TARGETED KAPOSI’S SARCOMA ASSOCIATED HERPESVIRUS ORFEOME INTERACTIONS BY RIBOSOME DISPLAY

Kaposi’s sarcoma associated herpesvirus (KSHV, HHV-8) is the etiological agent of Kaposi’s sarcoma (KS), and is tightly associated with primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD). KSHV has a large dsDNA circular genome of about 165kb that reside in the nucleus of infected cells and encodes over 80 viral genes. These genes include viral genes unique to KSHV, viral genes homologous to other human herpes viruses and homologous to cellular genes that were pirated by the virus. One approach to uncover the functions of viral genes is through protein-protein interaction (PPI). While many previous PPI studies revealed many important viral host interactions they were mainly based on screens of one viral protein against a pool of cellular proteins. Based on the growing knowledge of key cellular proteins and protein modifications, we thought to perform the opposite screen by choosing important cellular protein/complex and identify the viral protein/s interactors. We have chosen the Ribosome Display, a method that couples phenotype to genotype by preventing the release of newly synthesized protein from the ribosome and the RNA that coded for its translation. Therefore, following binding of a pool of proteins to immobilized bait the bound proteins with their encoded RNA can be isolated to serve as a template for cDNA and sequencing or PCR amplification. We have cloned the KSHV ORFeome (84 genes) into a Ribosome Display expression system. For the proof of concept we have initially performed the KSHV OFReome screen with SUMO-2, a protein that four KSHV proteins have already shown to interact with. Our initial screen detected the known interactors and identified several novel interactors.