Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma (KS), a vascular cancer commonly found in AIDS patients. Despite extensive studies, the mechanism of KSHV-induced cellular transformation and tumorigenesis remains undefined. This critical gap of knowledge has impeded the development of effective intervention measures. We have shown that KSHV can efficiently infect and transform primary rat embryonic metanephric mesenchymal precursor cells (MM). KSHV-infected MM (KMM) induce KS-like tumors in nude mice. Using reverse genetics, we have found that a cluster of 10 KSHV pre-microRNAs (pre-miRs) is required for KSHV-induced cellular transformation and tumorigenesis. Encouraged by these findings, we have recently made significant progresses in infecting and transforming human primary cells with KSHV. Specifically, we have found that KSHV can also infect, immortalize and transform human primary mesenchymal stem cells (MSC), the human version of MM. This collaborative application between the University of Southern California and Nanjing Medical University proposes to extend these exciting paradigm-shifting discoveries with the objective to refine the MSC model, and use both the MM and MSC models to identify the specific miRs that mediate KSHV cellular transformation and tumorigenesis, and define the essential cellular genes and pathways that are targeted by these miRs. Based on our preliminary results, we have formulated a working hypothesis that KSHV can infect and transform MSC, and specific KSHV miRs manipulate essential cell growth and survival pathways, contributing critically to KSHV-induced cellular transformation and tumorigenesis. Therefore, the proposed project will refine the model of KSHV cellular transformation of human primary MSC (Aim 1); determine the contribution of individual viral pre-miRs and miRs to KSHV-induced cellular transformation and tumorigenesis by genetic complementation (Aim 2); determine the effect of individual viral pre-miRs and miRs on cellular transformation, and identify those that regulate cell growth, survival, angiogenesis and invasion (Aim 3); and delineate the mechanism of viral miRs- mediated KSHV-induced cellular transformation and tumorigenesis by identifying the essential direct targets (Aim 4). This collaborative project takes advantage of the expertise of the US and China laboratories. These works are significant and innovative because they will, for the first time, show that KSHV can truly transform human primary cells, define the functions and mechanisms of action of KSHV miRs in cellular transformation and tumorigenesis, and identify potential novel targets for developing innovative prognostic and therapeutic approaches. The study will also establish a novel paradigm of oncogenesis mediated by viral subversion of the miR pathway, thus providing insights into the oncogenesis of other cancers.