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.

Author Summary

Kaposi's sarcoma-associated herpesvirus is the etiological agent of KS and two lymphoproliferative diseases: multicentricCastleman's disease and primary effusion lymphomas (PEL). KSHV tumors are the most prevalent AIDS malignancies and within Sub-Saharan Africa KS is the most common cancer in males, both in the presence and absence of HIV infection. KSHV encodes 12 miRNA genes whose function is largely unknown. Viral miRNAs are incorporated into RISCs, which regulate gene expression mostly by binding to 3′UTRs of mRNAs to inhibit their translation and/or induce degradation. The small subset of viral miRNA targets identified to date suggests that these small posttranscriptional regulators target important cellular pathways involved in pathogenesis and tumorgenesis. Using Ago HITS-CLIP, a technique which combines UV cross-linking, immunoprecipitation of Ago-miRNA-mRNA complexes, and high throughput sequencing, we performed a detailed analysis of the KSHV miRNA targetome in two commonly studied PEL cell lines, BCBL-1 and BC-3 and identified 1170 and 950 putative miRNA targets, respectively. This data set provides a valuable resource to decipher how KSHV miRNAs contribute to viral biology and pathogenesis.