Studies of KSHV have been impeded because of the lack of an efficient infection system. Consequently, the development of a genetic manipulation system is elusive. Over 90 KSHV genes or ORFs have been identified, and many of them have been cloned and examined in tissue culture, however, their functions in KSHV infection and the pathogenesis of KSHV-related malignancies remain unclear. To facilitate KSHV studies, we have recently developed a genetic approach to isolate infectious KSHV. The entire KSHV has been cloned into bacteria artificial chromosome (BAC) and reconstituted in mammalian cells. Cells infected by the recombinant KSHV (BAC36) can be efficiently induced into lytic replication to produce highly infectious virions. BAC36 virions efficiently infect human primary endothelial cells (>90%) and transform them into Kaposi's sarcoma-like vascular spindle cells. This cell model recapitulates both the viral and cellular aspects of KSHV infection in KS tumor cells. Thus, we have established a cell model and an efficient bacteria-mammalian shuttle system for KSHV infection, replication, cellular transformation and genetic analysis.
Characterizations of KSHV infection and replication have mostly been performed with KSHV-infected PEL cell lines, for which appropriate controls are difficult to identify for some experiments. Most of these studies have been restricted to the examination of viral latency and reactivation. Only a limited number of studies have investigated KSHV primary infection so far; however, they have been carried out using systems that have inefficient viral infection. Our cell model is ideal for defining the virology and cellular biology of KSHV primary, latent and lytic infection, and cellular transformation. Currently, we are determining the expression patterns of viral and cellular genes during KSHV infection and replication in human primary endothelial cells using a KSHV microarray recently developed in our laboratory and Genechips from Affymetrics in combination with real-time RT-PCR technology. Simultaneously, we are also examining the alterations of cellular phenotypes including cell adhesion and migration, cellular proliferation, cell cycle progression, and apoptosis. These studies should provide insight into the molecular events of KSHV latent and lytic replication, and viral regulation of cellular pathways in human primary endothelial cells, and further define the cell model for the examination of KSHV infection, replication, and transformation.