Homotypic interactions of the infectious bursal disease virus proteins VP3, pVP2, VP4, and VP5: mapping of the interacting domains.

Infectious bursal disease virus (IBDV), a nonenveloped double-stranded RNA virus of chicken, encodes five proteins. Of these, the RNA-dependent RNA polymerase (VP1) is specified by the smaller genome segment, while the large segment directs synthesis of a nonstructural protein (VP5) and a structural protein precursor from which the capsid proteins pVP2 and VP3 as well as the viral protease VP4 are derived. Using the recently redefined processing sites of the precursor, we have reevaluated the homotypic interactions of the viral proteins using the yeast two-hybrid system. Except for VP1, which interacted weakly, all proteins appeared to self-associate strongly. Using a deletion mutagenesis approach, we subsequently mapped the interacting domains in these polypeptides, where possible confirming the observations made in the two-hybrid system by performing coimmunoprecipitation analyses of tagged protein constructs coexpressed in avian culture cells. The results revealed that pVP2 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. Interestingly, this domain is distinct from two other interaction domains occurring in this internal capsid protein: while binding to VP1 has been mapped to the carboxy-terminal end of the protein, interaction with the genomic dsRNA segments has been suggested to occur just upstream thereof. No interaction sites could be assigned to the VP4 protein; any deletion applied abolished its self-association. Finally, one interaction domain was detected in the central, most hydrophobic region of VP5, supporting the idea that this virulence determinant may function as a membrane pore-forming protein in infected cells.