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Viruses are obligate intracellular parasites that require the host cell replication, transcription, and translation machinery for reproduction. Each viral group provides a unique series of viral-cellular interactions. Studies have provided insight not only into viral replication and control of host functions, but also into cellular functions such as eukaryotic replication, transcription, and translation as well as the regulation of these events. This unit presents a protocol for flow cytometric monitoring of viral infection and quantitating viral-cellular events. The availability of monoclonal and/or polyclonal antibodies directed to both viral and cellular proteins offers the ability to assay a specific molecule in the intact fixed cell and the opportunity to correlate viral events with cellular processes such as progression through the cell cycle.

Simian virus 40 (SV40) infection of quiescent monkey kidney cells stimulates two successive rounds of cellular DNA synthesis without an intervening mitosis. This uncoupling of S phase and mitosis indicates that SV40 modulates pathways regulating the G2-to-M phase transition. To examine the integrity of mitotic initiation pathways in infected cells that have bypassed mitosis, SV40-infected CV-1 cells were treated with okadaic acid (OA), a known inducer of premature mitosis in other cell types. OA treatment triggered the appearance of the mitotic marker MPM-2 in SV40-infected CV-1 cells progressing through either the first (diploid) or second (tetraploid) S phases. These results demonstrate that a subset of mitotic pathways are intact but inactive in SV40-infected cells that have bypassed mitosis and initiated tetraploid S phase.

To determine the role of SV40 T antigen in stimulating multiple rounds of DNA synthesis in permissive cells, CV-1 cells were transfected with plasmids expressing mutant or wt T antigen in the presence or absence of the SV40 origin of replication. Induction of cells with > G2 DNA content (tetraploid DNA content) and levels of T antigen protein were detected and analyzed by flow cytometry. The mutant T antigen proteins demonstrated the expected phenotypes as determined by immunoprecipitation. Elevated levels of T antigen protein were detected in each transfection, but full-length T antigen alone was responsible for the tetraploid DNA content. The studies show that full-length T antigen with point mutations to reduce binding to the cellular proteins p53 and/or Rb were capable of inducing > G2 DNA content though the induction by these mutants was greatly enhanced by the presence of the SV40 origin of replication. Truncated T antigen (aa 1-259) could induce cells with tetraploid DNA content only in the presence of the SV40 origin of replication and the absence of Rb binding. These studies suggest that multiple functions of T antigen are involved in the stimulation of the second round of cellular DNA synthesis.

Previous experiments with the simian virus 40 mutant tsA357R-K (tsA30) demonstrated a T-antigen function that is required for production of cells with a greater-than-G2-phase DNA content. In this study, temperature shift experiments indicated that the temperature-sensitive function of tsA357R-K, which is necessary for entry into the greater-than-G2 phase, is not required in G1 or S but must be supplied in the G2 phase.

Infection of primary to tertiary mouse embryo fibroblasts or mouse kidney cells with polyoma virus leads to stimulation of cellular DNA synthesis. When either confluent or growing mouse cells were infected, the monolayer cells were found to accumulate cells with a DNA content of S and G2/M phases of the cell cycle as assayed by flow cytometry. A similar pattern of DNA content was also observed in cells in the supernatant, which are probably cells replicating virus and dying. When compared with control cells, the infected monolayer and supernatant cells exhibited a population (5-27%) with a > G2 DNA content. The increase in DNA content of these > G2 cells was calculated to be an average of 26.7%, which is probably due to viral DNA. Polyoma contrasts with another papovavirus, SV40, which stimulates cells into DNA synthesis, with the majority of cells attaining a > G2/tetraploid DNA content, suggesting that there are differences in polyploidization between these two viruses.

Simian virus 40 (SV40) infection of monkey kidney cells induces successive rounds of cellular DNA synthesis without intervening mitosis. To gain an understanding of the mechanisms responsible for disruption of cell cycle control during lytic infection, pRB phosphorylation and cell cycle distribution were examined following SV40 infection of CV-1 cells. The hypophosphorylated pRB present in confluent CV-1 cells was phosphorylated within 14 h following SV40 infection. Phosphorylated pRB then remained the predominant form as cells progressed from late G1 through S phase. Hypophosphorylated pRB reappeared as cells moved through G2 and acquired a tetraploid (> G2) DNA content. The reappearance of hypophosphorylated pRB in a population with decreasing numbers of cells in G1 phase and increasing numbers of cells in > G2 suggests that accumulation of hypophosphorylated pRB may be involved in T antigen-induced tetraploidy.

Infection of quiescent CV-1 cells with simian virus 40 mutant tsA30 at 37 degrees C resulted in the induction of two rounds of cellular DNA synthesis in T-antigen-positive cells, as previously described for wild-type simian virus 40. Following infection with tsA30 at 40.5 degrees C, T-antigen-positive cells were induced into S phase and reached a diploid G2 DNA content; however, a second S phase was not initiated. The failure of tsA30-infected CV-1 cells to enter tetraploid S phase at 40.5 degrees C identifies a T-antigen function, distinct from T-antigen functions responsible for stimulation of cell DNA synthesis, which is required for initiation of a second round of DNA synthesis without mitosis.

Doxorubicin does not kill L929 cells at concentrations that profoundly reduce clonogenic survival. Instead, the cell and nuclear volume progressively increase for at least 1 week following drug exposure leading to the production of characteristic giant cells. The increase in nuclear volume is due to continued DNA synthesis and increase in chromosome number without entry into mitosis. The implications of this finding for in vitro chemosensitivity assays and for the mechanism of doxorubicin cytotoxicity are discussed.

Simian virus 40 (SV40) is capable of inducing cellular DNA synthesis in permissive and nonpermissive cells. Utilizing flow cytometry, we analyzed the DNA content changes in two diploid human cell strains and two monkey cell lines. The osteogenesis imperfecta (OI) human skin fibroblasts were induced into DNA synthesis, and within one to two cell generations, a polyploid cell population was produced. With WI-38 phase II cells, a similar pattern of increased cycling of cells into DNA synthesis was observed; however, the majority (approximately 60%) of the cells were blocked in the G2 + M phase of the cell cycle. At later time intervals, an increase in the G1 population was demonstrated. The two monkey cell lines responded to SV40 virus with an accumulation of cells in the G2 + M phase of the cell cycle. Thus, two diploid human cell strains exhibited different cell cycle kinetics early after infection with SV40 virus. The one strain (WI-38) behaved similarly to the two monkey cell lines studied. The other strain (OI) responded similarly to nonpermissive (transforming) cells infected with SV40 virus.

The difference in DNA content of peripheral lymphocytes from normal males, normal females, and an individual with a 48 (xxxy) chromosome constitution was determined by rapid flow microfluorometric techniques. A similar comparison was performed using tissue culture fibroblasts derived from an individual with a 49 (xxxxy) chromosome constitution and WI-38 cells as a normal control. Less than 60 min were required to isolate the lymphocytes, to stain the cells fluorescently, and to measure the increased DNA content. The measured increase in DNA content is consistent with chromosome DNA analyses and chromosome length measurements.

The infection of secondary cultures of Chinese hamster cells with simian virus 40 (SV40) induces the appearance of cells with polyploid deoxyribonucleic acid (DNA) content or chromosomal component within one cell generation. The mechanism of this phenomenon was studied by the use of 5-bromodeoxyuridine (BUdR) incorporation as a DNA density marker. When cultures were treated with (14)C-BUdR and colcemide and harvested at 48 hr postinfection, only hybrid and light DNA molecules were found in control cultures, whereas in infected cultures there were also heavy molecules. The proportion of heavy DNA synthesized during the experimental period varied from 13 to 25%. It was determined by DNA-DNA hybridization that the heavy DNA consisted of cellular DNA. In radioautographic experiments, it was shown that, under the conditions used, a fraction of the infected cell population twice replicated its complete DNA content. Analysis of the kinetics indicated that the heavy DNA resulted from the reinitiation of DNA synthesis after the initial replication of the entire cell DNA. It was concluded that, after infection with SV40, a fraction of the Chinese hamster cell population undergoes two cycles of DNA synthesis without intervening mitosis.

Infection of primary or secondary cultures of Chinese hamster embryo cells with simian virus 40 at a multiplicity of 20 to 50 induced synthesis of the virus-specific intranuclear T antigen in 80 to 90% of the cells within 48 to 72 hr. In the infected cultures, 30 to 50% more cells were recruited into deoxyribonucleic acid (DNA) synthesis than in the controls, whether or not the cultures were confluent. The newly synthesized DNA was mostly cellular, since little virus was produced (as shown by various techniques: immunofluorescence for viral antigen, virus growth curves, and isolation of viral DNA from infected cultures). Transformed cells could be detected a few weeks after infection and produced tumors when inoculated into irradiated animals. Chromosomal changes were observed soon after infection (24 hr). Initially, there was a marked increase in the proportion of polyploid cells (8 to 14%), most of which were chromosomally normal. In a few weeks, a large majority of the infected population was polyploid (30 to 50%). Thus, the polyploid cells have the ability to proliferate. Evidence is presented to suggest that polyploid cells arise by stimulation of cells in the G(1), G(2), or S phases to undergo two or more successive periods of DNA synthesis without an intervening mitosis. With a subsequent loss or redistribution of chromosomal material, this may lead eventually to a biologically transformed cell; thus, it is suggested that the initial event(s) relevant to transformation occurs at the level of control of cellular DNA synthesis.

Simian virus 40 deoxyribonucleic acid (DNA) became associated in an alkaline-stable form with the DNA of Chinese hamster embryo cells at 15 to 20 hr post-infection, at the time when cell DNA synthesis and T antigen were induced. The integration process was not inhibited by d-arabinosyl cytosine and was only partially inhibited by cycloheximide.