The C-terminal domain of rotavirus NSP5 is essential for its multimerization, hyperphosphorylation and interaction with NSP6.
Rotavirus NSP5 is a non-structural phosphoprotein with putative autocatalytic kinase activity, and is present in infected cells as various isoforms having molecular masses of 26, 28 and 30-34 kDa. We have previously shown that NSP5 forms oligomers and interacts with NSP6 in yeast cells. Here we have mapped the domains of NSP5 responsible for these associations. Deletion mutants of the rotavirus YM NSP5 were constructed and assayed for their ability to interact with full-length NSP5 and NSP6 using the yeast two-hybrid assay. The homomultimerization domain was mapped to the 20 C-terminal aa of the protein, which have a predicted alpha-helical structure. A deletion mutant lacking the 10 C-terminal aa (DeltaC10) failed to multimerize both in yeast cells and in an in vitro affinity assay. When transiently expressed in MA104 cells, NSP5 became hyperphosphorylated (30-34 kDa isoforms). In contrast, the DeltaC10 mutant produced forms equivalent to the 26 and 28 kDa species, but was poorly hyperphosphorylated, suggesting that multimerization is important for this proposed activity of the protein. The interaction domain with NSP6 was found to be present in the 35 C-terminal aa of NSP5, overlapping the multimerization domain of the protein, and suggesting that NSP6 might have a regulatory role in the self-association of NSP5. NSP6 was also found to interact with wild-type NSP5, but not with its mutant DeltaC10, in cells transiently transfected with plasmids encoding these proteins, confirming the relevance of the 10 C-terminal aa for the formation of the heterocomplex.
Torres-Vega
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MA
Departamento de Genética y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, Cuernavaca, Morelos 62250, Mexico.
González
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RA
Duarte
M
M
Poncet
D
D
López
S
S
Arias
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CF
D000818:Animals; D002460:Cell Line; D005814:Genes, Viral; D006801:Humans; D008970:Molecular Weight; D010766:Phosphorylation; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012401:Rotavirus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017384:Sequence Deletion; D020798:Two-Hybrid System Techniques; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C071649:gene 11 protein, Rotavirus B