Kinetics and functional studies on interaction between the replicase proteins of Tomato Bushy Stunt Virus: requirement of p33:p92 interaction for replicase assembly.
The assembly of the functional replicase complex via protein:protein and RNA:protein interactions among the viral-coded proteins, host factors and the viral RNA on cellular membranes is a key step in the replication process of plus-stranded RNA viruses. In this work, we have characterized essential interactions between p33:p33 and p33:p92 replication proteins of Tomato bushy stunt virus (TBSV), a tombusvirus with a non-segmented, plus-stranded RNA genome. Surface plasmon resonance (SPR) measurements with purified recombinant p33 and p92 demonstrate that p33 interacts with p92 in vitro and that the interaction requires the S1 subdomain, whereas the S2 subdomain plays lesser function. Kinetic SPR analyses showed that binding of S1 subdomain to the C-terminal half of p33 takes place with moderate binding affinity in the nanomolar range whereas S2 subdomain binds to p33 with micromolar affinity. Using mutated p33 and p92 proteins, we identified critical amino acid residues within the p33:p92 interaction domain that play essential role in replication and the assembly of the tombusviral replicase. In addition, we show that interaction takes place between replication proteins of TBSV and the closely related Cucumber necrosis virus but not between TBSV and the more distantly related Turnip crinkle virus, suggesting that selective protein interactions might prevent the assembly of chimeric replicases carrying replication proteins from different viruses during mixed infections.
Rajendran
K S
KS
Department of Plant Pathology, University of Kentucky, 201F Plant Science Building, Lexington, KY 40546, USA.
Nagy
Peter D
PD
D019943:Amino Acid Substitution; D015152:Blotting, Northern; D047468:Immunoprecipitation; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000502:physiology*; D020349:Surface Plasmon Resonance; D017864:Tombusvirus / Q000502:physiology*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D015003:Yeasts / Q000737:chemistry
D021122:Protein Subunits; D012333:RNA, Messenger; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D012324:RNA-Dependent RNA Polymerase