Construction of a KSHV-GFP Recombinant Virus and the
Development of a System to Assess Gene functions of KSHV

Ke Lan, MD, PhD

Kaposi's sarcoma associated herpesvirus(KSHV), also designated HHV8 was discovered in 1994. Epidemiological studies have demonstrated that KSHV is the causative agent for KS. For instance, DNA sequences of this virus were consistently found in KS lesions of all epidemiological forms. Furthermore, serological assays suggest that KSHV is not ubiquitous but is closely associated with those people at risk for developing KS. In addition to KS tumors, KSHV is also found in B-cell primary effusion lymphomas or body cavity based lymphomas and in some forms of Multicentric Castleman Disease, a B-cell lymphoproliferative disorder. The discovery of the genome of KSHV was a major breakthrough in our understanding of the pathogenesis of these diseases. Unique among human herpesvirus, KSHV encodes a number of cytokines, cyclin homologues, G proteins, moreover, unlike Epstein-Barr virus encoded EBNA2, EBNA3 and LMPs, which are latent genes, the KSHV cytokine signaling genes are expressed in lytic stage, so lytic replication of the virus appears to be important not only for viral propagation but also for viral pathogenicity, however, the proteins and genomic elements that control activation and completion of the viral lytic cycle are only partly understood. In addition, most data regarding the transforming potential of this virus have been derived from experiments with single viral gene products, nothing is known about the function of these gene products in the context of the whole virus. In particular, it remain unclear whether all or only certain viral transforming proteins are required for cellular transformation. The major goal of this project is to construct a KSHV-GFP recombinant virus by homologous recombination strategy. This KSHV-GFP recombinant virus will facilitate tracking the infected cells during the process of lytic primary infection and further to elucidate the mechanism of interaction between virus and host cells. Moreover, based on this recombinant virus, a series KSHV mutants can be made for analysis of the functions of specific genes.