| Kaposi's Sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL) and a subset of multicentric Castleman's disease (MCD). It is classified as a gamma-herpesvirus, along with Epstein Barr virus (EBV) and Herpesvirus Saimiri (HVS). In 2004 it was shown that EBV encoded 5 microRNAs (miRNAs). MiRNAs are short 22+/-3 nt single stranded RNA molecules that have been demonstrated to control cell differentiation and development. By binding to the RNA induced silencing complex (RISC), miRNAs are able to target the 3'UTRs of messenger RNAs and induce either mRNA degradation or translational silencing. We hypothesized that KSHV, being in the same family as EBV, would also contain miRNAs and that these miRNAs would target cellular transcripts. Using positional cDNA cloning from latently and lytically infected cell lines, we cloned 10 miRNAs unique to KSHV. Interestingly, all of the miRNAs were located within the latency associated region of the viral genome. To investigate how these miRNAs function, we performed a search for cellular targets of the miRNAs using Affymetrix microarray analysis of 293 cells stably expressing the KSHV miRNA cluster. We found 81 genes with significant expression changes of which 8 showed down-regulation between 4- and 20-fold. We confirmed miRNA-dependent inhibition for three of these genes, one of which, Thrombospondin 1 (THBS1), also showed decreased protein levels in response to miRNA expression. THBS1 is a potent inhibitor of angiogenesis and cell migration, and was reported to be reduced in KS lesions. Using sequence-specific miRNA inhibitors we revealed that the THBS1 gene is being regulated by several KSHV miRNAs in combination. Additionally, we showed that THBS1-dependent activation of TGF-beta was significantly decreased by KSHV miRNAs. These results show that KSHV produces miRNAs and that they are able to target and control cellular genes to provide a more favorable environment for pathogenesis of the virus. |
