In a recent communication we presented experimental evidence that poxvirus genes coding for early functions were transcribed from protein-coated viral genomes (poxvirus cores) in vivo. ' We demonstrated that poxvirus-specific RNA was synthesized by cores under conditions where protein synthesis had been arrested prior to virus infection of cultured HeLa cells. Therefore the DNA-dependent RNA polymerase utilized by the virus either pre-exists in the uninfected cell and becomes associated with the viral core after infection, or the RNA polymerase is an integral part of the poxvirus virion.The goals of the present study, therefore, were as follows: (1) to prepare poxvirus cores in a purified state and to study their ability to synthesize virusspecific RNA under controllable in vitro conditions, and (2) to see if a DNA-dependent RNA polymerase is an integral part of the poxvirus virion.Materials and Methods.-Virus infection procedure: HeLa S3 cells were grown in spinner cultures and the Utrecht strain of rabbit poxvirus (RP) was used exclusively. The virological procedures have been described previously.2 3Preparation of the cytoplasmic fraction from infected cells: One liter of a growing culture of HeLa cells (4 X 106 cells per ml) was treated with streptovitacin A (15 pg/ml) 30 min prior to infection with RP virus (50 PFU per cell). Following virus adsorption the infected cells were incubated in the presence of streptovitacin A for an additional 60 min. The infected cells were then harvested by centrifugation, washed once with standard saline solution, and resuspended at a concentration of 2 X 107 cells/ml in a solution of 10-2 M Tris-HCl, pH 9.0, containing 5 X 10-3 M MgCl2, 10-2 M KC1, and 5 X 10-3 M 2-mercaptoethanol. The cells were allowed to swell in this solution at 5oC for 5 min prior to disruption with a Dounce homogenizer. A nuclearfree cytoplasmic fraction was then prepared by centrifugation as previously described.' Poxvirus cores were purified from the crude cytoplasmic fraction described above according to the following procedure:(1) Aliquots (5 ml) of the infected cytoplasmic fraction were sonicated briefly (15 sec, MSE ultrasonic disintegrator at full power) and each was then layered over 26 ml of a 36% w/v solution of sucrose in 0.05 M Tris-HCl, pH 8.5, containing 5 X 10-3 M 2-mercaptoethanol. These suspensions were centrifuged in the SW 25.1 head of the Spinco model L ultracentrifuge (80 min, 50C, 15,000 rpm). After centrifugation the supernatant solution was carefully removed with a Pasteur pipette and the pellet was resuspended in 0.05 M Tris-HCl, pH 8.5, containing 5 X 10-3 M 2-mercaptoethanol and sonicated briefly. The centrifugation through 36% sucrose was repeated and the pellet was resuspended as described above.(2) Occasionally poxvirus cores were further purified by banding in a 25-45% w/v linear sucrose gradient containing 0.05 M Tris-HCl, pH 8.5, and 5 X 10-3 M 2-mercaptoethanol. The sucrose gradient was centrifuged in the Spinco 25.1 SW head (15,000 rpm, 45 min, 50C). A major opalescen...

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The uncoating of the poxvirus is a process of considerable interest since it requires protein synthesis,1' 2 yet it takes place when the viral genome is believed to be inactive. It has been postulated that uncoating is mediated by proteins coded by the host cell genome, and induced by incoming virus particles.2' 3 The observation that actinomycin D inhibits uncoating is consistent with the hypothesis that a gene is derepressed.2' 3

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