Kaposi'S Sarcoma-Associated Herpesvirus Microrna Mutants Modulate Cancer Hallmark Phenotypic Differences In Human Endothelial Cells

Kaposi's sarcoma (KS) results from the transformation of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected endothelial cells. The contribution of the KSHV microRNAs (miRNAs) to the process of oncogenesis in endothelial cells has not been fully elucidated. To better understand the contributions of individual miRNAs to oncogenesis-related cellular phenotypes, we used KSHV miRNA knockout mutants, each lacking one of the 12 miRNA genes. An additional mutant lacked all miRNAs. Since KSHV infection causes a variety of phenotypic changes in endothelial cells, we tested the mutants for their ability to effect such changes in telomeraseimmortalized vein endothelial (TIVE) cells infected with each of the mutant viruses. Wild type- and mutant-infected as well as uninfected cells were evaluated for perturbations to proliferation, migration, tubule formation, and glycolysis. We found broad variation between the different viruses in these aspects. With respect to proliferation rate, Delta miR-K12-3, Delta miR-K12-8, and Delta miR-K12-11 showed significant impairment. Cells infected with Delta miR-K12-11 had reduced migration. In tubule formation, the Delta miR-K12-5, -6, and -7 viruses were deficient. At the same time, cells infected with the Delta miR-K12-10 virus showed dysregulated glycolysis. By combining these observations with previously published KSHV miRNA targetome lists from ribonomics data, we were able to functionally validate a number of new miRNA targets in specific pathways. As proof of concept, miR-K12-3 was shown to target cathepsin D, a strong promoter of apoptosis. Taken together, the results demonstrate that KSHV miRNAs play different roles in inducing the phenotypic changes that are characteristic of transformed cells.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) causes Kaposi's sarcoma (KS). The contribution of KSHV microRNAs (miRNAs) to oncogenesis is not fully understood. This is particularly true for human endothelial cells, the cell type from which KS tumors are derived. Here, we used a panel of KSHV miRNA knockout viruses to shed light on the roles of individual miRNAs in the process of transformation. Latently infected endothelial cells were studied for phenotypic changes related to cancer, including proliferation, migration, angiogenesis, glycolysis, and apoptosis. The mutant-infected cell lines displayed a wide range of phenotypes in these selected measures of oncogenesis, which differed from those of wild-type-infected cells and from each other. These results indicate that KSHV miRNAs contribute to different aspects of oncogenesis and that each one has a unique role to play.