Negative regulation of mitotic promoting factor by the checkpoint kinase chk1 in simian virus 40 lytic infection

Lytic infection of African green monkey kidney (CV-1) cells by simian virus 40 (SV40) is characterized by stimulation of DNA synthesis leading to bypass of mitosis and replication of cellular and viral DNA beyond a 4C DNA content. To define mechanisms underlying the absence of mitosis, the expression levels of upstream regulatory molecules of mitosis-promoting factor (MPF) were compared in parallel synchronized cultures of SV40-infected and uninfected CV-1 cells. The DNA replication/damage checkpoint kinase Chk1 was phosphorylated in both uninfected and SV40-infected cultures arrested at G(1)/S by mimosine, consistent with checkpoint activation. Following release of uninfected cultures from G(1)/S, Chk1 phosphorylation was lost even though Chk1 protein levels were retained. In contrast, G(1)/S-released SV40-infected cultures exhibited dephosphorylation of Chk1 in S phase, followed by an increase in Chk1 phosphorylation coinciding with entry of infected cells into >G(2). Inhibitors of Chk1, UCN-01 and caffeine, induced mitosis and abnormal nuclear condensation and increased the protein kinase activity of MPF in SV40-infected CV-1 cells. These results demonstrate that SV40 lytic infection triggers components of a DNA damage checkpoint pathway. In addition, chemical inhibition of Chk1 activity suggests that Chk1 contributes to the absence of mitosis during SV40 lytic infection.     

Simian virus 40 prevents activation of M-phase-promoting factor during lytic infection.

Simian virus 40 (SV40) infection stimulates confluent cultures of monkey kidney cells into successive rounds of cellular DNA synthesis without intervening mitosis. As an initial step in defining the mechanisms responsible for viral inhibition of mitosis, M-phase-promoting factor (MPF) was examined in SV40-infected CV-1 cells passing from G2 phase into a second S phase. MPF is a serine-threonine protein kinase that is essential for mitosis in eukaryotic cells. In SV40-infected cells exiting G2 phase, there was a reduced amount of MPF-associated H1 kinase activity relative to that of uninfected cells passing through mitosis. Both subunits of MPF, cyclin B and the p34cdc2 catalytic subunit, were present and in a complex in infected cells. In uninfected cultures, passage through mitosis was associated with the dephosphorylation of the p34cdc2 subunit, which is characteristic of MPF activation. In contrast, the p34cdc2 subunit remained in the tyrosine-phosphorylated, inactive form in SV40-infected cells passing from G2 phase into a second S phase. These results suggest that although the MPF complex is assembled and modified normally, SV40 interferes with pathways leading to MPF activation.     

Induction of Cellular Deoxyribonuleic Acid Synthesis by Simian Virus 40

The incorporation of (3)H-thymidine ((3)H-dT) into deoxyribonucleic acid (DNA) has been studied in uninfected confluent monolayer cultures of monkey kidney and mouse kidney cells, simian virus 40 (SV40)-infected cells, and in SV40-transformed mouse kidney cells. Radioautographic measurements revealed that during the period from 28 to 51 hr after productive SV40 infection of monkey kidney cultures about 80% of the cells synthesized DNA, compared to about 16% in uninfected cultures. At 28 to 43 hr after abortive SV40 infection of mouse kidney cultures, 24 to 37% of the cells synthesized DNA, compared to about 6 to 8% in uninfected cultures. The infected monkey kidney and mouse kidney cultures, respectively, incorporated about 5 to 10 times and 3 to 5 times as much (3)H-dT into DNA as did uninfected cultures. Moreover, the net DNA synthesized by SV40-infected monkey kidney cultures, estimated by colorimetric methods, substantially exceeded that of uninfected cultures.Nitrocellulose chromatography and band centrifugation experiments were performed to elucidate the kinds of DNA synthesized in the cultures. In uninfected monkey kidney cultures and at 2 to 12 hr after SV40 infection, almost all of the (3)H-dT labeled DNA sedimented more rapidly than SV40 DNA, and the radioactive DNA was denatured by heating for 12 min at 100 C (cellular DNA). Almost all of the labeled DNA obtained from abortively infected mouse kidney cultures and from SV40-transformed cells also had the properties of cellular DNA. However, approximately one-third to one-half of the labeled DNA obtained from monkey kidney cultures 28 to 51 hr after infection sedimented more slowly than cellular DNA and was not denatured by the heating (SV40 DNA). It is concluded that cellular DNA synthesis was induced during either the productive or abortive SV40 infections.     

Differential increase in topoisomerase II in simian virus 40-infected cells.

The time course of expression of topoisomerase I, topoisomerase II, and simian virus 40 (SV40) large tumor (T) antigen was determined in whole-cell extracts of uninfected versus SV40-infected TC7 cells. After a minor increase, the level of topoisomerase I remained fairly constant throughout the time course in both uninfected and SV40-infected cells. In contrast, the level of topoisomerase II increased markedly in SV40-infected cells but not in uninfected cells following the appearance of SV40 T antigen.     

Distinct patterns of MCM protein binding in nuclei of S phase and rereplicating SV40-infected monkey kidney cells.

BACKGROUND: Simian Virus 40 (SV40) infection of growth-arrested monkey kidney cells stimulates S phase entry and the continued synthesis of both viral and cellular DNA. Infected cells can attain total DNA contents as high as DNA Index, DI = 5.0-6.0 (10-12C), with host cell DNA representing 70-80% of the total. In this study, SV40-infected and uninfected control cells were compared to determine whether continued DNA replication beyond DI = 2.0 was associated with rebinding of the minichromosome maintenance (MCM) hexamer, the putative replicative helicase, to chromatin. METHOD: Laser scanning cytometry was used to measure the total expression per cell and the chromatin/matrix-association of two MCM subunits in relation to DNA content. RESULTS: MCM2 and MCM3 proteins that were associated with the chromatin/matrix fraction in G1 phase of both uninfected and SV40-infected cells were gradually released during progression through S phase. However, in SV40-infected cells that progressed beyond DI = 2.0, chromatin/matrix-associated MCM2 and MCM3 remained at the low levels observed at the end of S phase. Rereplication was not preceded by an obvious rebinding of MCM proteins to chromatin, as was observed in G1 phase. CONCLUSIONS: The rereplication of host cell DNA in the absence of the reassociation of MCM proteins with chromatin indicates that SV40 infection induces a novel mechanism of licensing cellular DNA replication.     

Inhibition of simian virus 40 DNA synthesis in Yaba virus-preinfected cells.

The effect of Yaba virus preinfection on DNA synthesis in SV40-infected Jinet cells was studied. Time-course synthesis studies were conducted using the incorporation of labeled thymidine. Yaba virus preinfection resulted in the inhibition of SV40 DNA synthesis when the elapsed time between Yaba virus and SV40 infections was three days. This inhibition was demonstrated by hybridization studies and sedimentation analysis. In addition, the usual stimulation of cellular DNA synthesis induced by SV40 infection was inhibited. This inhibition occurred at a time in Yaba virus infection when no cytoplasmic Yaba virus-specific DNA synthesis occurred.     

Simian Virus 40 Deoxyribonucleic Acid Replication: I. Effect of Cycloheximide on the Replication of SV40 Deoxyribonucleic Acid in Monkey Kidney Cells and in Heterokaryons of SV40-transformed and Susceptible Cells

Infectious deoxyribonucleic acid (DNA) was extracted from green monkey kidney (CV-1) cultures at various times after the cultures were infected with simian virus 40 (SV40) at input multiplicities of 0.01 and 0.1 plaque-forming unit (PFU) per cell. A pronounced decrease in infectious DNA was observed from 3 to 16 hr after virus infection, suggesting that structurally altered intracellular forms may have been generated early in infection. Evidence is also presented that SV40 DNA synthesis requires concurrent protein synthesis. DNA replication was studied in the presence and absence of cycloheximide in: (i) SV40-infected and uninfected cultures of CV-1 cells; (ii) cultures synchronized with 1-β-d-arabinofuranosylcytosine (ara-C) for 24 to 30 hr prior to the addition of cycloheximide; and (iii) in heterokaryons of SV40-transformed hamster and susceptible monkey kidney cells. DNA synthesis was determined by pulse-labeling the cultures with ³H-thymidine at various times from 24 to 46 hr after infection. In addition, the total infectious SV40 DNA was measured. Addition of cycloheximide, even after early proteins had been induced, grossly inhibited both SV40 and cellular DNA syntheses. The activities of thymidine kinase, DNA polymerase, deoxycytidylate deaminase, and thymidylate kinase were measured; these enzyme activities remained high for at least 9 hr in the presence of cycloheximide. SV40 DNA prelabeled with ³H-thymidine before the addition of cycloheximide was also relatively stable during the time required for cycloheximide to inhibit further DNA replication.     

Reinitiation of host DNA synthesis in senescent human diploid cells by infection with simian virus 40

Human diploid fibroblasts, TIG-1, cease to proliferate at about 60-62 population doubling level. In their senescent state used in this study, the percentage of nuclei labeled by [3H]thymidine for 48 h was around 1-2% in fresh medium containing 5-40% fetal bovine serum. The percentage of labelled nuclei increased up to 10-fold after infection with SV40. This increase reflects stimulation of cell DNA synthesis because: 1. The increase also occurred when ts A900 was used for infection at the non-permissive temperature, under these conditions viral DNA synthesis is inhibited; 2, the increase paralleled the stimulation of [3H]thymidine incorporation into DNA in a Hirt-precipitate fraction from SV40-infected cells. UV-irradiated SV40 had reduced ability to induce DNA synthesis. A viable deletion mutant of SV40, d1940, had almost the same activity to induce cell DNA synthesis as did wild-type SV40. Equilibrium density gradient centrifugation analysis of DNA labelled with 5-bromodeoxyuridine (BrdU) supported semiconservative replication rather than repair synthesis. We conclude that a considerable fraction of human diploid cells in a senescent population initiate host DNA replication by infection with SV40, although these cells cannot be stimulated with fetal bovine serum.     

Simian virus 40 and polyoma virus induce synthesis of heat shock proteins in permissive cells.

During the lytic infection of monkey and mouse cells with simian virus 40 and polyoma virus, respectively, the preferentially increased synthesis of two host proteins of 92,000 and 72,000 Mr was observed by 15 to 20 h after infection besides the general stimulation of most cellular proteins. The incubation of uninfected monkey and mouse cell cultures for 30 to 60 min at 43.5 degrees C induced the enhanced synthesis of at least three proteins of 92,000, 72,000 and 70,000 Mr, the last one being the major heat shock protein of mammalian cells. Two-dimensional gel electrophoresis and partial proteolytic digestion confirmed that the same 92,000- and 72,000-Mr proteins are stimulated by virus infection and thermal treatment. In simian virus 40-infected CV-1 cells, we also observed the weak stimulation of a 70,000-Mr protein comigrating in gel electrophoresis with the major heat shock protein. The 92,000-, 72,000- and 70,000-Mr proteins of monkey cells are structurally very similar to the corresponding proteins of mouse cells. In immunoprecipitations, no specific association of these proteins to simian virus 40 T antigens was noticed.     

Characterization of a 54K dalton cellular SV40 tumor antigen present in SV40-transformed cells and uninfected embryonal carcinoma cells.

SV40 infection or transformation of murine cells stimulated the production of a 54K dalton protein that was specifically immunoprecipitated, along with SV40 large T and small t antigens, with sera from mice or hamsters bearing SV40-induced tumors. The same SV40 anti-T sera immunoprecipitated a 54K dalton protein from two different, uninfected murine embryonal carcinoma cell lines. These 54K proteins from SV40-transformed mouse cells and the uninfected embryonal carcinomas cells had identical partial peptide maps which were completely different from the partial peptide map of SV40 large T antigen. An Ad2+ND4-transformed hamster cell line also expressed a 54K protein that was specifically immunoprecipitated by SV40 T sera. The partial peptide maps of the mouse and hamster 54K protein were different, showing the host cell species specificity of these proteins. The 54K hamster protein was also unrelated to the Ad2+ND4 SV40 T antigen. Analogous proteins immunoprecipitated by SV40 T sera, ranging in molecular weight from 44K to 60K, were detected in human and monkey SV40-infected or -transformed cells. A wide variety of sera from hamsters and mice bearing SV40-induced tumors immunoprecipitated the 54K protein of SV40-transformed cells and murine embryonal carcinoma cells. Antibody produced by somatic cell hybrids between a B cell and a myeloma cell (hybridoma) against SV40 large T antigen also immunoprecipitated the 54K protein in virus-infected and -transformed cells, but did not do so in the embryonal carcinoma cell lines. We conclude that SV40 infection or transformation of mouse cells stimulates the synthesis or enhances the stability of a 54K protein. This protein appears to be associated with SV40 T antigen in SV40-infected and -transformed cells, and is co-immunoprecipitated by hybridomas sera to SV40 large T antigen. The 54K protein either shares antigenic determinants with SV40 T antigen or is itself immunogenic when in association with SV40 large T antigen. The protein varies with host cell species, and analogous proteins were observed in hamster, monkey and human cells. The role of this protein in transformation is unclear at present.     

Temperature-Sensitive Simian Virus 40 Mutant Defective in a Late Function

A temperature-sensitive simian virus 40 (SV40) mutant, tsTNG-1, has been isolated from nitrosoguanidine-treated and SV40-infected African green monkey kidney (CV-1) cultures. Replication of virus at the nonpermissive temperature (38.7 C) was 3,000-fold less than at the permissive temperature (33.5 C). Plaque formation by SV40tsTNG-1 deoxyribonucleic acid (DNA) on CV-1 monolayers occurred normally at 33.5 C but was grossly inhibited at 38.7 C. The time at which virus replication was blocked at 38.7 C was determined by temperature-shift experiments. In shift-up experiments, cultures infected for various times at 33.5 C were shifted to 38.7 C. In shift-down experiments, cultures infected for various times at 38.7 C were shifted to 33.5 C. All cultures were harvested at 96 hr postinfection (PI). No virus growth occurred when the shift-up occurred before 40 hr PI. Maximum virus yields were obtained at 96 hr PI when the shift-down occurred at 66 hr, but only about 15% of the maximum yield was obtained when the shift-down occurred at 76 hr PI. These results indicate that SV40tsTNG-1 contains a conditional lethal mutation in a late viral gene function. Mutant SV40tsTNG-1 synthesized T antigen, viral capsid antigens, and viral DNA, and induced thymidine kinase activity at either 33.5 or 38.7 C. The properties of the SV40 DNA synthesized in mutant-infected CV-1 cells at 33.5 or 38.7 C were very similar to those of SV40 DNA made in parental virus-infected cells, as determined by nitrocellulose column chromatography, cesium-chloride-ethidium bromide equilibrium centrifugation, and by velocity centrifugation in neutral sucrose gradients. Mutant SV40tsTNG-1 enhanced cellular DNA synthesis in primary cultures of mouse kidney cells at 33.5 and 38.7 C and also transformed mouse kidney cultures at 36.5 C. SV40tsTNG-1 was recovered from clonal lines of transformed cells after fusion with susceptible CV-1 cells and incubation of heterokaryons at 33.5 C, but not at 38.7 C.     

Stimulation of non-histone chromosomal protein synthesis in simian virus 40-infected simian cells.

The pattern of synthesis of non-histone chromosomal proteins in simian virus (SV) 40-infected African green monkey kidney cells was analyzed by polyacryl-amide gel electrophoresis to see whether the changes in chromosomal protein metabolism are involved in the viral-induced synthesis of cellular DNA and mRNA. During the prereplicative phase of infection, the rate of histone synthesis was decreased until 15 h postinfection, whereas that of non-histone protein synthesis was increased after 5 h postinfection and reached a maximum at 10 to 15 h postinfection when viral-induced synthesis of cellular DNA and mRNA began to be observed. Stimulation of non-histone protein synthesis was also observed in the infected cells treated with cytosine arabinoside and was dependent on the multiplicity of infection. Stimulation occurred in almost all species of non-histone proteins. These results suggest that the stimulation of non-histone protein synthesis is caused by an early SV40 function and occurs prior to the viral-induced synthesis of cellular DNA and mRNA. During the replicative phase of infection, a marked increase in the rate of synthesis was observed in the non-histone proteins with molecular weights of about 48,000, 35,000, and 23,000, which were subsequently found to be SV40 capsid proteins.     

Effect of SV40 infection on [3H]uridine incorporation.

More [3H]uridine was incorporated into RNA of SV40-infected than into uninfected cells 31 h after infection. When the specific activity of the uridine triphosphate pools in infected and uninfected cells was equated by the addition of appropriate amounts of exogenous unlabelled uridine, no difference in the rate of [3H]uridine incorporation into RNA was observed. Although no difference in [3H]uridine entry or phosphorylation was demonstrable, the apparently smaller pools of endogenous RNA precursors in infected cells resulted in less isotope dilution and thus to synthesis of uridine triphosphate and RNA of higher specific activity.     

Enlargement of Escherichia coli After Bacteriophage Infection I. Description of the Phenomenon

Cycloheximide addition at various times from 24 to 36 hr after virus infection markedly inhibits the rate of simian virus 40 (SV40) deoxyribonucleic acid (DNA) synthesis in monkey kidney (CV-1) cultures. To determine whether superhelical (form I) SV40 DNA was synthesized in the cycloheximide-inhibited cultures, extracts were prepared by the method of Hirt from cultures labeled with (3)H-thymidine ((3)H-dT) and were analyzed by cesium chloride-ethidium bromide (CsCl-EtBr) equilibrium centrifugation and by velocity sedimentation in neutral sucrose gradients. When control or cycloheximide-treated cultures were labeled for 2 or 4 hr with (3)H-dT at 36 or 37 hr after infection, 71 to 83% of the radioactivity soluble in 1 m NaCl was detected in closed-circular SV40 DNA (form I). Cycloheximide treatment did not generate an increase of higher multiple circular forms of SV40 DNA. In pulse-chase experiments with or without cycloheximide treatment, radioactivity first appeared in nicked molecular forms sedimenting faster than open-circular SV40 DNA (form II), and then was chased into superhelical form I SV40 DNA. These results suggest that in cycloheximide-treated SV40-infected cultures: (i) polynucleotide ligase concentrations are adequate, and (ii) duplication errors causing formation of circular oligomers of SV40 DNA are not enhanced.     

Maintenance of DNA methylation level in SV40-infected human fibroblasts during their in vitro limited proliferative life span

Methylation level as expressed by the molar ratio of 5-methylcytosine content to the combined content of cytosine and 5-methylcytosine was determined by HPLC and uv adsorption of cellular DNA extracted from SV40-infected and pretransformed MRC-5 human diploid fibroblasts (HDFs) during their limited in vitro life span. The level decreased slightly during early passages, and then was maintained within a certain range in the subsequent pretransformed stage of serial passages. When HDFs were treated with 5-aza-2'-deoxycytidine (5-aza-CdR) at an effective concentration shortly after the SV40 infection, the level decreased and then increased or was maintained again within a certain range in the subsequent pretransformed state. The proliferative life span potential of SV40-infected HDFs was not significantly decreased by the 5-aza-CdR treatment. These results are in contrast to the established observations for uninfected HDFs, that methylation level decreases during serial passages, and that, after treatment with 5-aza-CdR, the level, as well as the proliferative life span, is decreased in comparison to untreated populations. These results show that SV40-infected pretransformed HDFs are in an intermediate state between normal finite growth and an established permanent line, in that they retain limited in vitro cell proliferation, while acquiring the ability to maintain methylation levels.