A microRNA encoded by Kaposi sarcoma-associated herpesvirus promotes B-cell expansion in vivo.

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.

Common microRNA signatures in cardiac hypertrophic and atrophic remodeling induced by changes in hemodynamic load.

BACKGROUND: Mechanical overload leads to cardiac hypertrophy and mechanical unloading to cardiac atrophy. Both conditions produce similar transcriptional changes including a re-expression of fetal genes, despite obvious differences in phenotype. MicroRNAs (miRNAs) are discussed as superordinate regulators of global gene networks acting mainly at the translational level. Here, we hypothesized that defined sets of miRNAs may determine the direction of cardiomyocyte plasticity responses. METHODOLOGY/PRINCIPAL FINDINGS: We employed ascending aortic stenosis (AS) and heterotopic heart transplantation (HTX) in syngenic Lewis rats to induce mechanical overloading and unloading, respectively. Heart weight was 26±3% higher in AS (n = 7) and 33±2% lower in HTX (n = 7) as compared to sham-operated (n = 6) and healthy controls (n = 7). Small RNAs were enriched from the left ventricles and subjected to quantitative stem-loop specific RT-PCR targeting a panel of 351 miRNAs. In total, 153 miRNAs could be unambiguously detected. Out of 72 miRNAs previously implicated in the cardiovascular system, 40 miRNAs were regulated in AS and/or HTX. Overall, HTX displayed a slightly broader activation pattern for moderately regulated miRNAs. Surprisingly, however, the regulation of individual miRNA expression was strikingly similar in direction and amplitude in AS and HTX with no miRNA being regulated in opposite direction. In contrast, fetal hearts from Lewis rats at embryonic day 18 exhibited an entirely different miRNA expression pattern. CONCLUSIONS: Taken together, our findings demonstrate that opposite changes in cardiac workload induce a common miRNA expression pattern which is markedly different from the fetal miRNA expression pattern. The direction of postnatal adaptive cardiac growth does, therefore, not appear to be determined at the level of single miRNAs or a specific set of miRNAs. Moreover, miRNAs themselves are not reprogrammed to a fetal program in response to changes in hemodynamic load.

A global analysis of evolutionary conservation among known and predicted gammaherpesvirus microRNAs.

MicroRNAs (miRNAs) are small, noncoding RNAs which posttranscriptionally regulate gene expression. The current release of the miRNA registry lists 16 viruses which encode a total of 146 miRNA hairpins. Strikingly, 139 of these are encoded by members of the herpesvirus family, suggesting an important role for miRNAs in the herpesvirus life cycle. However, with the exception of 7 miRNA hairpins known to be shared by Epstein-Barr virus (EBV) and the closely related rhesus lymphocryptovirus (rLCV), the known herpesvirus miRNAs show little evidence of evolutionary conservation. We have performed a global analysis of miRNA conservation among gammaherpesviruses which is not limited to family members known to encode miRNAs but includes also those which have not been previously analyzed. For this purpose, we have performed a computational prediction of miRNA candidates of all fully sequenced gammaherpesvirus genomes, followed by sequence/structure alignments. Our results indicate that gammaherpesvirus miRNA conservation is limited to two pairs of viral genomes. One is the already-known case of EBV and rLCV. These viruses, however, share significantly more miRNAs than previously thought, as we identified and experimentally verified 10 novel conserved as well as 7 novel nonconserved rLCV pre-miRNA hairpins. The second case consists of rhesus rhadinovirus (RRV), which is predicted to share at least 9 pre-miRNAs with the closely related Japanese macaque herpesvirus (JMHV). Although several other gammaherpesviruses are predicted to encode large numbers of clustered miRNAs at conserved genomic loci, no further examples of evolutionarily conserved miRNA sequences were found.

The interaction of the gammaherpesvirus 68 orf73 protein with cellular BET proteins affects the activation of cell cycle promoters.

Infection of mice with murine gammaherpesvirus 68 (MHV-68) provides a valuable animal model for gamma-2 herpesvirus (rhadinovirus) infection and pathogenesis. The MHV-68 orf73 protein has been shown to be required for the establishment of viral latency in vivo. This study describes a novel transcriptional activation function of the MHV-68 orf73 protein and identifies the cellular bromodomain containing BET proteins Brd2/RING3, Brd3/ORFX, and BRD4 as interaction partners for the MHV-68 orf73 protein. BET protein members are known to interact with acetylated histones, and Brd2 and Brd4 have been implicated in fundamental cellular processes, including cell cycle regulation and transcriptional regulation. Using MHV-68 orf73 peptide array assays, we identified Brd2 and Brd4 interaction sites in the orf73 protein. Mutation of one binding site led to a loss of the interaction with Brd2/4 but not the retinoblastoma protein Rb, to impaired chromatin association, and to a decreased ability to activate the BET-responsive cyclin D1, D2, and E promoters. The results therefore pinpoint the binding site for Brd2/4 in a rhadinoviral orf73 protein and suggest that the recruitment of a member of the BET protein family allows the MHV-68 orf73 protein to activate the promoters of G(1)/S cyclins. These findings point to parallels between the transcriptional activator functions of rhadinoviral orf73 proteins and papillomavirus E2 proteins.

Kaposi's sarcoma-associated herpesvirus LANA-1 interacts with the short variant of BRD4 and releases cells from a BRD4- and BRD2/RING3-induced G1 cell cycle arrest.

The Kaposi's sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen 1 (LANA-1) is required for the replication of episomal viral genomes. Regions in its N-terminal and C-terminal domains mediate the interaction with host cell chromatin. Several cellular nuclear proteins, e.g., BRD2/RING3, histones H2A and H2B, MeCP2, DEK, and HP1alpha, have been suggested to mediate this interaction. In this work, we identify the double-bromodomain proteins BRD4 and BRD3/ORFX as additional LANA-1 interaction partners. The carboxy-terminal region of the short variant of BRD4 (BRD4S) containing the highly conserved extraterminal domain directly interacts with an element in the LANA-1 carboxy-terminal domain. We show that ectopically expressed BRD4S and BRD2/RING3 delay progression into the S phase of the cell cycle in epithelial and B-cell lines and increase cyclin E promoter activity. LANA-1 partly releases epithelial and B cells from a BRD4S- and BRD2/RING3-induced G1 cell cycle arrest and also promotes S-phase entry in the presence of BRD4S and BRD2/RING3. This is accompanied by a reduction in BRD4S-mediated cyclin E promoter activity. Our data are in keeping with the notion that the direct interaction of KSHV LANA-1 with BRD4 and other BRD proteins could play a role in the G1/S phase-promoting functions of KSHV LANA-1. Further, our data support a model in which the LANA-1 C terminus contributes to a functional attachment to acetylated histones H3 and H4 via BRD4 and BRD2, in addition to the recently demonstrated direct interaction (A. J. Barbera, J. V. Chodaparambil, B. Kelley-Clarke, V. Joukov, J. C. Walter, K. Luger, and K. M. Kaye, Science 311:856-861, 2006) of the LANA-1 N terminus with histones H2A and H2B.