n-Butyrate, a cell cycle blocker, inhibits the replication of polyomaviruses and papillomaviruses but not that of adenoviruses and herpesviruses.

Small DNA viruses are dependent on the interaction of early proteins (such as large T antigen) with host p53 and Rb to bring about the G1-to-S cell cycle transition. The large DNA viruses are less dependent on host regulatory genes since additional early viral proteins (such as viral DNA polymerase, DNA metabolic enzymes, and other replication proteins) are involved in DNA synthesis. A highly conserved domain of large T antigen (similar to the p53-binding region) exclusively identifies papovavirus, parvovirus, and papillomaviruses from all other larger DNA viruses and implies a conserved interaction with host regulatory genes. In this report, we show that 3 to 6 mM butyrate, a general cell cycle blocker implicated in inhibition of the G1-to-S transition, inhibits DNA replication of polyomavirus and human papillomavirus type 11 but not the replication of larger DNA viruses such as adenovirus types 2 and 5, herpes simplex virus type 1, Epstein-Barr virus, and cytomegalovirus, which all bypass the butyrate-mediated cell cycle block. This butyrate effect on polyomavirus replication is not cell type specific, nor does it depend on the p53 or Rb gene, as inhibition was seen in fibroblasts with intact or homozygous deleted p53 or Rb, 3T6 cells, keratinocytes, C2C12 myoblasts, and 3T3-L1 adipocytes. In addition, butyrate did not inhibit expression of polyomavirus T antigen. The antiviral effect of butyrate involves a form of imprinted state, since pretreatment of cells with 3 mM butyrate inhibits human papillomavirus type 11 DNA replication for at least 96 h after its removal. Butyrate, therefore, serves as a molecular tool in dissecting the life cycle of smaller DNA viruses from that of the larger DNA viruses in relation to the cell cycle.     

Polyomavirus large and small T antigens cooperate in induction of the S phase in serum-starved 3T3 mouse fibroblasts.

The induction of an S phase in the host cell is a prerequisite for the lytic replication cycle of polyomavirus. This function was attributed to proteins coded for by the early region of the viral DNA, the T antigens. A consideration of the role of the T antigens in the initiation of a mitogenic response of the host cell has to take into account the recent discovery that virus adsorption is sufficient to induce the synthesis of proteins which are known to appear early after quiescent cells are stimulated by the addition of serum, namely fos, jun, and myc (J. Zullo, C.D. Stiles, and R.L. Garcea, Proc. Natl. Acad. Sci. USA 84:1210-1214, 1987; G. M. Glenn and W. Eckhart, J. Virol. 64:2193-2201, 1990). This induction is followed by an initiation of DNA synthesis. It is therefore important to dissociate the effects of the T antigens on the host cell from those of virus adsorption. To do so, we used dexamethasone-regulated versions of the large and small T antigens of polyomavirus stably integrated into the genome of Swiss 3T3 cells to study their function in S-phase induction. When the production of the large or small T antigen in serum-starved 3T3 mouse fibroblasts was activated, only a small fraction of cells was able to leave G0/G1 despite the synthesis of considerable amounts of the respective T antigen. Activation of both T antigens within the same cell, on the other hand, resulted in S-phase induction in a notable percentage of cells, suggesting that the two proteins cooperate in this activity. Polyomavirus T antigens appear to bypass the pathway of growth regulation involving the activation of c-fos. These results are discussed in relation to other known functions of the two virally coded proteins.     

Infection of cells with human cytomegalovirus during S phase results in a blockade to immediate-early gene expression that can be overcome by inhibition of the proteasome

Cells infected with human cytomegalovirus (HCMV) after commencing DNA replication do not initiate viral immediate-early (IE) gene expression and divide before arresting. To determine the nature of this blockade, we examined cells that were infected 24 h after release from G(0) using immunofluorescence, laser scanning cytometry, and fluorescence-activated cell sorting (FACS) analysis. Approximately 40 to 50% of the cells had 2N DNA content, became IE(+) in the first 12 h, and arrested. Most but not all of the cells with >2N DNA content did not express IE antigens until after mitosis. To define the small population of IE(+) cells that gradually accumulated within the S and G(2)/M compartments, cells were pulsed with bromodeoxyuridine (BrdU) just prior to S-phase infection and analyzed at 12 h postinfection for IE gene expression, BrdU positivity, and cell cycle position. Most of the BrdU(+) cells were IE(-) and had progressed into G(2)/M or back to G(1). The majority of the IE(+) cells in S and G(2)/M were BrdU(-). Only a few cells were IE(+) BrdU(+), and they resided in G(2)/M. Multipoint BrdU pulse-labeling revealed that, compared to cells actively synthesizing DNA at the beginning of the infection, a greater percentage of the cells that initiated DNA replication 4 h later could express IE antigens and proceed into S. Synchronization of the cells with aphidicolin also indicated that the blockade to the activation of IE gene expression was established in cells soon after initiation of DNA replication. It appears that a short-lived protein in S-phase cells may be required for IE gene expression, as it is partially restored by treatment with the proteasome inhibitor MG132.     

Microarray analysis of 1alpha,25-dihydroxyvitamin D3-treated MC3T3-E1 cells

The active form of Vitamin D, 1alpha,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], demonstrates potent antiproliferative actions on normal as well as on malignant cell types by blocking the transition from the G1- to the S-phase of the cell cycle. Key target genes for 1,25-(OH)(2)D(3) in this non-classic effect remain largely unknown. Therefore, this study aims to identify genes that, through changes in expression after 1,25-(OH)(2)D(3) treatment, contribute to the observed antiproliferative effect. cDNA microarrays containing 4600 genes were used to investigate changes in gene expression in MC3T3-E1 mouse osteoblasts at 6 and at 12h after treatment with 1,25-(OH)(2)D(3) (10(-8)M), preceding (6h) or coinciding with (12h) the G1/S block in these cells. Approximately one fifth of the genes that were significantly down-regulated after a 12h incubation period with 1,25-(OH)(2)D(3) were genes involved in the DNA replication process, a basic process for cell growth that starts at the end of G1-phase and continues in S-phase. Down-regulation of these genes by 1,25-(OH)(2)D(3) was confirmed by quantitative RT-PCR in MC3T3-E1. In conclusion, cDNA microarrays revealed that treatment of MC3T3-E1 cells with 1,25-(OH)(2)D(3) resulted in the down-regulation of DNA replication genes in parallel with the observed G1/S-arrest.     

Parvovirus-Induced Depletion of Cyclin B1 Prevents Mitotic Entry of Infected Cells

Parvoviruses halt cell cycle progression following initiation of their replication during S-phase and continue to replicate their genomes for extended periods of time in arrested cells. The parvovirus minute virus of mice (MVM) induces a DNA damage response that is required for viral replication and induction of the S/G2 cell cycle block. However, p21 and Chk1, major effectors typically associated with S-phase and G2-phase cell cycle arrest in response to diverse DNA damage stimuli, are either down-regulated, or inactivated, respectively, during MVM infection. This suggested that parvoviruses can modulate cell cycle progression by another mechanism. In this work we show that the MVM-induced, p21- and Chk1-independent, cell cycle block proceeds via a two-step process unlike that seen in response to other DNA-damaging agents or virus infections. MVM infection induced Chk2 activation early in infection which led to a transient S-phase block associated with proteasome-mediated CDC25A degradation. This step was necessary for efficient viral replication; however, Chk2 activation and CDC25A loss were not sufficient to keep infected cells in the sustained G2-arrested state which characterizes this infection. Rather, although the phosphorylation of CDK1 that normally inhibits entry into mitosis was lost, the MVM induced DDR resulted first in a targeted mis-localization and then significant depletion of cyclin B1, thus directly inhibiting cyclin B1-CDK1 complex function and preventing mitotic entry. MVM infection thus uses a novel strategy to ensure a pseudo S-phase, pre-mitotic, nuclear environment for sustained viral replication.     

A new compound which reversibly arrests T lymphocyte cell cycle near the G1/S boundary

A novel cell cycle blocking agent profoundly suppressed the proliferation of mitogen-stimulated T lymphocytes. The carboxythiazole derivative arrested cells in the G1 phase of the cell cycle but did not inhibit the induction of cell surface receptors for either interleukin-2 or transferrin. The uncoupling of transferrin receptor expression from DNA synthesis indicated that a previously undefined restriction point in the cell cycle has been identified which occurs after transferrin receptor expression in late G1 and just prior to the initiation of DNA replication in S phase. T cells incubated in an inhibitory dose of the carboxythiazole derivative resumed cell cycle progression subsequent to its removal, indicating that the compound reversibly arrests cells at the late G1 restriction point. In contrast to other techniques which have been inefficient in achieving T cell synchronization, T cells released from the block mediated by the carboxythiazole compound progress through S phase with a considerable degree of synchrony.     

Nuclear DNA synthesis in vitro is mediated via stable replication forks assembled in a temporally specific fashion in vivo.

A cell-free nuclear replication system that is S-phase specific, that requires the activity of DNA polymerase alpha, and that is stimulated three- to eightfold by cytoplasmic factors from S-phase cells was used to examine the temporal specificity of chromosomal DNA synthesis in vitro. Temporal specificity of DNA synthesis in isolated nuclei was assessed directly by examining the replication of restriction fragments derived from the amplified 200-kilobase dihydrofolate reductase domain of methotrexate-resistant CHOC 400 cells as a function of the cell cycle. In nuclei prepared from cells collected at the G1/S boundary of the cell cycle, synthesis of amplified sequences commenced within the immediate dihydrofolate reductase origin region and elongation continued for 60 to 80 min. The order of synthesis of amplified restriction fragments in nuclei from early S-phase cells in vitro appeared to be indistinguishable from that in vivo. Nuclei prepared from CHOC 400 cells poised at later times in the S phase synthesized characteristic subsets of other amplified fragments. The specificity of fragment labeling patterns was stable to short-term storage at 4 degrees C. The occurrence of stimulatory factors in cytosol extracts was cell cycle dependent in that minimal stimulation was observed with early G1-phase extracts, whereas maximal stimulation was observed with cytosol extracts from S-phase cells. Chromosomal synthesis was not observed in nuclei from G1 cells, nor did cytosol extracts from S-phase cells induce chromosomal replication in G1 nuclei. In contrast to chromosomal DNA synthesis, mitochondrial DNA replication in vitro was not stimulated by cytoplasmic factors and occurred at equivalent rates throughout the G1 and S phases. These studies show that chromosomal DNA replication in isolated nuclei is mediated by stable replication forks that are assembled in a temporally specific fashion in vivo and indicate that the synthetic mechanisms observed in vitro accurately reflect those operative in vivo.     

The Cellular Protein MCM3AP Is Required for Inhibition of Cellular DNA Synthesis by the IE86 Protein of Human Cytomegalovirus

Like all DNA viruses, human cytomegalovirus (HCMV) infection is known to result in profound effects on host cell cycle. Infection of fibroblasts with HCMV is known to induce an advance in cell cycle through the G₀-G₁ phase and then a subsequent arrest of cell cycle in early S-phase, presumably resulting in a cellular environment optimum for high levels of viral DNA replication whilst precluding replication of cellular DNA. Although the exact mechanisms used to arrest cell cycle by HCMV are unclear, they likely involve a number of viral gene products and evidence points to the ability of the virus to prevent licensing of cellular DNA synthesis. One viral protein known to profoundly alter cell cycle is the viral immediate early 86 (IE86) protein - an established function of which is to initially drive cells into early S phase but then inhibit cellular DNA synthesis. Here we show that, although IE86 interacts with the cellular licensing factor Cdt1, it does not inhibit licensing of cellular origins. Instead, IE86-mediated inhibition of cellular DNA synthesis requires mini-chromosome-maintenance 3 (MCM3) associated protein (MCM3AP), which can cause subsequent inhibition of initiation of cellular DNA synthesis in a licensing-independent manner.     

Saccharomyces cerevisiae lacking Snm1, Rev3 or Rad51 have a normal S-phase but arrest permanently in G2 after cisplatin treatment

The role of Snm1, Rev3 and Rad51 in S-phase after cisplatin (CDDP) DNA treatment has been examined. When isogenic deletion mutants snm1 delta, rev3 delta and rad51 delta were arrested in G1 and treated with doses of CDDP causing significant lethality (<20% survival in the mutant strains), they progressed through S-phase with normal kinetics. The mutants arrested in G2 like wild-type cells, however they did not exit the arrest and reenter the cell cycle. This finding demonstrates that these genes are not required to allow DNA replication in the presence of damage. Therefore, Snm1, Rev3 and Rad51 may act after S to allow repair. At high levels of damage (<40% survival in wild-type cells) S-phase was slowed in a MEC1-dependent fashion. The cross-link incision kinetics of snm1 delta and rev3 delta mutants were also examined; both showed no deficiencies in incision of cross-linked DNA.     

Cisplatin DNA cross-links do not inhibit S-phase and cause only a G2/M arrest in Saccharomyces cerevisiae.

Cisplatin (CDDP) has been used as a DNA cross-linking agent to evaluate whether there is a specific cell cycle checkpoint response to such damage in Saccharomyces cerevisiae (S. cerevisiae). Fluorescent-activated cell sorting (FACS) analysis showed only a G2/M checkpoint, normal exit from G1 and progression through S-phase following alpha-factor arrest and CDDP treatment. Of the checkpoint mutants tested, rad9, rad17 and rad24, did not show increased sensitivity to CDDP compared to isogenic wild-type cells. However, other checkpoint mutants tested (mec1, mec3 and rad53) showed increased sensitivity to CDDP, as did controls with a defect in excision repair (rad1 and rad14) or a defect in recombination (rad51 and rad52). Thus, by survival and cell cycle kinetics, it appears that DNA cross-links do not inhibit entry into S-phase or slow DNA replication and that replication continues after cisplatin treatment in yeast.     

Influenza virus matrix protein M1 interacts with SLD5 to block host cell cycle

Influenza virus matrix 1 protein (M1) is highly conserved and plays essential roles at many stages of virus life cycle. Here, we used a yeast two‐hybrid system to identify the host protein SLD5, a component of the GINS complex, which is essential for the initiation of DNA replication in eukaryotic cells, as a new M1 interacting protein. M1 from several different influenza virus strains all interacted with SLD5. Overexpression of SLD5 suppressed influenza virus replication. Transient, stable, or inducible expression of M1 induced host cell cycle blockade at G0/G1 phase. Moreover, SLD5 partially rescued M1 expression‐ or influenza virus infection‐induced G0/G1 phase accumulation in cell lines and primary mouse embryonic fibroblasts. Importantly, SLD5 transgenic mice exhibited higher resistance and improved lung epithelial regeneration after virus infection compared with wild‐type mice. Therefore, influenza virus M1 blocks host cell cycle process by interacting with SLD5. Our finding reveals the multifunctional nature of M1 and provides new insight for understanding influenza virus–host interaction.     

Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells

DNA replication in eukaryotic cells is restricted to the S-phase of the cell cycle. In a cell-free replication model system, using SV40 origin-containing DNA, extracts from G1 cells are inefficient in supporting DNA replication. We have undertaken a detailed analysis of the subcellular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and therefore available for DNA replication. Cyclin A and cdk2 have been implicated in regulating DNA replication, and may be responsible for activating components of the DNA replication mitiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the replication proteins RPA (the eukaryotic single-stranded DNA binding protein) and DNA polymerase as well as cdk2, but lack cyclin A. On localizing these components in G1 cells we find that both RPA and DNA polymerase are present as nuclear proteins, while cdk2 is primarily cytoplasmic and there is no detectable cyclin A. An apparent change in the distribution of these proteins occurs as the cell enters S-phase. Cyclin A becomes abundant and both cyclin A and cdk2 become localized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 subunits of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a distinct punctate nuclear pattern. The initiation of DNA replication therefore most likely occurs by modification and activation of these replication initiation proteins rather than by their recruitment to the nuclear compartment.     

Genes encoding two essential DNA replication activation proteins,Cdc6 and Mcm3, exhibit very different patterns of expression in the tobacco BY-2 cell cycle

Very little is known about the expression patterns in plants of genes that encode proteins involved in the initiation of DNA replication. Partial cDNA sequences that encode Cdc6 and Mcm3 in tobacco have been isolated. The sequences were used as probes in northern blots which suggested that, in the cell cycle of synchronized tobacco BY-2 cells, expression of CDC6 is confined to late G(1) and S-phase whereas the expression of MCM3 is not confined to any particular cell cycle phase. These data were confirmed and extended by real-time PCR measurements of mRNA abundance through the cell cycle. CDC6 exhibits a very clear peak of expression in S-phase whereas MCM3, expressed at a much lower level than CDC6, is not cell-cycle-regulated. These patterns of cell cycle gene expression resemble those found in the fission yeast Schizosaccharomyces pombe rather than those in budding yeast or mammalian cells.     

S-phase-dependent cell cycle disturbances caused by Aleutian mink disease parvovirus.

We examined replication of the autonomous parvovirus Aleutian mink disease parvovirus (ADV) in relation to cell cycle progression of permissive Crandell feline kidney (CRFK) cells. Flow cytometric analysis showed that ADV caused a composite, binary pattern of cell cycle arrest. ADV-induced cell cycle arrest occurred exclusively in cells containing de novo-synthesized viral nonstructural (NS) proteins. Production of ADV NS proteins, indicative of ADV replication, was triggered during S-phase traverse. The NS+ cells that were generated during later parts of S phase did not undergo cytokinesis and formed a distinct population, termed population A. Formation of population A was not prevented by VM-26, indicating that these cells were arrested in late S or G2 phase. Cells in population A continued to support high-level ADV DNA replication and production of infectious virus after the normal S phase had ceased. A second, postmitotic, NS+ population (termed population B) arose in G0/G1, downstream of population A. Population B cells were unable to traverse S phase but did exhibit low-level DNA synthesis. Since the nature of this DNA synthesis was not examined, we cannot at present differentiate between G1 and early S arrest in population B. Cells that became NS+ during S phase entered population A, whereas population B cells apparently remained NS- during S phase and expressed high NS levels postmitosis in G0/G1. This suggested that population B resulted from leakage of cells with subthreshold levels of ADV products through the late S/G2 block and, consequently, that the binary pattern of ADV-induced cell cycle arrest may be governed merely by viral replication levels within a single S phase. Flow cytometric analysis of propidium iodide fluorescence and bromodeoxyuridine uptake showed that population A cells sustained significantly higher levels of DNA replication than population B cells during the ADV-induced cell cycle arrest. Therefore, the type of ADV-induced cell cycle arrest was not trivial and could have implications for subsequent viral replication in the target cell.     

Characterization of a ts mutant of BALB/3T3 cells and correction of the defect by in vitro addition of extracts from wild-type cells.

ts20 is a temperature-sensitive mutant cell line derived from BALB/3T3 cells. DNA synthesis in the mutant decreased progressively after an initial increase during the first 3 h at the restrictive temperature. RNA and protein synthesis increased for 20 h and remained at a high level for 40 h. Cells were arrested in S phase as determined by flow microfluorimetry, and DNA chain elongation was retarded as measured by fiber autoradiography. Infection with polyomavirus did not bypass the defect in cell DNA synthesis, and the mutant did not support virus DNA replication at the restrictive temperature. After shift down to the permissive temperature, cell DNA synthesis was restored whereas virus DNA synthesis was not. Analysis of virus DNA synthesized at the restrictive temperature showed that the synthesis of form I and replicative intermediate DNA decreased concurrently and that the rate of completion of virus DNA molecules remained constant with increasing time at the restrictive temperature. These studies indicated that the mutation inhibited ongoing DNA synthesis at a step early in elongation of nascent chains. The defect in virus and cell DNA synthesis was expressed in vitro. [3H]dTTP incorporation was reduced, consistent with the in vivo data. The addition of a high-salt extract prepared from wild-type 3T3 cells preferentially stimulated the incorporation of [3H]dTTP into the DNA of mutant cells at the restrictive temperature. A similar extract prepared from mutant cells was less effective and was more heat labile as incubation of it at the restrictive temperature for 1 h destroyed its ability to stimulate DNA synthesis in vitro, whereas wild-type extract was not inactivated until incubated at that temperature for 3 h.