Intracellular distribution of rotavirus structural proteins and virus-like particles expressed in the insect cell-baculovirus system.
The production of virus-like particles (VLP) is of interest to several fields. However, little is known about their assembly when they are expressed in insect cells, as it occurs in conditions different to those of native virus. Knowledge of the localization of recombinant proteins and of the site of accumulation of VLP can increase the understanding of VLP assembly and be useful for proposing production strategies. In this work, the rotavirus proteins VP6 and the fusion protein GFPVP2 were expressed in High Five insect cells. Recombinant proteins and rotavirus-like particles (RLP) were located and visualized by confocal, epifluorescence and electron microscopy. Single-layered (sl) RLP (conformed by GFPVP2) accumulated in the cytoplasm as highly ordered aggregates. In contrast, VP6 formed fibrillar structures composed of various tubes of VP6 that were not associated to microtubules. Coexpression of GFPVP2 and VP6 altered the distribution of both proteins. VP6 formed aggregates, even when all other conditions of individual protein expression remained unchanged. Double-layered (dl) RLP were observed in dense zones of the cytoplasm, but were not in ordered aggregates. It was determined that the assembly of both slRLP and dlRLP occurs intracellularly. Accordingly, strategies for the optimum assembly of dlRLP should guarantee that each cell produces both recombinant proteins.
Mena
Jimmy A
JA
Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apdo. Postal 510-3, Cuernavaca, Morelos CP 62250, Mexico.
Ramírez
Octavio T
OT
Palomares
Laura A
LA
D000818:Animals; D000956:Antigens, Viral / Q000378:metabolism*; D016367:Baculoviridae / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005822:Genetic Vectors / Q000378:metabolism; D007313:Insecta / Q000166:cytology / Q000378:metabolism / Q000821:virology; D008854:Microscopy, Electron; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012401:Rotavirus / Q000201:enzymology / Q000378:metabolism*
D000956:Antigens, Viral; D036022:Capsid Proteins; D011994:Recombinant Proteins; C442545:VP2 protein, Rotavirus; C061332:VP6 protein, Rotavirus