Deletion mapping of functional domains in the rotavirus capsid protein VP6.
VP6, the major capsid protein of rotavirus, oligomerizes into trimers that constitute the intermediate shell of the virions. In order to map functional domains in this protein, we introduced seven internal in-frame deletions within the coding region of gene 6 of human rotavirus strain Wa. Regions of homology among the VP6 proteins of group A and group C rotaviruses were targeted for deletion mutagenesis. The mutant VP6 proteins were expressed in mammalian cells using the recombinant vaccinia virus system and were examined for their ability to oligomerize into trimers as well as to assemble into double-layered virus-like particles upon coexpression with the rotavirus core protein VP2. Deletions that abolished trimerization defined a domain (residues 246 to 314) that maps within a larger region previously found to be critical for oligomerization (amino acids 105 to 328). When the capacity of each mutant to assemble into double-layered virus-like particles was analysed, three different assembly phenotypes were observed. Phenotype I was represented by two deletion mutants lacking residues 246 to 250 and 308 to 314 that produced particles with efficiencies similar to that of wild-type VP6. Phenotype II, characterized by a moderate decrease in the efficiency of particle assembly with respect to that of wild-type VP6, included two mutants with deletions at the C terminus of the protein. Phenotype III was exhibited by three mutants whose abilities to assemble into double-layered virus-like particles were drastically impaired. Two of these mutants define a previously unidentified assembly domain (amino acids 122 to 147) at the N terminus of rotavirus VP6.
Affranchino
J L
JL
Centro de Virología Animal (CEVAN-CONICET), Buenos Aires, Argentina. jluis@cevan.sld.ar
González
S A
SA
D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral; D002213:Capsid / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D036022:Capsid Proteins; D002460:Cell Line; D002522:Chlorocebus aethiops; D005822:Genetic Vectors; D006801:Humans; D046911:Macromolecular Substances; D008322:Mammals; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012401:Rotavirus / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D014162:Transfection; D014616:Vaccinia virus
D000956:Antigens, Viral; D036022:Capsid Proteins; D046911:Macromolecular Substances; D011994:Recombinant Proteins; C061332:VP6 protein, Rotavirus