MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally regulate gene expression by binding to 3-untranslated regions (3UTRs) of target mRNAs. Kaposi's sarcoma-associated herpesvirus (KSHV), a virus linked to malignancies including primary effusion lymphoma (PEL), encodes 12 miRNA genes, but only a few regulatory targets are known. We found that KSHV-miR-K12-11 shares 100% seed sequence homology with hsa-miR-155, an miRNA frequently found to be up-regulated in lymphomas and critically important for B-cell development. Based on this seed sequence homology, we hypothesized that both miRNAs regulate a common set of target genes and, as a result, could have similar biological activities. Examination of five PEL lines showed that PELs do not express miR-155 but do express high levels of miR-K12-11. Bioinformatic tools predicted the transcriptional repressor BACH-1 to be targeted by both miRNAs, and ectopic expression of either miR-155 or miR-K12-11 inhibited a BACH-1 3UTR-containing reporter. Furthermore, BACH-1 protein levels are low in cells expressing either miRNA. Gene expression profiling of miRNAexpressing stable cell lines revealed 66 genes that were commonly down-regulated. For select genes, miRNA targeting was confirmed by reporter assays. Thus, based on our in silico predictions, reporter assays, and expression profiling data, miR-K12-11 and miR-155 regulate a common set of cellular targets. Given the role of miR-155 during B-cell maturation, we speculate that miR-K12-11 may contribute to the distinct developmental phenotype of PEL cells, which are blocked in a late stage of B-cell development. Together, these findings indicate that KSHV miR-K12-11 is an ortholog of miR-155.

MicroRNAs (miRNAs) are small, noncoding regulatory RNA molecules that bind to 3 untranslated regions (UTRs) of mRNAs to either prevent their translation or induce their degradation. Previously identified in a variety of organisms ranging from plants to mammals, miRNAs are also now known to be produced by viruses. The human gammaherpesvirus Epstein-Barr virus has been shown to encode miRNAs, which potentially regulate both viral and cellular genes. To determine whether Kaposi's sarcoma-associated herpesvirus (KSHV) encodes miRNAs, we cloned small RNAs from KSHV-positive primary effusion lymphoma-derived cells and endothelial cells. Sequence analysis revealed 11 isolated RNAs of 19 to 23 bases in length that perfectly align with KSHV. Surprisingly, all candidate miRNAs mapped to a single genomic locale within the latencyassociated region of KSHV. These data suggest that viral and host cellular gene expression may be regulated by miRNAs during both latent and lytic KSHV replication.MicroRNAs (miRNAs) range from 19 to 24 nucleotides (nt) in length and are small, noncoding RNA molecules which bind complementary mRNAs to regulate gene expression at the posttranscriptional level. In plants, miRNAs bind precisely to complementary sequences within the 3Ј untranslated region (UTR) of a target gene and induce mRNA degradation through the RNA interference pathway. Most animal miRNAs have limited complementarity to their target sequences within the 3Ј UTR and either degrade mRNA via the RNA interference pathway or down-regulate translation by a mechanism not yet understood. In human cells, over 235 miRNAs have been identified to date (for review, see references 1 and 4). Targets and functions of very few miRNAs have been experimentally determined thus far, yet some molecules, such as human hsamiR-14 and hsa-miR-181, are known to have roles in fundamental biological processes like apoptosis, cell proliferation, and hematopoiesis (6, 9).Most recently, miRNAs have been identified and isolated in the gammaherpesvirus Epstein-Barr virus (EBV) (21) and predicted for the human immunodeficiency virus using in silico methods (5). Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), also called human herpesvirus type 8 (HHV-8), is another gammaherpesvirus. The virus is associated with KS and two lymphoproliferative diseases: primary effusion lymphomas (PELs) and a subset of multicentric Castleman's disease (7,8,26). In this report, we demonstrate that KSHV, like EBV, encodes miRNAs.Cloning of small RNAs from KSHV-infected cells. To determine whether KSHV encodes miRNAs, we generated small RNA libraries by positional cDNA cloning from a primary effusion lymphoma-derived cell line (BCBL-1) undergoing either latent or tetradecanoyl phorbol acetate (TPA)-induced lytic KSHV infection (23). Additionally, we cloned small RNAs from a telomerase-immortalized endothelial cell line latently infected with KSHV (TIVE-LTC) (F. Q. An and R. Renne, data to be published elsewhere). Cloning was performed as described in reference 16, with minor modific...

MicroRNAs (miRNAs) are 19 to 23 nucleotide–long RNAs that post-transcriptionally regulate gene expression. Human cells express several hundred miRNAs which regulate important biological pathways such as development, proliferation, and apoptosis. Recently, 12 miRNA genes have been identified within the genome of Kaposi sarcoma–associated herpesvirus; however, their functions are still unknown. To identify host cellular genes that may be targeted by these novel viral regulators, we performed gene expression profiling in cells stably expressing KSHV-encoded miRNAs. Data analysis revealed a set of 81 genes whose expression was significantly changed in the presence of miRNAs. While the majority of changes were below 2-fold, eight genes were down-regulated between 4- and 20-fold. We confirmed miRNA-dependent regulation for three of these genes and found that protein levels of thrombospondin 1 (THBS1) were decreased >10-fold. THBS1 has previously been reported to be down-regulated in Kaposi sarcoma lesions and has known activity as a strong tumor suppressor and anti-angiogenic factor, exerting its anti-angiogenic effect in part by activating the latent form of TGF-β. We show that reduced THBS1 expression in the presence of viral miRNAs translates into decreased TGF-β activity. These data suggest that KSHV-encoded miRNAs may contribute directly to pathogenesis by down-regulation of THBS1, a major regulator of cell adhesion, migration, and angiogenesis.