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