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

Abstract

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.

The coronavirus avian infectious bronchitis virus requires the cell nucleus and host transcriptional factors

“…The generation of pyrimidine dimers in nucleic acid molecules by this treatment is known to block normal host DNA replication and RNA transcription, and the uv doses employed in our experiments would be expected to strongly inhibit both DNA and RNA synthesis (cf. Hruby et al, 1979). Thus, although uv irradiation simultaneously inhibits a number of cellular biosynthetic activities, it is clear that radiationdamaged BHK-21 cells with functionally impaired nuclei are unable to support active replication of infectious IBV particles.…”

“…We have also examined ultraviolet radiation as an inhibitor of cellular DNA function which is known to compromise replication of animal viruses such as influenza (Barry, 1964;Rott et al, 1965) or vaccinia virus (Hruby et al, 1979) to establish whether growth of IBV is also sensitive to these treat-…”

Template-Dependent Initiation of Sindbis Virus RNA Replication In Vitro

“…Viruses and cells. vTF7-3, a vaccinia virus recombinant which expresses the T7 polymerase (7), was propagated as previously described (12). The growth conditions for BSC40 cells (12) and BHK-21 cells (16) have been described previously.…”

Molecular dissection of cis-acting regulatory elements from 5'-proximal regions of a vaccinia virus late gene cluster

“…Cells and virus. Growth, purification, and plaque titration of VV (strain WR) were carried out essentially as previously described (17). BSC-40 cells were grown in Earle minimum essential medium (GIBCO Laboratories), supplemented with 10% heat-inactivated fetal bovine serum, 2 mM Lglutamine at 37°C, 5% C02, and 95% humidity.…”