In vitro reconstitution of an intermediate assembly stage of vaccinia virus

A novel method is described which facilitates the in vitro assembly of one step in the life cycle of vaccinia virus, the formation of the spherical immature virus (IV). For this, advantage was taken of the ability of rifampicin to reversibly block the assembly of the IV. Rifampicin-treated, vaccinia virus-infected HeLa cells were permeabilized with streptolysin O (SLO) and the endogenous cytosol was allowed to exit the cells at 4 degrees . Subsequently, exogenous cytosol from infected or uninfected HeLa cells as well as an ATP-regenerating system were added and the cells were incubated for different times at 37 degrees in the absence of rifampicin. The preparations were then evaluated by thin section EM. Our data show that in the presence of infected or uninfected cell cytosol and ATP a significant fraction of cells could reconstitute IV assembly in vitro. Under no conditions were we able to reconstitute any later stages of assembly. The potential of this system for the in vitro reconstitution of viral assembly in general is discussed.