ASSEMBLY OF VIROPLASM AND VIRUS-LIKE PARTICLES OF ROTAVIRUS BY A SEMLIKI-FOREST-VIRUS REPLICON

In this study we have used an expression system based on Semliki Fores t virus (SFV) to study assembly and intracellular localization of cert ain capsid proteins of rotavirus in neurons and mammalian epithelial c ells. The complete genes of vp2 (vp2A) and vp6 (vp6A) of group A rotav irus (SA-11) and gene 5 encoding vp6 (vp6C) of porcine group C rotavir us (strain Cowden/AmC-1) were inserted into an SFV expression replicon . Transfection of BHK-21 cells with in vitro-made SFV transcripts resu lted in a high level of expression of the heterologous genes. Cotransf ection with helper RNA encoding the SN structural proteins, but lackin g the genomic RNA packing signal, resulted in production of recombinan t infectious virus. Immunological and biochemical analysis revealed th at vp6 was expressed to high levels in primary neurons and mammalian e pithelial cells and that vp6 was retained as an authentic homotrimer, stabilized by noncovalent interactions with native antigenic determina nts. Thin section electron microscopy analysis revealed that vp6 alone assembled into viroplasm-like structures in the cytoplasm. While coex pression of vp2 and vp6 of group A rotavirus resulted in formation of single-shelled-like particles, no evidence of intracellular assembly w as found, suggesting that other viral proteins are required for intrac ellular formation of single-shelled particles. A notable observation w as that the vp6 proteins of group A and C rotaviruses showed different immunofluorescence patterns in BHK-21 cells; vp6C displayed an intens e punctate immunofluorescence pattern, while vp6A was characterized by a pronounced filamentous staining in close vicinity to the cytoskelet on. (C) 1998 Academic Press.