Molecular Interactions of theInfectious Bursal Disease Virus Proteins

Infectious bursal disease virus (IBDV), a member of the family Birnaviridae (Dobos et al., 1979), is the causative agent of a highly contagious immunosuppressive disease in young chickens. IBDV multiplies rapidly in developing B lymphocytes in the bursa of Fabricius, leading to immunosuppression. This increases susceptibility to infections with opportunistic pathogens and reduces the growth rate of surviving animals. IBDV is a double-stranded RNA (dsRNA) virus. Its two genome segments are encapsidated together with multiple copies of the viral RNA-dependent RNA polymerase, VP1, in a single-shelled capsid that is composed of VP2 and VP3. The other (non-structural) viral proteins of IBDV are VP4, a protease, and VP5, which is not essential for viral replication in vitro but important for virus-induced pathogenicity . Viral proteins generally function by interactions with viral and/or host cell proteins. Information about these interactions is thus essential for understanding the infection process. The aim of this thesis therefore was to initiate making the inventory of interactions essential to the IBDV life cycle. To this end, we employed the then recently developed yeast two-hybrid system. First, we focussed on the homo- and heteromeric interactions of the known viral proteins. A heterologous interaction between VP1 and VP3, and homologous interactions of VP2, VP3, VP4, VP5, and possibly of VP1, were found by co-expression of the fusion proteins in Saccharomyces cerevisiae. The presence of the VP1-VP3 complex in IBDV-infected cells was confirmed by co-immunoprecipitation studies. Kinetic analyses showed that the complex of VP1 and VP3 is formed in the cytoplasm and eventually is released into the cell-culture medium, indicating that VP1-VP3 complexes are present in mature virions. Then, we identified the domains responsible for these interactions. The VP1 interacting domain of VP3 was mapped to the extreme carboxy-terminal domain of the polypeptide. This interaction appeared to be crucial for the production of infectious progeny. These investigations also revealed that VP3 additionally binds to both the viral dsRNA segments. The mapping of the individual contact sites on the self-interacting viral proteins VP2, VP3, VP4 and VP5 revealed that VP2 possesses multiple interaction domains, consistent with available structural information about this external capsid protein. VP3-VP3 interactions were mapped to the amino-terminal part of the polypeptide. No interaction sites could be assigned to the VP4 protein; any deletion applied abolished its self-association. And for VP5, one interaction domain was detected in its central, most hydrophobic region, supporting the idea that this virulence determinant may function as a membrane pore-forming protein in infected cells. Finally, we performed a yeast two-hybrid search for candidate cellular proteins of the bursa of Fabricius interacting with VP1, VP2, VP3 and VP5. We found that several host cell proteins are able to form complexes with the viral proteins of IBDV in yeast cells. A specific interaction of VP1 with the carboxy-terminal domain of eukaryotic initiation factor 4AII (eIF4AII), a key component in the initiation of eukaryotic translation of both capped and uncapped mRNAs, was confirmed by co-immunoprecipitation. The biological relevance of the potential VPg-eIF4AII interaction is discussed. It is now clear that VP3 is the key organizer of the birnavirus structure as it maintains critical interactions with all components of the viral particle: itself, VP2, VP1 and the two genomic dsRNAs. Different domains in the protein are responsible for these different interactions. The association of eIF4AII with IBDV VP1 will require future studies to confirm and establish the functional significance of this interaction for viral multiplication.