Vaccinia Virus Replication I. Requirement for the Host-Cell Nucleus

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Abstract
Cytochalasin B-induced enucleation techniques were used to examine the ability of vaccinia virus to replicate in the absence of the host-cell nucleus in several mammalian cell lines. Virus-infected enucleated cells (cytoplasts) prepared from BSC-40 [African green monkey kidney], CVC and L [mouse fibroblast] 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 [35S]methionine pulse-labeled proteins synthesized in vaccinia virus-infected cytoplasts demonstrated that early and late viral gene products were expressed at high levels and with the proper temporal sequence. Vaccinia virus cytoplasmic DNA synthesis, as measured by [3H]thymidine incorporation, peaked at 3 h postinfection and was 70-90% of control levels in cytoplasts. 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 .mu.g of rifampin/ml from 0-8 h to increase the pools of viral precursors, followed by subsequent removal of the drug, resulted in a 3-fold increase virus yield. This treatment had no effect on virus-infected cytoplasts. Vaccinia virus morphogenesis was studied by EM in thin sections of virus-infected cells and cytoplasts prepared at various times during a single-step growth cycle. Although early virus morphogenetic forms appeared, there was no subsequent DNA condensation or particle maturation in the cytoplasts. Vaccinia virus probably requires some factor or function from the host-cell nucleus in order to mature properly and produce infectious progeny virus.