Host DNA Synthesis Research Articles

Growth patterns of temperature-sensitive mutants of Western equine encephalitis virus in cultured Aedes albopictus (mosquito) cells.

Several temperature-sensitive (ts) mutants of Western equine encephalitis virus (WEEV) have been isolated previously from persistently infected cultures of mosquito cells and divided into three groups: early passage RNA- mutants, early passage RNA+ mutants and late passage RNA- mutants (Maeda et al., 1979(. The growth patterns of these groups, as well as of several ts mutants isolated after chemical mutagenesis and of wild-type (wt) WEEV, have been compared in BHK cells and in two strains of mosquito cells. The late passage ts mutants grew much better in mosquito cells than either the wt WEEV or the chemically induced mutants. When mosquito cells were co-infected with a late passage mutant (A125) and Wt WEEV, infectious virions of both parental types as well as phenotypically mixed particles were produced. Infection of mosquito cells with WEEV resulted in a slight suppression of host DNA and protein synthesis during the acute stage of the infection (the first 1 or 2 days). Virus growth in a line of cloned mosquito in which WEEV produced a cytopathic infection (c.p.e.) was analysed with the result that the viruses could be divided into two groups: one in which wt WEEV, chemically induced ts mutants and early passage RNA+ mutants all induced maximal c.p.e., and another in which late passage RNA- mutants and one early passage RNA- mutant induced very little c.p.e., but released much more infections virus into the culture fluid. Electron microscopy showed that in these cloned mosquito cells infected with a virus of the first group, large amounts of virus accumulated on or in the plasma membrane.

Coordinate control of host DNA synthesis and measles virus production of persistently infected BHK-21 cells.

Measles virus, upon infection, caused cytopathic effects in BHK-21 cells while it readily established persistent infection in a mutant strain of BHK-21 cells which possessed a temperature sensitive defect in DNA synthesis (BN2). This finding demonstrated that host function can be involved in allowing the establishment of persistent infection. When BN2 and infected BN2 cells were incubated at the non-permissive temperature (39.5 degrees C), DNA synthesis was inhibited in a similar manner in both cells types, suggesting that viral functions do not interfere with the expression of the host mutation. The ts mutation which blocked the DNA synthesis also caused inhibition of viral yield, viral antigen expression and viral RNA synthesis at 39.5 degrees C. The results suggest that continued DNA synthesis is necessary for the expression of measles functions.

The morphology of staphylococcal bacteriophage K and DNA metabolism in infected Staphylococcus aureus.

The morphology and dimensions of bacteriophage K particles were determined by electron microscopy. This virus had an icosahedral head (approx. 70 nm diam.) and a long (210 nm) thin (15 nm) contractile tail which terminated in a complex basal appendage. The precise dimensions of the particles were dependent on the negative stain employed. The buoyant densities of the K virus particle and its DNA were 1 X 479 g/ml and 1 X 689 g/ml respectively. The DNA had a base composition of 30% G + C, a contour length of 16 X 1 micron and a calculated mol. wt. of 33 X 10(6). With Staphylococcus aureus (NCTC 9318) as host, the latent period was 25 min, the eclipse period 14 min and the average burst size 60 p.f.u./bacterium. Infection resulted in inhibition of host DNA synthesis and degradation of the bacterial DNA: the products were used for tee synthesis of phage DNA. The kinetics of DNA synthesis is infected and uninfected bacteria were examined. There was no initial cessation of DNA synthesis in the infected bacteria.

Inhibition of host DNA synthesis by adenovirus infection is reversed at elevated temperatures.

Infection of KB cells by adenovirus normally results in a drastic reduction of host DNA synthesis at 33 to 37 degrees C. This inhibition was largely reversed by raising the temperature to 41 degrees C. Inhibition was reinstated if the temperature is lowered.

Visualization of genetic recombination intermediates of human adenovirus type 2 DNA from infected HeLa cells.

The study of recombination in prokaryotes has been facilitated by the availability of recombinational mutants and simple genetic elements such as phages and plasmids. These small but defined molecules of DNA have been especially useful for electron microscopic analysis of structural detail of molecules undergoing recombination both in vivo and in vitro. A limitation in the structural analysis of plasmid recombination is the absolute number of recombining molecules which can be identified and analysed amidst a background of nonrecombining molecules. This limitation would be of even greater consequence in studies of genetic recombination in animal cells. We therefore chose virus-infected animal cells as a model system for the study of the molecular mechanism of genetic recombination in higher organisms. HeLa cells infected with adenovirus serotype 2 (Ad-2) offer several advantages for studying recombination: (1) the virus contains a small and well characterized genome of about 35 kilobases; (2) a large amount of Ad-2 DNA is accumulated during lytic infection and host DNA synthesis is suppressed; (3) Ad-2 recombines at a very high frequency; and (4) similar to phages, animal viruses and Ad-2 in particular are believed to use many of the host cell's enzymes in necessary metabolic processes, presumably including recombination. In this study we used electron microscopic techniques to visualize the structures of in vivo Ad-2 DNA recombination intermediates. Molecules were observed with structures at putative cross-over points which were consistent with the molecular mechanism of recombination proposed by Holliday. In addition, we observed Ad-2 DNA molecules engaged in recombination which were simultaneously serving as templates for replication and/or transcription. To the best of our knowledge, this is the first visualization of in vivo recombination intermediates of discrete DNA molecules isolated from eukaryotic cells.

Cell cycle-dependent replication of the DNA of minute virus of mice, a parvovirus

The formation of double-stranded viral DNA was examined in synchronized cells infected with minute virus of mice in early G 1 phase. In the infected cells, a minimum of 50–100 copies of the input single-stranded DNA have been converted to a double-stranded form by mid S phase. In well-synchronized cells, the amount of double-stranded viral DNA detected during G 1 is on the order of a few copies per cell or less. When cells are infected in the presence of the thymidine analog, 5-bromodeoxyuridine, viral DNA synthesis is inhibited. However, 5-bromodeoxyuridine does not inhibit host DNA synthesis nor does it prevent replication of viral DNA if added to the infected cells in late S phase. Viral DNA replication first becomes resistant to 5-bromodeoxyuridine inhibition at the beginning of S phase. As 5-bromodeoxyuridine appears to specifically block early steps in viral DNA synthesis but not the subsequent replication of the DNA, the conversion of the input viral genome to a double-stranded form which undergoes further replication appears to be an S phase-specific event.

Synthesis of thymine and alpha-putrescinylthymine in bacteriophage phi W-14-infected Pseudomonas acidovorans

Host DNA synthesis stopped about 10 min after the infection of Pseudomonas acidovorans with bacteriophage phi W-14, but host DNA was not degraded to acid-soluble fragments. The synthesis of host but not of phage DNA was inhibited by 5-fluorodeoxyuridine. The nucleotide pools of infected cells did not contain dTTP, and infection resulted in the appearance of dTTPase activity. Although ornithine labeled the alpha-putrescinylthymine residues of phi W-14 DNA, ornithine-labeled nucleotides were not detected in infected cells. A new deoxynucleoside triphosphate did appear in infected cells, but it was not labeled by ornithine. It is concluded that the thymine and alpha-putrescinylthymine in phi W-14 DNA are synthesized at the polynucleotide level.

The problem of host DNA synthesis in murine cytomegalovirus-infected cells

Murine cytomegalovirus (MCMV) infection in mouse fibroblasts resulted in an early inhibition in thymidine incorporation into cell DNA. In contrast [32P]phosphate incorporation was only slightly inhibited, while the incorporation of deoxycytidine, deoxyadenosine, and deoxyguanosine continued unabated. The limiting factor in thymidine incorporation was a decrease in uptake by the infected cells. Uptake of the other nucleosides, however, was either normal or enhanced. Nuclear monolayers, derived from infected cells, incorporated thymidine triphosphate into cell DNA at the same rate as nuclei of uninfected cells. Measurement of total DNA content of the cells revealed no difference between infected and control cultures. Thus MCMV neither inhibits nor stimulates cellular DNA synthesis.

The induction of cellular DNA synthesis in G1-arrested BKH21 cell infected with adenovirus type 2.

G1-arrested BHK21 cells infected with adenovirus type 2 were studied to determine the effect of infection on host DNA synthesis in a productive cycle of infection. At various intervals following infection, analysis of the intracellular DNA was carried out by zonal sedimentation centrifugation through alkaline sucrose gradients. In addition to material which sedimented to the position of the virus markers (34S), a class of high mol. wt. DNA (40 to 100S) was found to be synthesized beginning approx. 13 h p.i. and continuing up to about 35 h p.i. DNA--DNA hybridization studies on this newly synthesized DNA showed it to be cellular DNA. When this material was density labelled with 5-bromodeoxyuridine and centrifuged to equilibrium through alkaline CsCl gradients, it was found to be the product of semi-conservation replication and not of repair synthesis.

The isolation of SV40 tsA/deletion, double mutants, and the induction of host DNA synthesis

Simian virus 40 mutants were constructed that contain both a tsA mutation leading to temperature sensitivity of the 92K T-antigen, and deletions of 20–200 base pairs leading to a loss in the expression of the 20K t-antigen. As expected, these mutants were temperature sensitive for viral growth and viral DNA replication in lytically infected cells. At nonpermissive temperatures, the ts/deletion mutants stimulated the incorporation of nucleosides into host DNA as did the tsA mutant alone. This induction of incorporation by the tsA mutants resulted from semiconservative DNA replication, not repair synthesis. At 200 μg/ml caffeine the induction of host DNA by A 209 was inhibited by 30 to 50%, whereas induction by the ts/deletion mutants was abolished.

Enhanced replication of herpes simplex virus type 1 in BHK-21 cells transformed by hamster sarcoma virus.

Untransformed and hamster sarcoma virus-transformed BHK-21 cells were compared for their ability to support productive infection with herpes simplex virus type 1. In both types of cells, infection with herpes simplex virus resulted in a lytic, productive cycle. However, inhibition of host cell RNA and DNA synthesis, transcription and replication of viral DNA, and production of infectious virus began earlier and reached higher levels in transformed cells.

Simian virus 40 small t antigen is not required for the maintenance of transformation but may act as a promoter (cocarcinogen) during establishment of transformation in resting rat cells

Simian virus 40 deletion mutants affecting the 20,000-dalton (20K) t antigen and tsA mutants rendering the 90K T antigen temperature sensitive, as well as double mutants containing both mutations, induced host DNA synthesis in resting rat cells at the restrictive temperature. Nonetheless, the deletion mutants and double mutants did not induce transformation in resting cells even at the permissive temperature. On the other hand, the deletion mutants did induce full transformants when actively growing rat cells were infected; the transformants grew efficiently in agar and to high saturation densities on platic. The double mutants did not induce T-antigen-independent (temperature-insensitive) transformants which were shown previously to arise preferentially from resting cells. Thus, small t antigen was dispensable for the maintenance of the transformed phenotype in T-antigen-dependent rat transformants (transformants derived from growing cells) and may play a role in the establishment of T-antigen-independent transformants. We attempt to establish a parallel between transformation induced by chemical carcinogens and simian virus 40-induced transformation.

DNA synthesis in temperature-sensitive mutants of the cell cycle infected by polyoma virus and adenovirus.

tsAF8 cells are a temperature-sensitive (ts) mutant of BHK cells that are arrested in G1 at the nonpermissive temperature. When made quiescent by serum restriction, they can be stimulated to enter S phase by 10% serum at 34 degrees C but not at 40.6 degrees C. The same results can be obtained if quiescent cells are infected with polyoma virus or adenovirus 12 instead of serum. However, adenovirus 2 infection stimulates DNA synthesis in tsAF8 cells at both 34 degrees C and 40.6 degrees C. The DNA synthesized after adenovirus 2 infection has been shown to be cellular DNA by CsCl density centrifugation. By density labeling it can be shown that adenovirus 2-induced DNA synthesis is due to semiconservative replication. The difference between adenovirus 2 and polyoma (or serum) is also evident with another ts mutant of BHK cells, ts13 cells. These results open the possibility of identifying the viral or cellular mechanism at the basis of this difference in the induction of host DNA synthesis between adenovirus 2 and polyoma or serum.

Induction of host DNA synthesis and DNA polymerase by DNA-negative temperature-sensitive mutants of human cytomegalovirus

Four DNA-negative temperature-sensitive ( ts) mutants of human cytomegalovirus, belonging to different complementation groups, were studied for their ability to induce cell DNA synthesis and DNA polymerase in permissive human embryo lung (HEL) and nonpermissive rabbit lung (RL) cells. These is mutants stimulated host cell DNA synthesis in HEL and RL cells and DNA polymerase activity in HEL and RL cells at permissive (33.5°) and nonpermissive temperatures (39.5°). Salt stimulation of induced DNA polymerase activity was used to distinguish between virus and cell DNA polymerase from HEL cells. DNA polymerase activity was stimulated by 100 m M (NH 4) 2SO 4 at either 33.5 or 39.5° in cultures infected with three of the mutants ( ts 9, is 153, and ts 155). However, DNA polymerase activity was not stimulated by 100 m M (NH 4) 2SO 4, in cultures infected with one of the ts mutants ( ts 13). These data suggest that at least four cistrons control the synthesis of virus DNA and that virus DNA synthesis is not required for the induction of cell DNA synthesis.

Anatomy of herpes simplex virus DNA. XI. Apparent clustering of functions effecting rapid inhibition of host DNA and protein synthesis

Herpes simplex virus type 2 (HSV-2) strains inhibit the synthesis of both DNA and protein of the host cell more rapidly than HSV-1 strains. Several intertypic HSV-1 X HSV-2 recombinants and parental strains were examined for their ability to inhibit rapidly the synthesis of host protein and DNA. The two functions cosegregated in all of eight recombinants tested and are therefore controlled by the same gene or by different genes in the same region of the viral DNA.

Studies on Polyoma Virus DNA Replication in Synchronized C3H2K Cells

In G1-arrested cells infected between 1 and 12 h after having been stimulated by fresh serum to progress to S phase, polyoma virus DNA synthesis proceeded in the first half of S phase, and virus and whole cellular DNA accumulated at about the same time. However, in cells infected later than 14 h after serum stimulation, virus DNA synthesis was shifted to the next S phase. Thus, a permissive cell attains competence for polyoma virus DNA replication at a precise moment during an S phase initiated by fresh serum, which can efficiently replace the early virus host DNA stimulation function. When cells were incubated in serum that had lost its capacity to stimulate host DNA synthesis by pre-absorption with growing cells, normal yields of polyoma DNA could nevertheless be observed, which shows that extensive replication of host DNA does not seem to be an obligatory condition for virus DNA replication.

Effects of Nucleoside Analogs on Epstein-Barr Virus-Induced Transformation of Human Umbilical Cord Leukocytes and Epstein-Barr Virus Expression in Transformed Cells

Two methods of assay, measuring (i) stimulation of host cell DNA synthesis by [(3)H]thymidine incorporation and (ii) morphological transformation in microtiter plates, were employed to determine what effect treatment during infection with adenine arabinoside or 5-iodo-5'-amino-2',5'-dideoxyuridine has on Epstein-Barr virus (EBV) (B95-8)-induced transformation of human umbilical cord leukocytes. It was found that adenine arabinoside inhibited EBV-induced transformation in a dose-dependent manner in both assays, beginning at drug concentrations (<2 mug/ml) which had little effect on either spontaneous or phytohemagglutinin-induced host cell DNA synthesis. Adenine arabinoside was more effective in inhibiting morphological transformation than EBV-induced host DNA synthesis. Adenine arabinoside treatment was also effective in reducing both EB viral capsid antigen expression and production of biologically active extracellular transforming virus in EBV-transformed cells. In contrast, 5-iodo-5'-amino-2',5'-dideoxyuridine, which inhibited herpes simplex virus replication, had little effect on EBV-induced transformation as measured by either method of assay. However, 5-iodo-5'-amino-2',5'-dideoxyuridine was found to be effective in inhibiting viral capsid antigen expression and production of extracellular transforming virus in EBV-transformed cells.

Stimulation of host DNA synthesis and induction of early antigens by ultraviolet light irradiated human cytomegalovirus

The ultraviolet (UV-)sensitivity of the human cytomegalovirus (HCMV) genes coding for very early complement fixing and early antigens in human embryonic fibroblasts (HEF) and mouse embryonic fibroblasts (MEF) and the relation of these genes to the ability of the virus to stimulate host cell DNA synthesis were investigated. After 14 minutes of UV-irradiation of the virus inoculum only the very early complement fixing nuclear antigen (CMNA) developed in the HEF cells and only the early cytoplasmic antigen(s) was present in the MEF. In both HEF and MEF, host cell DNA synthesis was stimulated. We conclude that the ability of HCMV to stimulate host DNA synthesis is an early function of the viral genome and shows a high resistance to UV-irradiation. There is no direct correlation, however, between the ability of the virus to stimulate host cell DNA synthesis and the genes which code for the CMNA or for early cytoplasmic antigens.

Effect of spermidine on bacteriophage P22 infection

The effect of spermidine on phage P22 infection of Salmonella typhimurium has been found to depend on the time of addition of spermidine with respect to the time of addition of the phage and also on the composition of the growth medium. If spermidine was added prior to or within a short time after infection, the cells survived. Under this condition the invading DNA appeared to remain trapped in the cell membrane, and there was no expression of the phage genome. If spermidine was added after the initiation of the infective process, the replication of the phage was inhibited but the cells did not survive. If spermidine was added after DNA synthesis was over, there was no effect of spermidine on phage multiplication. Spermidine was found to affect phage DNA synthesis but not host DNA synthesis.

Suppression of the Synthesis of Cellular Macromolecules by Herpes Simplex Virus

Synthesis of cellular protein was substantially inhibited within 1 h of infection with herpes simplex virus, type 2, strain G (HSV-2). The inhibition also occurred, although no virus-specific protein synthesis was detected, after infection with u.v. irradiated virus and in cytoplasts that had been enucleated before infection. The inhibitory activity could not be distinguished from infectivity by dilution, sedimentation or reaction with gamma-globulin. HSV-2 also suppresssed the synthesis of Sendai virus proteins, but not those specified by HSV-1. Host protein synthesis was no more sensitive than virus protein synthesis to an increased concentration of NaCl in the medium, nor could the suppression of host synthesis be prevented by adding excess MgCl2 to the medium or by omitting CaCl2 or NaCl. It was accompanied by the breakdown of polyribosomes, which also occurred in the presence of cycloheximide but not at 4 degrees C. The breakdown yielded ribosomes that were sensitive to a high salt concentration, unlike those produced by treatment of polyribosomes with RNase. The synthesis of cellular DNA and RNA was also inhibited following infection with u.v.-inactivated virus. It is concluded that the suppression of host protein synthesis (and probably also of host DNA and RNA synthesis) is caused by a constituent of the infecting virus particles. The mechanism is obscure but probably does not depend on the leakage out of the cell of Mg2+ or into the cell or Ca2+ or Na+ ions, nor on the specific inhibition of initiation of host polypeptide chains, nor on RNase-like attack on host polyribosomes.