Mapping the assembly pathway of Bluetongue virus scaffolding protein VP3.
The structure of the Bluetongue virus (BTV) core and its outer layer VP7 has been solved by X-ray crystallography, but the assembly intermediates that lead to the inner scaffolding VP3 layer have not been defined. In this report, we addressed two key questions: (a) the role of VP3 amino terminus in core assembly and its interaction with the transcription complex (TC) components; and (b) the assembly intermediates involved in the construction of the VP3 shell. To do this, deletion mutants in the amino terminal and decamer-decamer interacting region of VP3 (DeltaDD) were generated, expressed in insect cells using baculovirus expression systems, and their ability to assemble into core-like particles (CLPs) and to incorporate the components of TC were investigated. Deletion of the N-terminal 5 (Delta5N) or 10 (Delta10N) amino acids did not affect the ability to assemble into CLPs in the presence of VP7 although the cores assembled using the 10 residue mutant (Delta10N) deletion were very unstable. Removal of five residues also did not effect incorporation of the internal VP1 RNA polymerase and VP4 mRNA capping enzyme proteins of the TC. Removal of the VP3-VP3 interacting domain (DeltaDD) led to failure to assemble into CLPs yet retained interaction with VP1 and VP4. In solution, purified DeltaDD mutant protein readily multimerized into dimers, pentamers, and decamers, suggesting that these oligomers are the authentic assembly intermediates of the subcore. However, unlike wild-type VP3 protein, the dimerization domain-deleted assembly intermediates were found to have lost RNA binding ability. Our study emphasizes the requirement of the N-terminus of VP3 for binding and encapsidation of the TC components, and defines the role of the dimerization domain in subcore assembly and RNA binding.
Kar
Alak Kanti
AK
Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Ghosh
Mrinal
M
Roy
Polly
P
D001820:Bluetongue virus / Q000502:physiology* / Q000648:ultrastructure; D036022:Capsid Proteins / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D019281:Dimerization; D009154:Mutation; D011994:Recombinant Proteins / Q000096:biosynthesis; D020079:Terminal Repeat Sequences; D014158:Transcription, Genetic; D014758:Viral Core Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D019065:Virus Assembly
D036022:Capsid Proteins; D011994:Recombinant Proteins; C074954:VP3 protein, Bluetongue virus; D014758:Viral Core Proteins; C066862:VP7 protein, orbivirus; D012321:DNA-Directed RNA Polymerases