ORFeome-based arrays in eukaryotic expression vectors - a new approach to screen for the function of viral proteins

摘要

Since its first description in 1994 by Yuan Chang and Patrick Moore, Kaposi’s sarcoma-associated herpesvirus (KSHV) or Human Herpesvirus 8 (HHV-8) has emerged as a pathogen of international public health importance. It has been detected in biopsies of all forms of Kaposi’s sarcoma (KS), irrespective of geographic origin, age, or gender of the patient. Moreover, KSHV has been shown to be associated with two other diseases, multicentric Castleman’s disease (MCD) and primary effusion lymphoma (PEL).In comparison to alpha and beta-herpesvirinae, the understanding of KSHV-related pathogenesis has been hampered by inefficient virus replication in vitro, poor cell culture systems and the lack of an animal model. Thus, many basic questions concerning the biology of KSHV infection remain open. For example, the primary target cell of KSHV and the function of more than 50% of the viral proteins are still unknown. Since the investigation of viral gene functions by virus mutants did not prove to be very efficient for KSHV, a system for a genome-wide screening of viral gene functions by cloning the complete KSHV ORFeome (all open reading frames) and by generating KSHV arrays in a variety of different expression vectors was established in this project. Very often viruses regulate cellular signalling pathways, which favour viral infection and replication in the host cells. The SRE is a transcription factor binding site present in promoters of many genes involved in cell growth and transformation. In this study, a genome-wide screen for KSHV genes inducing the SRE element and AP-1 was performed. A strong induction of SRE by the latency-associated nuclear antigen 1 (LANA-1) was observed. LANA-1 is a multifunctional protein which interacts with the p53 and RB tumor suppressor proteins. This study reveals several novel functions of LANA-1. LANA-1 led to an activation of the ERK-1/2 MAP kinase, but also bound to the Mediator, a multi-subunit transcriptional coactivator complex for RNA polymerase II, via the ARC92/ACID1 subunit. Since LANA-1 interacted with SRF, one of the two transcription factors binding to the bipartite SRE element, a model for LANA-1 as an adaptor between specific transcription factors and the basal transcriptional machinery was hypothesized.