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

Dennis E. Hruby, Linda A. Guarino, Joseph R. Kates
1979 Journal of Virology  
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
more » ... reduction of the growth temperature and initial multiplicity of infection, did not improve virus growth in cytoplasts. Sodium dodecyl sulfatepolyacrylamide gel analysis of the [35S]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 [3H]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 ,jg 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. Vaccinia virus, a large complex DNA-containing poxvirus, has been thought to replicate solely in the cytoplasm of susceptible host cells (23). This idea was based on several lines of evidence: vaccinia virus contains or codes for many of the enzymes necessary for DNA synthesis, transcription, and modification (18); virus replicative factories are located exclusively in the cytoplasm (11); and DNA synthesis, as measured by autoradiography, occurs in vaccinia virus-infected enucleated L cells (24). However, other experiments have shown that pretreatment of host cells with either mitomycin C or actinomycin D to inhibit host nuclear functions causes a marked diminution in subsequent vaccinia virus growth (15, 26) . Also, there have been indications that some virus-specific DNA synthesis may occur within the nucleus of poxvirus-infected cells (9, 13, 29) . Pennington and Follett (22) studied the replication of vaccinia virus in enucleated BSC-1 cells and found that the production of infectious progeny virus was completely blocked. This was accompanied by a considerable reduction in the levels ofvirus-specific DNA synthesis, protein synthesis, and mature particle formation, and also in the size and number of virus factories. Similar observations have been made concerning the inability of an iridovirus, African swine fever virus, to replicate in Vero cytoplasts (20) . It should be pointed out that replication of cytoplasmic RNA viruses such as vesicular stomatitus virus and Semliki Forest virus is essentially normal in enucleated cells (8). With the recent evidence demonstrating that another group of cytoplasmic DNA viruses, the icosahedral viruses such as frog virus 3, require the nucleus for viral DNA replication and transcription (10), it was of interest to reexamine the so-called cytoplasmic mode of poxvirus replication and the relationship of the apparent nuclear 705 on May 10, 2020 by guest
doi:10.1128/jvi.29.2.705-715.1979 fatcat:ffga42w7zbhdrm7sfwqjr7nud4