A microRNA Encoded by Kaposi Sarcoma-Associated Herpesvirus Promotes B-Cell Expansion <em>In Vivo</em>

<div><p>The human gammaherpesvirus Kaposi sarcoma-associated herpesvirus is strongly linked to neoplasms of endothelial and B-cell origin. The majority of tumor cells in these malignancies are latently infected, and latency genes are consequently thought to play a critical role in virus-induced tumorigenesis. One such factor is kshv-miR-K12-11, a viral microRNA that is constitutively expressed in cell lines derived from KSHV-associated tumors, and that shares perfect homology of its seed sequence with the cellular miR-155. Since miR-155 is overexpressed in a number of human tumors, it is conceivable that mimicry of miR-155 by miR-K12-11 may contribute to cellular transformation in KSHV-associated disease. Here, we have performed a side-by-side study of phenotypic alterations associated with constitutive expression of either human miR-155 or viral miR-K12-11 in bone marrow-derived hematopoietic stem cells. We demonstrate that retroviral-mediated gene transfer and hematopoietic progenitor cell transplantation into C57BL/6 mice leads to increased B-cell fractions in lymphoid organs, as well as to enhanced germinal center formation in both microRNA-expressing mouse cohorts. We furthermore identify Jarid2, a component of Polycomb repressive complex 2, as a novel validated target of miR-K12-11, and confirm its downregulation in miR-K12-11 as well as miR-155 expressing bone marrow cells. Our findings confirm and extend previous observations made in other mouse models, and underscore the notion that miR-K12-11 may have arisen to mimic miR-155 functions in KSHV-infected B-cells. The expression of miR-K12-11 may represent one mechanism by which KSHV presumably aims to reprogram naïve B-cells towards supporting long-term latency, which at the same time is likely to pre-dispose infected lymphocytes to malignant transformation.</p> </div

Retroviral constructs for ectopic miRNA expression.

<p>A) Seed sharing between viral and cellular miRNAs. Shown is an alignment of human (hsa) and murine (mmu) miR-155 orthologues, the KSHV-encoded miR-K12-11 and the MDV-encoded miR-M4. Nucleotides that are conserved between hsa- and mmu-miR-155 are shown on dark gray background, and nucleotides that are identical in two or more miRNAs are highlighted in light gray. The seed region (nts. 2–8) that is considered to be the most critical region for base-pairing between miRNA and target sites is marked. B) Schematic depiction of y-retroviral miRNA expression vectors. The precursor hairpins of kshv-miR-K12-11 and hsa-miR-155 are placed downstream of the GFP gene and transcribed from the LTR (long terminal repeat) promoter. C) Endogeneous and ectopic miRNA expression in transduced NIH 3T3 cells, and B-cell lymphoma lines, respectively. miRNA expression levels were analyzed by real-time stem-loop RT PCR in GFP⁺-sorted transduced NIH 3T3 cells or the indicated B cell lines. BCBL-1 and Raji were used as positive and normalization controls for kshv-miR-K12-11 or hsa-miR-155 expression, respectively, and relative expression levels in these cell lines was set to 1. All experiments were done in triplicate.</p

Enhanced GC development in miRNA-expressing mice.

<p>A) H&E staining and immunohistochemical detection if PNA indicate increased GC formation in miRNA-expressing mice. White arrows in H&E stained sections indicate the GC. PNA staining was used to determine the GC number and relative GC area per section. Shown are representative sections from one mouse of each cohort. B) Statistical analysis of the average number of GCs (left) and the relative total GC area (right) in the mouse cohorts. Enumeration of GCs and determination of total GC area was performed as described in the material and methods section.</p

Expansion of B-cells in spleens of miRNA-expressing mice.

<p>Shown are analyses of GFP⁺ gated spleenocytes. A) Flow cytometry analysis reveals an expansion of B-cells in miRNA-expressing mice using the B-cell marker CD19. Left: histograms of one representative mouse each of each cohort. Gate P1 indicates CD19⁺ fraction. Right: dot plots of all analyzed mice (control gfp: n = 15; kshv-miR-K12-11: n = 23; hsa-miR-155: n = 15) reveal a significant shift towards the B-cell population miRNA-expressing mice. B) Flow cytometry analysis reveals an expansion of B-cells in miRNA-expressing mice using the B-cell marker B220. Left: histograms of one representative mouse per cohort. Gate P1 indicates the B220⁺ fraction. Right: dot plots of all analyzed mice (control gfp: n = 16; kshv-miR-K12-11: n = 25; hsa-miR-155: n = 16) reveal an expansion of B-cells among the GFP⁺ splenocytes. C) The number of T-cells is decreased in the miRNA-expressing mice cohorts, as indicated by flow cytometry analysis of the T-cell marker CD3 (control gfp: n = 15; kshv-miR-K12-11: n = 19; hsa-miR-155: n = 16).</p

Increased fraction of pre B-cells in BM cells of miRNA-expressing mice.

<p>The immuno-phenotypic profile of BM cells harvested from tibiae and femora was characterized by flow cytometry. A) Decrease of myeloid population in miRNA-expressing mice. The graph represents flow cytometry results of GFP⁺ gated BM cells (total number of mice: control gfp: n = 12; kshv-miR-K12-11: n = 16; hsa-miR-155: n = 13). The result show a decrease of myeloid cell populations (CD11b⁺Gr1⁺) in the miRNA-expressing mice compared to control mice. B) Increase of B-cell population in the BM cells of miRNA-expressing mice. GFP⁺ gated BM cells from miRNA-expressing mice reveal a slight increase of B-lineage cells compared to GFP control mice (B220⁺ (left graph; (control gfp: n = 13; kshv-miR-K12-11: n = 11; hsa-miR-155: n = 16)) and CD19⁺ (right graph; (control gfp: n = 10; kshv-miR-K12-11: n = 9; hsa-miR-155: n = 13)). C) CD43 expression evaluated by flow cytometry on GFP⁺/CD19⁺ gated BM cells reveals a significant increase of the pre B-cell population (CD19⁺CD43⁻) in the BM of miRNA-expressing mice, indicating a shift toward the pre B-cell fraction. Left: histograms of one representative mouse each of miRNA-expressing and control mice. Gate P1 indicates the CD43⁻ population. Right: dot plots of all analyzed mice (control gfp: n = 10; kshv-miR-K12-11: n = 9; hsa-miR-155: n = 13).</p

Decreased expression levels of miR-155 and kshv-miR-K12-11 target transcripts in miRNA expressing BM cells.

<p>Real-time RT PCR analysis was carried out using total RNA derived from flow sorted GFP⁺ BM cells from two mice of each mouse cohort. The mRNA levels for fos, c-myb and Jarid2 were normalized to endogenous level of actin, GAPDH and RPLP.</p

High Risk HPVs do not encode canonical microRNAs.

<p><b>(A)</b> Coverage plots of miDGE DNA-seq and small RNA-seq across the genomes of HPV18 and HPV17. Filled green plots at the top of each panel show DNA-seq coverage, the three plots underneath show mapped small RNA-seq from: <i>PV</i>: HEK293T-cells transfected with our papillomavirus miDGE library, <i>PV filtered</i>: same reads as in PV, but filtered to eliminate low-complexity reads <i>JMRV</i>: Serving as a negative control, derived from 293T-cells transfected with our JMRV miDGE library. JMRV read counts were normalized to correct for different sequencing depths between PV and JMRV miDGE experiments (see total read counts in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007156#ppat.1007156.t001" target="_blank">Table 1</a>). Asterisk indicates a previously purported miRNA candidate region suggested in the literature [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007156#ppat.1007156.ref039" target="_blank">39</a>], which is nonspecific (detected in the negative control JMRV miDGE analysis, lower plot) and can be eliminated by removing low complexity reads (center plot). <b>(B)</b> RNA-seq coverage for the most abundantly mapped HPV in 303 tumors in the TCGA CESC project [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007156#ppat.1007156.ref050" target="_blank">50</a>]. Each of the 303 libraries are represented on the X-axis (sorted based on Y-axis value). Y-axis indicates the percentage of the positions in the HPV genome with read mappings. Libraries with > = 50% coverage (213 libraries) were used for subsequent analysis. <b>(C)</b> Percentages of TCGA cervical squamous cell carcinoma (CESC) libraries with miRDeep2 miRNA identifications for each set of reference sequences. Number of libraries examined is 213. <b>(D)</b> Number of unique miRDeep2 miRNA identifications across TCGA CESC libraries for each set of reference sequences. Number of libraries examined is 213. <b>(E)</b> Percentages of Qian <i>et al</i>. [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007156#ppat.1007156.ref040" target="_blank">40</a>] libraries with miRDeep2 miRNA identifications for each set of reference sequences. Number of libraries examined is 12. <b>(F)</b> Number of unique miRDeep2 miRNA identifications across Qian <i>et al</i>. libraries for each set of reference sequences. Number of libraries examined is 12. <b>(G)</b> Raw read counts of all small RNAs mapping to the indicated reference sequences for each library from Qian <i>et al</i>.</p

miDGE identifies PV-encoded miRNA candidates.

<p><b>(A)</b> Neighbor-joining tree calculated on alignment of L1 nucleotide sequences of the better-covered papillomaviruses (n = 73 with >95% DNA coverage) included in miDGE library. Color indicates genus membership, with miRNA-encoding papillomaviruses in bold and select high risk cancer-associated papillomaviruses in italics. <b>(B)</b> Small RNA coverage distribution of top-scoring miDGE miRNA candidates that were predicted by MiRDeep2. <b>(C)</b> Structures of PV pre-miRNAs were predicted by minimal free energy folding using the RNAfold algorithm. The positions of mature miRNAs observed in small RNA-seq libraries are indicated in red. <b>(D)</b> The position of identified viral pre-miRNAs is denoted by the hairpin shape. The identified seed sequence matches are noted at their respective positions with the sequences of the miRNA and potential targets.</p

Validation of miDGE via confirmation of miRNAs encoded by Japanese Monkey Herpesvirus (JMHV).

<p><b>(A)</b><i>Top</i>: Coverage plot of DNA-seq of expression fragment library containing a cosmid with ~40kb of the Japanese Monkey Herpesvirus (JMRV) genome. <i>Center and bottom</i>: small RNA coverage plots mapping of small RNA-seq reads from HEK293T cells transfected with the expression library (<i>center</i>) or from JMRV-infected rhesus primary fibroblasts (<i>bottom</i>). <b>(B)</b> Small RNA-seq coverage plot in linear (top panels) or logarithmic (bottom panels) across the miRNA-coding region. The genomic location of the 15 JMRV pre-miRNAs is indicated by black arrows at the top. <b>(C)</b> Detailed depiction of small RNA-seq coverage across the first three pre-miRNAs of the JMRV miRNA cluster.</p

Small RNA-seq read & mapping statistics.

<p>Small RNA-seq read & mapping statistics.</p