VIRUS-LIKE PARTICLE-INDUCED FUSION FROM WITHOUT IN TISSUE-CULTURE CELLS - ROLE OF OUTER-LAYER PROTEINS VP4 AND VP7

We recently described an assay that measures fusion from without induc ed in tissue culture cells by rotavirus, a nonenveloped, triple-protei n-layered member of the Reoviridae family (M. M. Falconer, J. M. Gilbe rt, A. M. Roper, H. B. Greenberg, and J. S. Gavora, J. Virol. 69:5582- 5591, 1995). The conditions required for syncytium formation are simil ar to those for viral penetration of the plasma membrane during the co urse of viral infection of host cells, as the presence of the outer-la yer proteins VP4 and VP7 and the cleavage of VP4 are required. Here we present evidence that virus-like particles (VLPs) produced in Spodopt era frugiperda Sf-9 cells from recombinant baculoviruses expressing th e four structural proteins of rotavirus can induce cell-cell fusion to the same extent as native rotavirus. This VLP-mediated fusion activit y was dependent on trypsinization of VP4, and the strain-specific phen otype of individual VP4 molecules was retained in the syncytium assay similar to what has been seen with reassortant rotaviruses. We show th at intact rotavirus and VLPs induce syncytia with cells that are permi ssive to rotavirus infection whereas nonpermissive cells are refractor y to syncytium formation. This finding further supports our hypothesis that the syncytium assay accurately reflects very early events involv ed in viral infection and specifically the events related to viral ent ry into the cell. Our results also demonstrate that neither viral repl ication nor rotavirus proteins other than VP2, VP6, VP4, and VP7 are r equired for fusion and that both VP4 and VP7 are essential. The combin ation of a cell-cell fusion assay and the availability of recombinant VLPs will permit us to dissect the mechanisms of rotavirus penetration into host cells.