KSHV biology, such as apoptosis, cell cycle regulation, lymphocyte proliferation, and immune evasion, thus further supporting a role in KSHV pathogenesis and potentially tumorigenesis. A limited number of viral transcripts were also enriched by HITS-CLIP including vIL-6 expressed only in a subset of PEL cells during latency. Interestingly, Ago HITS-CLIP revealed extremely high levels of Ago-associated KSHV miRNAs especially in BC-3 cells where more than 70% of all miRNAs are of viral origin. This suggests that in addition to seed match-specific targeting of cellular genes, KSHV miRNAs may also function by hijacking RISCs, thereby contributing to a global de-repression of cellular gene expression due to the loss of regulation by human miRNAs. In summary, we provide an extensive list of cellular and viral miRNA targets representing an important resource to decipher KSHV miRNA function.

Partial Text

Kaposi’s sarcoma-associated herpesvirus (KSHV) or Human Herpesvirus type 8 (HHV-8) is associated with Kaposi’s sarcoma (KS) and two lymphoproliferative disorders: primary effusion lymphomas (PEL) and a subset of multicentricCastleman’s disease (MCD) [1]–[3]. In KS tumors and PEL viral gene expression is highly restricted to the latency-associated region which encodes four proteins and the viral microRNAs(miRNA). MiRNAs are 21 to 23 nucleotide (nt) long, non-coding RNAs that preferentially bind to 3′UTRs of mRNAs to prevent translation and/or induce degradation (for review see [4]). The first viral miRNAs were identified in 2004 in Epstein-Barr virus (EBV)-infected Burkitt’s lymphoma cells [5] and subsequently more than 140 miRNAs have been identified in all herpes viruses studied thus far with the exception of Varicella Zoster virus (for review see [6], [7]). The 12 KSHV miRNA genes [8]–[11] can each give rise to two different mature products [12], miR and miR*. MiR-K12-10 is moreover edited [13] bringing the total number of mature miRNAs to 25. KSHV miRNAs are expressed during the latent phase of infection and expression has been detected in tissues and biopsies of classical and AIDS-associated KS as well as in PEL and MCD [14]–[16]. Since aberrant expression of miRNAs is associated with many human diseases including cancer [17], it was hypothesized early on that KSHV-encoded miRNAs may contribute to pathogenesis and/or tumorigenesis by de-regulating host cellular gene expression. Until recently, only a small number of target genes have been identified mainly by combining bioinformatics predictions with gene expression profiling and 3′UTR luciferase reporter assays in cells that either ectopically express viral miRNAs or in tumor cell lines in which viral miRNAs are inhibited by antagomir approaches [18]–[21]. Although limited in number, the initially reported targets immediately suggested that KSHVmiRNAs contribute to the regulation of pathogenesis-relevant processes such as angiogenesis, apoptosis, cell cycle control, endothelial cell differentiation, and immune surveillance (for review see [6], [7]). Moreover, one KSHV miRNA, miR-K12-11, shares the same seed sequence as human miR-155, one of the first “oncomirs” discovered [22], [23].MiR-K12-11 was shown to mimic miR-155 function to induce a splenic B cell expansion in a NOD/SCID mouse model [24].

Performing Ago HITS-CLIP on BCBL-1 and BC-3 cells produced a catalogue of putative cellular and viral miRNA targets (Table S3, S4). We carefully modified the original Ago HITS-CLIP protocol [25] by adding more stringent wash steps(see Text S1) to the Ago immunoprecipitation. In addition, we constructed both the miRNA and mRNA libraries from the 130 kDa complex, while previous studies isolated miRNAs from the 110 kDa complex [25]. Three biological replicates and two technical replicates were sequenced to monitor biological variation and assay reproducibility. Finally, we excluded from the targetome analysis all KSHV miRNAs that were recovered at very low levels (Figure 2A). For the miRNA libraries, which are less complex, cross-validation was very high across biological replicates(R2>0.9; Figure S1A, S1B). For the mRNA libraries reproducibility was lower (R2≥0.53; Figure S1E, F) but well within the observed variation of previously published studies [25], [34]. Variability from the complex experimental procedure (see also below) and the small amount of Ago-associated RNA extracted and sequenced yields libraries that are not 100% representative; hence biological replicates add to both stringency and depths of the targetome analysis.