Kaposi's Sarcoma-Associated Herpesvirus Encodes a Mimic of Cellular miR-23

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) expresses ϳ20 viral microRNAs (miRNAs) in latently infected cells. We have previously shown that two of these miRNAs function as mimics of the cellular miRNAs miR-155 and miR-142-3p. Two additional KSHV miRNAs, miR-K3؉1 and miR-K3, share perfect and offset 5= homology with cellular miR-23, respectively. Here, we report a single nucleotide polymorphism that causes miR-K3؉1 expression in a subset of KSHV-infected primary effusion lymphoma cell lines as a consequence of altered processing of the primary transcript by the Microprocessor complex. We confirm that miR-K3؉1 regulates miR-23 targets, which is expected because these miRNAs share the entire seed region (nucleotides 2 to 8). Surprisingly, we found that miR-K3 also regulates miR-23 targets, despite offset seed sequences. In addition, the offset homology of miR-K3 to miR-23 likely allows this viral miRNA to expand its target repertoire beyond the targets of miR-23. Because miR-23 is highly expressed in endothelial cells but expressed at only low levels in B cells, we hypothesize that miR-K3 may function to introduce miR-23-like activities into KSHV-infected B cells. Together, our data demonstrate that KSHV has evolved at least three distinct viral miRNAs to tap into evolutionarily conserved cellular miRNA-regulatory networks. Furthermore, our data allow fundamental insights into the generation and functional impact of miRNA 5= end variation.

Divergent target recognition by coexpressed 5′-isomiRs of miR-142-3p and selective viral mimicry

“…This is consistent with the previous observation that the miR-101 5 ′ -isomiRs have mostly similar targets (Cloonan et al 2011). Similarly, our earlier analysis of KSHV miR-K3/+1 has already demonstrated a remarkably strong target overlap between these two isomiRs (∼70%, Manzano et al 2013). In contrast, the divergent isomiRs miR-183/-1 and miR-BART10/-1 have only 11% and 27% shared PAR-CLIP binding sites, respectively (Fig.…”

“…Primers that were used for cloning or mutagenesis are detailed in Supplemental Tables S4, S5; primer sequences are listed in Supplemental Table S8. Reporter assays were conducted as described using mirVana miRNA mimics (Life Technologies) (Manzano et al 2013). To express miRc-RF9-3p, a 250-bp fragment centered on the predicted pre-miRc-RF9 was synthesized as a gBlock (IDT), PCR-amplified using primers 2079 and 2080, and inserted between XhoI and NotI sites of the published lentiviral vector pLCE .…”

“…Primer extension analyses were conducted as described using denaturing polyacrylamide gel-purified probes (Manzano et al 2013). Primers for miR-K10a, miR-142-3p, and 5S RNA have been previously published .…”

“…Two days after transfection, cells were harvested in 20 mM Tris pH 7.5, 100 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EDTA, and Complete EDTA-free protease inhibitors (Roche). Quantitative Western blotting using the Odyssey Fc Imaging System (LI-COR Biosciences) was performed as described (Manzano et al 2013) using anti-p190 (BD Biosciences, Cat. 610149), anti-cofilin 2 (EMD Millipore, Cat.…”

“…Like many other herpesviruses, KSHV expresses its own set of ∼20 mature miRNAs (Cai et al 2005;Pfeffer et al 2005;Samols et al 2005;Grundhoff et al 2006;. These microRNAs include known viral mimics of cellular miR-155 and miR-23 (Gottwein et al 2007;Skalsky et al 2007;Manzano et al 2013). One of the KSHV miRNA precursors is processed to two approximately equally abundant 5 ′ -isomiRs called miR-K10a and miR-K10a +1 ( Fig.…”