Interaction between the replicase proteins of Tomato bushy stunt virus in vitro and in vivo.
Tomato bushy stunt virus (TBSV), a tombusvirus with a non-segmented, plus-stranded RNA genome, codes for p33 and p92 replicase proteins. The sequence of p33 overlaps with the N-terminal domain of p92, which also contains the signature motifs of RNA-dependent RNA polymerases (RdRps) in its non-overlapping C-terminal portion. In this research, we demonstrate in vitro interactions between p33:p33 and p33:p92 using surface plasmon resonance analysis with purified recombinant p33 and p92. The sequence in p33 involved in the above protein-protein interactions was mapped to the C-terminal region, which also contains an RNA-binding site. Using the yeast two-hybrid assay, we confirmed that two short regions within p33 could promote p33:p33 and p33:p92 interactions in vivo. Mutations in either p33 or p92 within the short regions involved in p33:p33 and p33:p92 interactions decreased the replication of a TBSV defective interfering RNA in yeast, a model host, supporting the significance of these protein interactions in tombusvirus replication.
Department of Plant Pathology, University of Kentucky, Lexington 40546, USA.
K S
KS
Rajendran
Peter D
PD
Nagy
D000595:Amino Acid Sequence; D001665:Binding Sites; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance; D017864:Tombusvirus / Q000201:enzymology / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D014779:Virus Replication / Q000235:genetics; D015003:Yeasts
D012324:RNA-Dependent RNA Polymerase