Assembly of highly infectious rotavirus particles recoated with recombinant outer capsid proteins.
Assembly of the rotavirus outer capsid is the final step of a complex pathway. In vivo, the later steps include a maturational membrane penetration that is dependent on the scaffolding activity of a viral nonstructural protein. In vitro, simply adding the recombinant outer capsid proteins VP4 and VP7 to authentic double-layered rotavirus subviral particles (DLPs) in the presence of calcium and acidic pH increases infectivity by a factor of up to 10(7), yielding particles as infectious as authentic purified virions. VP4 must be added before VP7 for high-level infectivity. Steep dependence of infectious recoating on VP4 concentration suggests that VP4-VP4 interactions, probably oligomerization, precede VP4 binding to particles. Trypsin sensitivity analysis identifies two populations of VP4 associated with recoated particles: properly mounted VP4 that can be specifically primed by trypsin, and nonspecifically associated VP4 that is degraded by trypsin. A full complement of properly assembled VP4 is not required for efficient infectivity. Minimal dependence of recoating on VP7 concentration suggests that VP7 binds DLPs with high affinity. The parameters for efficient recoating and the characterization of recoated particles suggest a model in which, after a relatively weak interaction between oligomeric VP4 and DLPs, VP7 binds the particles and locks VP4 in place. Recoating will allow the use of infectious modified rotavirus particles to explore rotavirus assembly and cell entry and could lead to practical applications in novel immunization strategies.
Trask
Shane D
SD
Children's Hospital, Enders 673, 320 Longwood Ave., Boston, MA 02115, USA.
Dormitzer
Philip R
PR
D000818:Animals; D000956:Antigens, Viral / Q000235:genetics / Q000378:metabolism*; D002213:Capsid / Q000378:metabolism*; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D051379:Mice; D046529:Microscopy, Electron, Transmission; D016253:Microscopy, Immunoelectron; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012401:Rotavirus / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology / Q000648:ultrastructure; D014771:Virion / Q000378:metabolism / Q000648:ultrastructure; D019065:Virus Assembly / Q000235:genetics
D000956:Antigens, Viral; D036022:Capsid Proteins; D011994:Recombinant Proteins; C060339:VP4 protein, Rotavirus; C050779:VP7 protein, Rotavirus