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...

A rapid and specific technique to detect polyriboadenylic acid sequences in RNA is described. The method depends upon the ability of RNAs that contain poly(A) sequences to associate specifically with poly(U) that has been immobilized on fiberglass filters by ultraviolet irradiation. A high proportion of the transcripts synthesized in vivo and in vitro from the vaccinia virus genome contain poly(A) sequences and bind to the poly(U) filters. Similarly, DNA-like RNA from the nucleus and from the cytoplasmic polyribosomes of HeLa cells is rich in species that bind to poly(U) filters. Poly(U) immobilized on cellulose powder is useful to make columns with a high capacity for the binding and purification of poly(A)-containing RNAs.

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Using cytochalasin B-induced enucleation techniques, we examined the ability of vaccinia virus to replicate in the absence of the host-cell nucleus in several mammalian cell lines. It was found that virus-infected enucleated cells (cytoplasts) prepared from BSC-40, CVC, and L cells were incapable of producing infectious progeny virus. The nature of this apparent nuclear involvement was studied in detail in BSC-40 cells. Modulations designed to maximize cytoplast integrity and longevity, such as reduction of the growth temperature and initial multiplicity of infection, did not improve virus growth in cytoplasts. Sodium dodecyl sulfate-polyacrylamide gel analysis of the [ 35 S]methionine pulse-labeled proteins synthesized in vaccinia virus-infected cytoplasts demonstrated that both early and late viral gene products were being expressed at high levels and with the proper temporal sequence. Vaccinia virus cytoplasmic DNA synthesis, as measured by [ 3 H]thymidine incorporation, peaked at 3 h postinfection and was 70 to 90% of control levels in cytoplasts. However, in the cytoplasts this DNA was not converted to a DNase-resistant form late in infection, which was consistent with the failure to isolate physical particles from infected cytoplasts. Treatment of vaccinia virus-infected cells with 100 μg of rifampin/ml from 0 to 8 h to increase the pools of viral precursors, followed by subsequent removal of the drug, resulted in a threefold increase virus yield. This treatment had no effect on virus-infected cytoplasts. Finally, vaccinia virus morphogenesis was studied under an electron microscope in thin sections of virus-infected cells and cytoplasts which had been prepared at various times during a single-step growth cycle. It was apparent that, although early virus morphogenetic forms appeared, there was no subsequent DNA condensation or particle maturation in the cytoplasts. These results suggest that vaccinia virus requires some factor or function from the host-cell nucleus in order to mature properly and produce infectious progeny virus.