ABSTRACT
Satellite RNA ofBaMV (satBaMV), a single-stranded positive-sense RNA of 836 nucleotide [excluding 3′ poly(A) tail]containing a single open reading frame which encodes a nonstructural protein of 20 ku (P20), depends on BaMV for its replication and encapsidation. P20 is a nucleic-acid-binding protein, which facilitates the long distance movement of satBaMV in plants.Protein-protein interactions were analyzed by a bacterial two-hybrid system and pull-down assays to investigate whether P20 could self-associate and/or interact with the helper virus proteins. Self-interaction of P20 was the strongest among the viral protein-protein interactions detected in vivo and in vitro. Significant interactions of P20 with methyltransferase (MET) and capsid protein (CP) of BaMV were evidenced. Interactions among triple gene block protein 1, 2 and 3 (TGBp1, TGBp2 and TGBp3) of BaMV were also significant. BaMV CP also exhibited a strong self-interaction and strong affinity to TGBp1, TGBp2 and TGBp3. By deletion analysis,the minimal region delineated for the self-interaction of P20 was the N-terminal 15 amino acids which include the RNA binding motif of P20. N-terminal deletion resulted in the loss of self-interaction of P20. Deletion analysis also confirmed the importance of β-sheet structure of P20 in the P20-P20 interaction. P20 showed significant interactions with two host (Nicotiana benthamiana) proteins,cytochrome-C reductase and β-tubulins. The homotypic and heterotypic interactions of BaMV proteins and P20 in vivo thus suggest that the protein-protein interactions may directly exert an effect on the BaMV and satBaMV RNA movement as a protein complex in the infected host plants.