Ago HITS-CLIP Expands Understanding of Kaposi's Sarcoma-associated Herpesvirus miRNA Function in Primary Effusion Lymphomas

Abstract

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.

Kaposi sarcoma–associated herpesvirus: immunobiology, oncogenesis, and therapy

“…miRNAs are developmentally regulated and fine-tune lineage differentiation and cellular signaling. The targets of the KSHV miRNAs have been established through a series of comprehensive biochemical studies (128)(129)(130)(131). Thus far, miRNA studies have been constrained by sensitivity limits for detection of individual miRNAs and for the discovery of miRNA-target interactions.…”

Genomewide Mapping and Screening of Kaposi's Sarcoma-Associated Herpesvirus (KSHV) 3′ Untranslated Regions Identify Bicistronic and Polycistronic Viral Transcripts as Frequent Targets of KSHV MicroRNAs

“…Indeed, recent studies have shown that KSHV miRNAs regulate viral life cycle in addition to cell cycle, apoptosis, inflammation, angiogenesis, and immune evasion by targeting cellular genes . Several KSHV miRNAs directly target lytic genes, including ORF50, ORF56, ORF57, and ORF-K2 (19,34,40,41). However, whether any other KSHV genes and transcripts are targeted by the viral miRNAs remains unclear.…”

Viral noncoding RNAs: more surprises

“…KSHV miR-K12-1 also down-regulates p21, an inhibitor of cell cycle progression, to promote virally induced oncogenesis (Gottwein and Cullen 2010). Genome-wide AGO HITS-CLIP (Chi et al 2009) and photoactivatable-ribonucleoside-enhanced CLIP (PAR-CLIP) (Hafner et al 2010) studies revealed that targets of EBV and KSHV miRNAs are enriched in apoptosis and cell cycle pathways (Gottwein et al 2011;Haecker et al 2012;Riley et al 2012;Skalsky et al 2012). Studies of EBV genomic deletions revealed that several miRNAs expressed from the BHRF1 locus (miRBHRF1s) play critical roles in B-cell transformation by inhibiting apoptosis and promoting cell cycle progression (Seto et al 2010;Feederle et al 2011).…”