The Epstein-Barr Virus Nuclear Antigens 1 and 5; Study of virus-host cellular protein interactions

The Epstein-Barr virus (EBV) is the causative agent or cofactor in the aetiology of several human malignancies such as Burkitt’s lymphoma, Hodgkin’s disease, nasopharyngeal carcinoma (NPC) and lymphoproliferative disorder in immunocompromised patients. EBV is a lymphotropic γ-herpes virus infecting more than 90 percent of the population worldwide. Following acute infection the virus establishes a life-long latency in resting memory B cells. The virus is remarkable for the efficiency with which it causes proliferation and immortalization of the infected B cells through expression of several latent gene products. All of the viral EBNA proteins have been proposed to play a role in the control of gene expression in the EBV infected lymphoblastoid cell. The present thesis is mainly focused on further elucidating the molecular mechanisms of the EBNA1 and EBNA5 proteins using proteomic technologies as approach. In paper I we used an improved tandem affinity purification procedure for identification and characterization of factors in the EBNA5 interaction proteome. The majority of the 37 validated interactors could be assigned to one of three groups according to function: protein folding and degradation, pre mRNA processing, or ribosomal proteins, implicating functional relationships with EBNA5 in these processes. We also showed that EBNA5 is part of high molecular protein complexes, supporting the notion that functional units in the cell are not single proteins but well-structured complexes composed of multiple proteins i.e. modules. The previously reported repressor activity of EBNA5 was further investigated in paper II. The study identified the novel interactor BAG2 as a major target for the function of EBNA5 via the chaperone-mediated folding and proteasome-degradation pathways. Taken together, the results are consistent with the hypothesis that EBNA5 tune the balance between protein rescue and destruction in a way that disfavour the path of degradation. The constituents of the large macromolecular complex that initiates transcription from the viral C promoter were investigated in paper III. Using a DNA affinity procedure we showed that the transcription factors E2F1, ARID3A/Bright and Oct-2 binds the Cp as well as EBNA1 and oriPI, possibly facilitating long-distance promoter-enhancer interactions. While the study of genes and proteins continues to be important, looking at isolated components is not enough to understand most biological processes. Modularity has been proposed as a general principle for the molecular architecture of living systems. These assemblies interact with other large protein complexes, thus the proteins are part of a protein-protein interaction network inside the cell. A common feature of these interaction networks is that it contains junctions of proteins that are highly interconnected, also called hubs. Hubs have a tendency of being essential and involved in cancer development. Two central pathways in cancer biology are the Rb- and p53-pathways, which are targets for both EBNA1 and EBNA5 action. This is consistent with the hypothesis that several viral proteins target the same hubs in the host, which ensures the takeover of the cellular machineries essential for the viral infection and persistence processes, and contribute to the robustness of the viral infectious system

Molecular genetic characterisation of the Asc locus of tomato conferring resistance to the fungal pathogen Alternaria alternata f. sp. lycopersici

The Alternaria stem canker disease of tomato is caused by the fungal pathogen Alternaria alternata f. sp. lycopersici and its host-selective AAL-toxins. Resistance to the pathogen and insensitivity to the toxins are conferred by the Asc locus on chromosome 3L. Sensitivity to AAL-toxins is a relative character; the toxins inhibit development of all tested tomato tissues but susceptible cultivars are much more sensitive than resistant cultivars. In addition to tomato, some other plant and animal species are sensitive to the toxins as well. The likely mode of action of AAL-toxins is interference with sphingolipid biosynthesis by specific inhibition of ceramide synthase activity. To molecularly isolate Asc, transposon tagging and positional cloning strategies are applied. As a first step, transposon insertions and restriction fragment length polymorphism (RFLP) markers are identified in proximity of the Asc locus. Subsequently, the transposons are used to inactivate Asc by insertion mutagenesis, and the RFLP markers are used to identify yeast artificial chromosomes (YACs) with tomato DNA inserts. Once an Asc-insertion mutant and/or a YAC encompassing Asc has been obtained, physical isolation and characterisation of Asc will be conceivable. Elucidation of the molecular role of Asc will illuminate the specificity of host recognition by Alternaria alternata f. sp. lycopersici.

A longelivety assurance gene homolog of tomato mediates resistance to Alernaria Alternata f.sp.Lycopersici toxins and fumonisin B1

The phytopathogenic fungus Alternaria alternata f. sp. lycopersici (AAL) produces toxins that are essential for pathogenicity of the fungus on tomato (Lycopersicon esculentum). AAL toxins and fumonisins of the unrelated fungus Fusarium moniliforme are sphinganine-analog mycotoxins (SAMs), which cause inhibition of sphingolipid biosynthesis in vitro and are toxic for some plant species and mammalian cell lines. Sphingolipids can be determinants in the proliferation or death of cells. We investigated the tomato Alternaria stem canker (Asc) locus, which mediates resistance to SAM-induced apoptosis. Until now, mycotoxin resistance of plants has been associated with detoxification and altered affinity or absence of the toxin targets. Here we show that SAM resistance of tomato is determined by Asc-1, a gene homologous to the yeast longevity assurance gene LAG1 and that susceptibility is associated with a mutant Asc-1. Because both sphingolipid synthesis and LAG1 facilitate endocytosis of glycosylphosphatidylinositol-anchored proteins in yeast, we propose a role for Asc-1 in a salvage mechanism of sphingolipid-depleted plant cells