Initiation Of Cellular DNA Synthesis Research Articles

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.

Sarcolectin: Complete purification for molecular cloning

A great variety of vertebrate cells contain detecamounts of lectins, able to stimulate the initiation of cellular DNA synthesis. One of them, sarcolectin (SCL) can block interferon (IFN) action, by inhibiting the synthesis and the expression of the IFN dependent secondary proteins. As a result, the IFN-induced antiviral state is abolished in the cells, which likely facilitates their replication. We identified a major 65 kDa and a minor 55 kDa protein, which could carry these cellular functions. Their purification, especially that of the 65 kDa, was difficult, because of the proximity of albumin. We devised therefore a two-step primary separation, followed by a four-step final purification, which are reported here. The purification was controlled by high pressure liquid chromatography (HPLC), SDS-PAGE electrophoresis and identified by Western blots. We found that only the minor 55 kDa protein can be considered as being sarcolectin, while the major 65 kDa band results from the binding of some SCL molecules to albumin. The major biological functions, namely, stimulation of DNA synthesis and cell agglutination were preserved to the end of the last purification step. This work is requisite for establishing the molecular structure of SCL by recombinant DNA technology.

Chromosomal localization of the human proliferating cell nuclear antigen (PCNA) gene to or close to 20p12 by in situ hybridization

The proliferating cell nuclear antigen (PCNA/cyclin) is described as a nuclear protein and plays a key role in the initiation of cellular DNA synthesis and regulation of cell cycle progression. Using in situ hybridization, the human PCNA gene is localized on chromosome 20 at or close to 20p12.

Suppression of block to entry into S phase in cell-cycle mutants of rat 3Y1 fibroblasts after transformation by adenovirus type 12

Four temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts, belonging to separate complementation groups, cease to proliferate in the G1 phase of the cell cycle at a restrictive temperature (39.8°). These is mutants were transformed at a permissive temperature with adenovirus type 12 (Ad12), its E1 A gene, or in203S mutant of Ad12 which has a mutation in the E1 A 13 S mRNA unique region. We examined whether the proliferation of the transformed cells would be blocked in the G1 phase, at 39.8°. One mutant did not cease to proliferate at 39.8° after transformation with either Ad1 2 or E1 A. In two other mutants, Ad12-transformed cells did not cease to proliferate at 39.8°, whereas E1A-transformed cells did not survive at 39.8°, though they did continue to enter the S phase. Analysis of transcription of the viral early genes in the transformants of one of the latter two mutants suggests that the expression of other viral early genes, in addition to E1 A, is required for cell proliferation, in addition to entry into S phase. In the fourth mutant, both Ad12- and E1A-transformed sublines did not cease to enter the S phase but cells readily detached from the dishes. These results suggest that (1)function(s)of the E1A gene alone is sufficient for Ad12 to suppress the inhibition of the initiation of cellular DNA synthesis caused by four different cellular is defects, (2) functions of Ad12 early genes other than, or in addition to, E1 A are necessary for suppression of the inhibition of cell proliferation (and/or for survival) in two of the four is mutants, and (3) in the case of one other is mutant, E1 A alone overcomes the is defect and allows for the entire cell proliferation.

Induction of cellular DNA synthesis by adenovirus type 12 in a set of temperature-sensitive mutants of rat 3Y1 fibroblasts blocked in G1 phase

Four temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts, which represent separate complementation groups, cease to proliferate predominantly with a 2C DNA content, either at 39.8° (temperature arrest), or at 33.8° at a confluent cell density (density arrest). When infected at 39.8° with adenovirus type 12 (Ad 12), cells of all four is mutants in both arrest states entered the S phase, thereby suggesting that Ad 12 overcomes the four independent functional blocks to cellular entry into S phase. Results of experiments using Ad12 El-region mutants suggest that the El A gene product(s) is indispensable to overcoming the is block, whereas the El B product(s) may be dispensable. The cell killing observed in 3Y1 cells infected with wild-type Ad12 did not occur in infection with one of the El-region mutants with a 6-bp insertion in the El A 13 S mRNA unique region. When infected with this mutant at 39.8°, two is mutants of 3Y1 (3Y1tsF121 and 3Y1tsG125) in both arrested states proliferated through at least one generation. Another mutant (3Y1tsD123) was accelerated to die following entry into the S phase. In the other mutant (3YltsH203), the cell number was either unchanged (temperature arrest) or was increased less than twofold and then decreased (density arrest). The findings with the latter two mutant lines suggest that induction of cellular DNA synthesis is not sufficient for the subsequent proliferation of the infected cells, and that the Adl 2 gene function(s) does not directly rescue the primary lesions in these is mutants but does overcome some of the blocks to concomitantly occurring events. In the former two mutant lines, however, Ad1 2 gene function(s) may directly rescue the is lesions. We propose that the Ad12 gene product(s) can overcome blocks to the initiation of cellular DNA synthesis but cannot overcome blocks to events related to cell survival.

Cellular integrity is required for inhibition of initiation of cellular DNA synthesis by reovirus type 3.

Synchronized HeLa cells, primed for entry into the synthesis phase by amethopterin, were prevented from initiating DNA synthesis 9 h after infection with reovirus type 3. However, nuclei isolated from synchronized cells infected with reovirus for 9 or 16 h demonstrated a restored ability to synthesize DNA. The addition of enucleated cytoplasmic extracts from infected or uninfected cells did not affect this restored capacity for synthesis. The addition of ribonucleotide triphosphates to nuclei isolated from infected cells stimulated additional DNA synthesis, suggesting that these nuclei were competent to initiate new rounds of DNA replication. Permeabilization of infected cells did not restore the ability of these cells to synthesize DNA. Nucleoids isolated from intact or permeabilized cells, infected for 9 or 16 h displayed an increased rate of sedimentation when compared with nucleoids isolated from uninfected cells. Nucleoids isolated from the nuclei of infected cells demonstrated a rate of sedimentation similar to that of nucleoids isolated from the nuclei of uninfected cells. The inhibition of initiation of cellular DNA synthesis by reovirus type 3 appears not to have been due to a permanent alteration of the replication complex, but this inhibition could be reversed by the removal of that complex from factors unique to the structural or metabolic integrity of the infected cell.

Butyrate inhibits mouse fibroblasts at a control point in the G1 phase.

Butyrate block 3T6 cells in the G1 phase of the cell cycle approximately 5--6 h prior to the start of the S phase. Serum factors are required before as well as after the butyrate-sensitive steps in G1 in order to allow cells to start DNA synthesis. 3T6 cells infected with SV40 or with polyoma virus are also blocked at the same stage in G1 in the presence of the fatty acid. However, events before as well as after the butyrate-sensitive step do not require serum in virus-infected cells. The sensitivity of the initiation of cellular DNA synthesis to increasing concentrations of butyrate is the same for serum-stimulated or for virus-infected cells. A similar and parallel effect on DNA synthesis is observed if cells are incubated in the presence of very small amounts of cycloheximide. After release of the cycloheximide-induced G1 arrest about 4--6 h have to pass before cells enter the S phase. Cells stably transformed by SV40 are considerably more resistant to low cycloheximide concentrations and to butyrate. These data are discussed in the light of the hypothesis that both low concentrations of cycloheximide and sodium butyrate block cells at a control point in G1 by interference with the synthesis of one or more rapidly turning over, cell cycle-specific proteins.

Sequential appearance of Epstein-Barr virus nuclear and lymphocyte-detected membrane antigens in B cell transformation.

One of the central questions of Epstein-Barr (EB) virus biology concerns the nature of the asymptotic carrier state1,2 whereby the virus, which in vitro transforms B cells into permanent lymphoblastoid cell lines with such efficiency3,4, is harboured for life in the B lymphoid tissues of all previously-infected (seropositive) individuals5. It seems highly probable that this persistent infection is primarily controlled by memory T cells (cytotoxic precursors) specifically primed to the EB virus-induced lymphocyte-detected membrane antigen LYDMA6–9. Thus the degree to which the infection can progress without interruption in individual B cells of the immune host will depend upon the timing of LYDMA expression relative to that of other viral gene functions. We now demonstrate that during EB virus-induced B cell transformation in vitro LYDMA is expressed on the cell surface soon after the appearance of the virus-associated nuclear antigen EBNA10 and coincident with, but not dependent upon, the initiation of cellular DNA synthesis. This suggests that in the lymphoid tissues of the infected host, immunological control over virus-B cell interactions can be exercised early in the cycle at the very onset of virus-induced cell proliferation.

T antigen and initiation of cell DNA synthesis in a temperature-sensitive mouse line transformed by an SV40ts a mutant and in heterokaryons of the transformed cells and chick erythrocytes

The role of SV40 gene A product in initiation of cellular DNA synthesis was investigated, using a mouse kidney line [mKSA207] transformed by SV40tsA207. mKSA207 cells were temperature sensitive for growth, lost SV40 T antigen (Tag) when incubated in low serum at 40degreeC, and accumulated Tag in the cytoplasm when fed 10% serum and incubated at the nonpermissive temperature (39.7degreeC). Following serum addition, the percentage of mKSA207 cells synthesizing DNA was essentially the same at nonpermissive (39.7 degrees C) and permissive temperatures (33.5degreeC). The cells entered S phase asynchronously at both temperatures, but most cells entered S within 16 h, and before Tag accumulated. mKSA207 synchronized by a double thymidine block also synthesized DNA at 39.7degreesC and entered a second S phase. Tag-depleted or Tag-synchronized mKSA207, when fused with chick erythrocytes (CE), activated CE DNA synthesis. At nonpermissive temperatures (39.7degreesC), 40% of CE nuclei in heterokaryons with Tag-depleted mKSA207 displayed 3H-thymidine--labeled nuclei 28--40 h after fusion, when only 12% of CE nuclei were Tag+. The experiments indicate that SV40 gene A product probably does not have a direct role as initiator of cellular DNA synthesis.

Synthesis of a class of DNA-binding proteins in synchronized untransformed and virus-transformed cells.

The synthesis of a classs of proteins with affinity for denatured DNA has been studied in synchronized cultures of the established hamster fibroblast line HIL-1 and its virus-transformed derivative NIL-1-hamster sarcoma virus. It has been found that the synthesis of a DNA-binding protein (P6, molecular weight 50 000) in synchronized untransformed NIL-1 cells follows a pattern different from that observed in the transformed cells. The protein is low in stationary cultures of NIL-1 and NIL-1-hamster sarcoma virus but increases after the cells are stimulated to grow, although the time of maximal P6 synthesis relative to cellular DNA synthesis is different in NIL-1 and NIL-1-hamster sarcoma virus. In contrast, the pattern of synthesis of two other DNA-proteins (P7 and P8') in essentially identical in synchronized untransformed and transformed cells. P7 is very low in resting cultures of both types of cells, but greatly increases early after the cells are stimulated to divide, while P8'is hish in stationary cultures and decreases slightly after the initiation of cellular DNA synthesis has occurred.

Inhibition of the initiation of cellular DNA synthesis after reovirus infection.

The synthesis of cellular DNA was measured in synchronized L cells after reovirus infection. Initiation of the synthetic phase of the cell cycle was completely inhibited in cells infected 8 h before the beginning of DNA synthesis.

A working hypothesis explaining the maintenance of the transformed state by SV40 and polyoma

A model is presented that attempts to explain how SV40 or polyoma maintains the transformed state or phenotype. It is proposed that SV40 or polyoma codes for a protein involved in the initiation of cellular DNA synthesis (positive control element). This viral directed cellular DNA synthesis is different in some way from normal cellular DNA synthesis. This difference, in addition to other factors (possibly a second viral coded protein), results in a permanent cell surface alteration. This same cell surface alteration is observed transiently (only during mitosis) in the normal cell cycle. The permanent cell surface change permits the synthesis or activation of the viral positive control element for cellular DNA replication by-passing the normal cellular control for the initiation of DNA synthesis and allowing the cell to enter a new S period. This results in loss of density growth and expression of the transformed phenotype. The model is presented as a working hypothesis that makes specific predictions for new experiments.

Suppression of cellular DNA synthesis in growing cells by ultraviolet-irradiated adenoviruses

Ultraviolet (UV)-irradiated adenovirus 2 and 12 suppressed cellular DNA synthesis in growing human embryo kidney cells. The analysis of the mechanism of the suppression revealed that RNA and protein syntheses were not suppressed, thymidine kinase and DNA polymerase were produced normally, cellular DNA was not degraded, and the chain-elongation of nascent DNA proceeded normally. The autoradiograms showed that the suppression was due to the decrease in the number of cells synthesizing DNA, not to the decrease in the rate of DNA synthesis in a nucleus. These observations indicated that the initiation of cellular DNA synthesis was suppressed by UV-irradiated adenovirus. The suppressing activity was found in the UV-irradiated complete virion fraction (density 1.34). Quantitative analyses with purified virions revealed that suppression of cellular DNA synthesis was dependent on multiplicity of infection and 10 3 cell-associated virions suppressed 90-80% of cellular DNA synthesis.

Viral Genome And Oncogenic Transformation: Nuclear And Plasma Membrane Events

This chapter deals with the nature of oncogenic viruses, the properties of the different fractions of viral genome, and the interaction of the viral genome and the target cell. It illustrates the modification of the nucleus during the transformation of the cell, and particularly the chromosome modifications, the antigenic modifications, and the regulation of the expression of the viral genome. The first established fact is the integration of the genetic information of viral origin into the stock of cellular genetic information. The exact site of this integration is not known, but it is well established that it takes place at the chromosome level. It has been shown that the important point of the transformation is that the virus does not undergo a complete vegetative cycle. The question is whether this incapacity to multiply is of viral origin or of cellular origin. The chapter shows two contradictory hypotheses: (1) one calls upon an inhibitor of cellular origin in the nonpermissive cells, (2) the other is in favor of a viral inability and would prove the viral deficiency in this type of cells. Once the integration is carried out, the interesting phenomena are the consequences of the expression of the genetic information of viral origin at the level of the cell. For a long time, the initiation of cellular DNA synthesis is considered as one of the essential actions of the viral genome in the transformed cell.

Induction of specific antitumor immunity in hamsters with hydroxylamine-inactivated SV40 virus

This chapter deals with the nature of oncogenic viruses, the properties of the different fractions of viral genome, and the interaction of the viral genome and the target cell. It illustrates the modification of the nucleus during the transformation of the cell, and particularly the chromosome modifications, the antigenic modifications, and the regulation of the expression of the viral genome. The first established fact is the integration of the genetic information of viral origin into the stock of cellular genetic information. The exact site of this integration is not known, but it is well established that it takes place at the chromosome level. It has been shown that the important point of the transformation is that the virus does not undergo a complete vegetative cycle. The question is whether this incapacity to multiply is of viral origin or of cellular origin. The chapter shows two contradictory hypotheses: (1) one calls upon an inhibitor of cellular origin in the nonpermissive cells, (2) the other is in favor of a viral inability and would prove the viral deficiency in this type of cells. Once the integration is carried out, the interesting phenomena are the consequences of the expression of the genetic information of viral origin at the level of the cell. For a long time, the initiation of cellular DNA synthesis is considered as one of the essential actions of the viral genome in the transformed cell.