Identiﬁcation and Molecular Characterization of the RNA Polymerase-Binding Motif of Infectious Bursal Disease Virus Inner Capsid Protein VP3

Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is the causative agent of one of the most important infectious poultry diseases. Major aspects of the molecular biology of IBDV, such as assembly and replication, are as yet poorly understood. We have previously shown that encapsidation of the putative virus-encoded RNA-dependent RNA polymerase VP1 is mediated by its interaction with the inner capsid protein VP3. Here, we report the characterization of the VP1-VP3 interaction. RNase A treatment of VP1- and VP3-containing extracts does not affect the formation of VP1-VP3 complexes, indicating that formation of the complex requires the establishment of protein-protein interactions. The use of a set of VP3 deletion mutants allowed the mapping of the VP1 binding motif of VP3 within a highly charged 16-amino-acid stretch on the C terminus of VP3. This region of VP3 is sufﬁcient to confer VP1 binding activity when fused to an unrelated protein. Furthermore, a peptide corresponding to the VP1 binding region of VP3 speciﬁcally inhibits the formation of VP1-VP3 complexes. The presence of Trojan peptides containing the VP1 binding motif in IBDV-infected cells speciﬁcally reduces infective virus production, thus showing that formation of VP1-VP3 complexes plays a critical role in IBDV replication. Competition assays with synthetic peptides. The synthetic peptides used in our study were H 2 N-GRLGRWIRTVSDEDLE-COOH (GRL) and H N-GALGA WIATVSDEDLE-COOH (GAL). GRL contains the 16 C-terminal residues of the VP3 protein. The sequence of GAL, used as a control for the competition assays, is identical to that of GRL, with the exception that the arginine residues have been replaced by alanine residues. Both peptides were puriﬁed by high-pressure liquid chromatography and analyzed for concentration and composition to verify their identity. For the competition assays, increasing amounts of each peptide were added to the VP1-VP3 complex in the in vitro binding assays. assays were as above. of replication with synthetic peptides. The synthetic peptides study were Biot-H 2 N-RQILIWFQNRRMKWKKGRLGRWIRT VSDEDLE-COOH (GRL-biot) and Biot-H 2 N-RQILIWFQNRRMKWKKGA LGAWIATVSDEDLE-COOH (GAL-biot). The 16 N-terminal residues of both peptides contain the penetratin peptide sequence corresponding to the cell internalization motif of the antennapedia protein from Drosophila melanogaster (7). This sequence facilitates the internalization of the peptides into IBDV- susceptible cells. The 16 C-terminal residues of GRL-biot and GAL-biot are identical to those described for the oligopeptides GRL and GAL, respectively. Both peptides were biotinylated to allow their detection by immunoﬂuorescence with a rhodamine-streptavidin conjugate. Peptides were puriﬁed by high-pres- sure liquid chromatography and analyzed for concentration and composition to verify their identity. Peptides were resuspended at a concentration of 5 mM in dimethyl sulfoxide. BSC-1 cell monolayers, grown in 60-mm-diameter dishes, were washed twice with phosphate-buffered saline, and then 1 ml of DMEM containing various peptide concentrations was added. As a control for these