Characterization of rotavirus NSP2/NSP5 interactions and the dynamics of viroplasm formation.
Viroplasms are discrete structures formed in the cytoplasm of rotavirus-infected cells and constitute the replication machinery of the virus. The non-structural proteins NSP2 and NSP5 localize in viroplasms together with other viral proteins, including the polymerase VP1, VP3 and the main inner-core protein, VP2. NSP2 and NSP5 interact with each other, activating NSP5 hyperphosphorylation and the formation of viroplasm-like structures (VLSs). We have used NSP2 and NSP5 fused to the enhanced green fluorescent protein (EGFP) to investigate the localization of both proteins within viroplasms in virus-infected cells, as well as the dynamics of viroplasm formation. The number of viroplasms was shown first to increase and then to decrease with time post-infection, while the area of each one increased, suggesting the occurrence of fusions. The interaction between NSP2 and a series of NSP5 mutants was investigated using two different assays, a yeast two-hybrid system and an in vivo binding/immunoprecipitation assay. Both methods gave comparable results, indicating that the N-terminal region (33 aa) as well as the C-terminal part (aa 131-198) of NSP5 are required for binding to NSP2. When fused to the N and C terminus of EGFP, respectively, these two regions were able to confer the ability to localize in the viroplasm and to form VLSs with NSP2.
Eichwald
Catherine
C
International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy.
Rodriguez
José Francisco
JF
Department of Biología Molecular y Celular, Centro Nacional de Biotecnología, Cantoblanco, 28049 Madrid, Spain.
Burrone
Oscar R
OR
International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy.
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics; D008322:Mammals; D018613:Microscopy, Confocal; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012401:Rotavirus / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D008164:Luminescent Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C079234:NS35 protein, rotavirus; C071649:gene 11 protein, Rotavirus B; D049452:Green Fluorescent Proteins