Intrinsic resistance to viral infection. Mouse macrophage restriction of herpes simplex virus replication

Macrophages isolated from mice resistant to acute (lethal) infection with a neurovirulent isolate of HSV-1 express intrinsic resistance to viral infection in vitro. Bone marrow (BM), spleen (S), peritoneal (P), and thioglycolate-stimulated peritoneal (Pthio) macrophages isolated from resistant C57BL/6 Cr (B6) mice consistently restrict HSV-1 macromolecular synthesis earlier in the viral replicative cycle than do macrophages isolated from the same tissue sources from more susceptible DBA/2Cr (D2) mice. B6-BM (BM macrophages from B6 mice) restrict HSV macromolecular synthesis at least at two points in the replicative cycle: 1) before the onset of alpha-protein synthesis and 2) between the onset of gamma 1 protein and DNA synthesis. D2-BM macrophages restrict HSV replication at about the time of DNA synthesis. B6-P macrophages restrict HSV replication shortly after gamma 1 protein synthesis, and D2-P macrophages inhibit the virus slightly later, but before DNA synthesis. B6-S macrophages restrict HSV replication at about the time of DNA synthesis, and D2-S macrophages inhibit replication after the onset of gamma 2 protein synthesis. Pthio macrophages are more permissive to HSV infection than BM, P, or S macrophages: restrictions in viral replication occur at the time of DNA synthesis in B6-Pthio macrophages, and after the onset of gamma 2 protein synthesis in D2-Pthio cells. These studies demonstrate that isolated macrophages from inbred mouse strains express intrinsic resistance to HSV infection that correlates with in vivo resistance to acute (lethal) infection. Intrinsic resistance to HSV-1 infection is due to restriction of viral macromolecular synthesis. HSV replication is inhibited in macrophages at multiple points in the viral growth cycle, depending on the tissue from which the cells are isolated.     

Viral and Host Deoxyribonucleic Acid Synthesis in Shope Fibroma Virus-infected Cells as Studied by Means of High-Resolution Autoradiography

Incorporation of (3)H-thymidine by BSC-1 cells infected with Shope fibroma virus was studied by means of high-resolution electron microscopic radioautography. One-hour pulses with the radioactive precursor were given at various times after infection, during a one-step growth cycle of the virus. In the cytoplasm of infected cells, reacted grains occurred over foci of viroplasm; these foci are believed to represent the true sites of viral deoxyribonucleic acid (DNA) replication. Shope fibroma virus DNA synthesis began before 3 hr postinfection, reached a maximum at 8 to 9 hr, and then declined rapidly. It was demonstrated that the decline in (3)H-thymidine uptake is correlated with the onset of viral morphogenesis. In comparison with the noninfected culture, the nuclear labeling, which reflects host DNA metabolism, was slightly reduced by 4 hr postinfection. Inhibition became more marked as infection progressed, and host DNA synthesis was almost completely suppressed in late stages of viral development.     

Investigations into the inhibition of DNA repair processes by detergents.

The inhibition of semi-conservative DNA replication and unscheduled DNA synthesis by three different detergents was investigated in mouse spleen cells. The investigations were made by following the kinetics of the incorporation of 3H-thymidine into the DNA, as well as by autoradiographic studies. At various concentrations different inhibition patterns were found and the significance of these findings is discussed.     

RNase H and replication of ColE1 DNA in Escherichia coli

Amber mutations within the rnh (RNase H) gene of Escherichia coli K-12 were isolated by selecting for bacteria capable of replicating in a sup+ background replication-defective cer-6 mutant of the ColE1 replicon. The cer-6 mutation is an alteration of one base pair located 160 nucleotides upstream of the unique replication origin of this plasmid. Subsequently, we determined the DNA alterations present within these mutants. ColE1 DNA replicated in rnh(Am) recA cells, indicating that (i) RNase H, which has been shown to be absolutely required for in vitro initiation of ColE1 DNA replication, is dispensable in vivo, and (ii) ColE1 replication in the absence of RNase H is not dependent on "stable DNA replication," which has been reported to be an alternative mode of chromosomal DNA replication. Another class of bacterial mutations was also isolated. These mutations, named herB, suppressed cer-6 replication in rnh+ bacteria. herB mutations mapped close to the polA gene on the E. coli chromosome and increased the activity of DNA polymerase I. These findings suggest that when the DNA polymerase I has an opportunity to initiate DNA synthesis before RNase H acts, the replication-defective cer-6 mutant or the wild-type ColE1 replicates in E. coli.     

DNA replication in mammalian cells after the action of physical, chemical or biological factors. VI. The recovery of DNA synthesis in a culture of irradiated cells

The kinetics of DNA synthesis restoration in cultured HeLa cells and in L-929 mouse fibroblasts irradiated by gamma-rays of 60Co with a dose of 10 Gy was studied. Early after irradiation the rate of DNA synthesis in HeLa cells measured with 3H-thymidine incorporation was seen to decrease. Two hours later the incorporation starts to increase to reach the control level 4 hours after irradiation and then becomes even higher than this level. The distribution of cells among phases of the cell cycle measured with flow cytometry undergoes changes. 4-6 hours after irradiation part of S-phase cells increased contributing presumably to the elevating of 3H-thymidine incorporation observed at this time. The restoration of the incorporation was suppressed by inhibitors of protein and RNA synthesis--cycloheximide and actinomycin D. It is suggested that the processes of restoration of DNA synthesis in irradiated cells can be of inducible nature. In irradiated HeLa and L-929 cells the restoration of DNA synthesis is resistant to novobiocin, an inhibitor of DNA replication.     

Repair Replication of HeLa Cell Deoxyribonucleic Acid in Cells Infected with Newcastle Disease Virus or Mengovirus or Treated with Puromycin

We examined repair replication of HeLa cell deoxyribonucleic acid (DNA) in cells infected with mengovirus or Newcastle disease virus or treated with puromycin. Cellular DNA was damaged by ultraviolet light and then pulse-labeled with (3)H-thymidine. Autoradiographic analysis of non-S-phase DNA synthesis (repair replication) showed that there was no inhibition of this process at a time when overall cellular DNA synthesis was severely inhibited by either virus infection or puromycin treatment.     

Participation of the intracellular enzymes in the control of the mutation process

Summary The influence of repair and replication on the frequency of spontaneous chromosome aberrations and of those induced by gamma-irradiation is reported.Using the technique of labelling DNA with radioactive ³H-thymidine and measuring the radioactivity of DNA isolated from embryos, the time of initiation and the duration of DNA synthesis in barley seeds was studied after the soaking of the seeds had begun. The average duration of each phase of the first DNA synthesis cycle in soaking barley seeds was found to be as follows: pre-DNA synthesis stage, 10–11 hrs; DNA synthesis stage, 8 hrs. After gamma-irradiation, the intensity of DNA synthesis decreased and the beginning of DNA synthesis was delayed.It was found that the inhibition of repair by caffeine led to an increase in the frequency of both spontaneous and induced chromosome aberrations. Caffeine enhanced several times the frequency of chromosome and chromatid aberrations at the time of the maximal activity of repair enzymes. During DNA replication, caffeine had a lower effect on the realization of premutational lesions.An inhibitor of DNA replication — hydroxyurea — had no influence on the frequency of spontaneous chromosome aberrations during the replication period, whereas after gamma-irradiation, hydroxyurea enhanced the frequency of aberrations mainly at the stage of DNA replication.The relatively small mutagenic action of both agents (caffeine and hydroxyurea) was observed during all stages of the cell cycle of germinating barley seeds.     

Cytotoxic activity of lymphocytes. VII. cellular origin of alpha-lymphotoxin.

To detect the cellular origins of alpha-lymphotoxin (alpha-LT), we cultured various subpopulations of human blood lymphocytes separated by erythrocyte-rosetting techniques with various mitogens. T cell-enriched subpopulations responded to PHA by increased 3H-thymidine uptake into DNA and large amounts of alpha-LT production. SPL and Con A-Sepharose stimulated DNA synthesis in T cell-enriched cultures if the macrophage content was greater than 1.5%; however, alpha-LT production was not induced by these two mitogens even when reconstituted with 10% macrophages. B and/or null cell-enriched populations severely depleted of T cells (less than 0.7% did not respond to PHA, SPL, or Con A-Sepharose. However, reconstitution to 5 or more percent in E-RFC allowed all three mitogens to stimulate DNA synthesis and alpha-LT production. The LT made by all cell populations 5 and 7 days after stimulation were equally neutralized by a heterologous antiserum to alpha-LT. These results show that human T and B and/or null cells, when appropriately stimulated, can produce alpha-LT.     

Stimulating effect of histones on DNA synthesis in the regenerating rat liver]

Effect of exogenous histones, nuclear globulins and acid proteins on DNA synthesis is studied in regenerating liver of rats in which the synthesis of their own proteins and thus DNA replication are inhibited by cycloheximide. In these conditions histones from regenerating rat liver are found to stimulate 3H-thymidine incorporation into DNA of hepatectomyzed rat liver. Nuclear globulins and acid proteins from regenerating liver, and histones from intact liver produced no stimulating effect on DNA sythesis.     

3H-thymidine incorporation into the macrophages in the process of eye regeneration in adult tritons]

Changes in the number of labelled macrophages in the regenerating eye cavity of the adult common newts were studied within 2 to 12 days following the injection of 3H-thymidine and removal of retina and lens from the eyes. A small number of labelled macrophages was found in the eye cavity (0,2--5.9%) at all regeneration stages under study upon the pulse 3H-thymidine incorporation. Their number rapidly increased and attained by the end of observation (12 days) 73%. These results suggest that mainly mature non-dividing forms of macrophages which completed the mitotic cycle S-phase in the places of their active reproduction migrate in the eye cavity. The sharp increase of the number of labelled macrophages in the eye cavity is determined by the migration of those macrophages the precursors of which were labelled as a result of both the pulse 3H-thymidine incorporation and reutilization of the labelled precursors of DNA synthesis. New portions of labelled macrophages migrate in the eye cavity within 2 to 4 days.     

Size distribution and molecular polarity of nascent DNA in a temperature-sensitive dna G mutant of Escherichia coli

Summary In the dna G t.s. strain BT 308, made lysogenic for the phage , nascent DNA was labelled by short pulses of ³H-thymidine, isolated and separated as a function of size by alkaline sucrose gradient sedimentation. The molecular polarity of the labelled DNA was then determined by hybridization to the separated strands of DNA.At 30° C, strand r DNA, made in the direction opposite that of fork movement, is synthesized in the form of short pieces. The first observable consequences of a shift to 42° C are the preferential inhibition of strand r synthesis and the small amount of strand r DNA which is made is recovered in long pieces of DNA rather than in short fragments. This indicates that the t.s. product, in strain BT 308, may be involved in the synthesis of the strand growing in the direction opposite that of replication fork movement.Newly synthesized strand l DNA, made in the same direction as replication fork movement, is found in long pieces in wild-type bacteria; it is found in pieces of intermediate size in strain BT 308 at 30° C as well as at 42° C. This indicates additional differences in the replication machinery between strain BT 308 and wild-type bacteria.     

Effect of arabinosyl cytosine on the level of DNA polymerase and thymidine kinase activity in PHA-stimulated human tonsillar lymphocytes

Summary The effect of arabinosyl cytosine (ara-C) was studied on the uptake, phosphorylation and incorporation of ³H-thymidine in human tonsillar lymphocyte cultures is described along with its effect on the level of DNA polymerase and thymidine kinase activities induced by phytohaemagglutinin (PHA). Freshly isolated tonsillar lymphocytes are stimulated cells with a remarkably high activity of DNA polymerase a and thymidine kinase. During in vitro culture, these stimulated cells are transformed to the resting state with low DNA polymerase and thymidine kinase activity. However, a new DNA synthesising cycle can be induced by PHA with maximum at 48 h.10^–6 M ara-C inhibited the incorporation of ³H-thymidine by 90–95%. This inhibition may be reversed by rinsing the cells. The inhibition of the transport of ³H-thymidine seems to be only a consequence of the inhibitory effect of ara-C on the DNA polymerisation reaction, because at 10 °C, where DNA synthesis was arrested, ara-C does not influence the uptake and the phosphorylation of ³H-thymidine.Ara-C (10^–6 M) abolished also the PHA induced elevation of DNA polymerase a and thymidine kinase activities without influencing protein synthesis of the cell. This supports a coordinated regulation mechanism between DNA synthesis and the synthesis of enzymes involved in DNA replication.     

Comparative effects of somatomedin C and insulin on the metabolism and growth of cultured human fibroblasts

At concentrations of 25 ng/ml in serum-free medium, somatomedin C (SM-C) and insulin stimulated 3H-thymidine incorporation in adult human fibroblasts 4- and 1.5-fold, respectively. The presence of 0.25% human hypopituitary serum (HHS), which by itself had little effect, enhanced the mitogenicity of both SM-C and insulin. Furthermore, 10(-7)M dexamethasone dramatically potentiated SM-C stimulation (70-fold) and insulin stimulation (28-fold) of 3H-thymidine incorporation. With dexamethasone and 0.25% HHS, significant stimulation of DNA synthesis was seen at 2.5 ng/ml for both SM-C and insulin. The effects of SM-C and insulin on 3H-thymidine incorporation were additive. These 3H-thymidine incorporation results were clearly supported by cell replication studies. On the other hand, SM-C and insulin had equivalent, nonadditive effects on RNA and protein synthesis and protein degradation. Half-maximal effects were seen for both peptides on all three metabolic processes at 2-5 ng/ml. In contrast to their synergism with SM-C in the stimulation of DNA synthesis and cell replication, HHS and dexamethasone did not enhance SM-C stimulation of RNA or protein synthesis or protein degradation. These data indicate that SM-C and insulin stimulate DNA, RNA, and protein synthesis, protein degradation, and cell replication in adult human fibroblasts at nanomolar concentrations, suggesting that each peptide is capable of acting through its own receptor. Both SM-C and insulin are also capable of synergism with low concentrations of serum and dexamethasone in the stimulation of DNA synthesis and cell replication. It is proposed that SM-C and insulin both participate in the regulation of cell growth and metabolism in vivo.     

Inhibition of macromolecular synthesis in cultured macrophages by Pseudomonas pseudomallei exotoxin

Pseudomonas pseudomallei exotoxin was found to be a potent inhibitor of protein and DNA synthesis in cultured macrophages. Inhibition of DNA synthesis occurred at toxin concentrations as low as 1-2 micrograms/ml and inhibition of 3H-thymidine uptake was almost complete at concentrations of 8 micrograms/ml or more. A close correlation between cell damage and inhibition by DNA synthesis was observed. For protein synthesis, inhibition was obtained at much lower doses (0.06-2.0 micrograms/ml) of the toxin. At similar toxin concentrations, DNA synthesis was marginally affected. Further, it was shown that protein synthesis inhibition occurred almost immediately after incubation, reaching its maximal inhibitory effect of 70% after 6 hr. DNA synthesis, however, was minimally affected by a similar toxin concentration even after 10 hr of incubation. The inhibition of macromolecular synthesis in macrophages by P. pseudomallei exotoxin may be relevant to its modulatory effect on the host defense mechanism.     

The effect of silica-treated macrophages on the synthesis of collagen and other proteins in vitro

Treatment with SiO₂ releases from peritoneal macrophages a soluble factor which stimulates the synthesis of collagen and other proteins in incubated slices of experimental granulation tissue. This factor can also be obtained by SiO₂-treatment from certain subcellular particles of intact macrophages. A similar agent is released from the macrophages by incubation with rheumatoid synovialtissue extract. Macrophages induced by paraffin or thioglycollate medium cannot be stimulated further by SiO₂. The SiO₂-treated macrophages have no effect on detached matrix-free cells from embryonic-chick tendon or granulation tissue. Another factor from macrophages, present in the 100000 g-supernatant of the homogenate, inhibits the synthesis of collagen in granuloma slices. The synthesis of DNA and RNA in slices is suppressed by the extract from intact macrophages but not affected by preparations obtained with SiO₂. The possible relevance of these findings to lysosomal actions, to the regulation of granuloma formation and to inflammation are discussed.     

花生种子萌发早期胚轴细胞DNA合成的特点

花生种子吸胀6h后胚轴DNA中有~3H-胸苷掺入。咖啡因和羟基脲均对6～12h的~3H—胸苷掺入具强烈的抑制作用;当12～24h时,咖啡因的抑制作用较大;但30h以后,羟基脲的抑制作用超过咖啡因。双链DNA放射性从种子吸胀9h后迅速上升,单链DNA放射性在吸胀12h后出现一个明显的峰。但在吸胀12h后,单链DNA形成和存在的时间是短暂的。     

Increase in DNA replication sites in cells held at the beginning of S phase

CHO cells were pulse labeled with ³H-thymidine after synchronization and blockage at the beginning of S phase for various intervals. The distribution of initiation sites for DNA replication and rates of chain growth were measured in autoradiographs prepared from these cells. Origins used for replication are widely distributed at or near the beginning of S phase, but usable origins increase continuously for many hours when FdU is used to block the synthesis of thymidylate. Potential origins are located about four microns apart, but in normal replication in these fibroblasts only one in 15 to 20 potential origins are used for initiation. On the other hand, when cells are held at the beginning of S phase for 12–14 h, about one-half of the potential origins are activated in part of the DNA and utilized when the cell is released from the block by supplying ³H-thymidine (10^–6M). Chain growth during a short pulse decreases with time of the blockage at what appears to be a linear rate. However, cells can replicate long continuous stretches of their DNA with only 2×10^–8M thymidine available in the medium for several hours when synthesis is blocked by FdU. The total amount of DNA replicated is, however, much less than when a concentration of 10^–6 M thymidine is supplied for the same period. The origins which are finally used under any experimental condition appear to be a random sample of the total potential origins which are distributed in a regular repeating sequence along the DNA at about 12 kilobase intervals.     

Role of replication protein A as sensor in activation of the S-phase checkpoint in Xenopus egg extracts

Uncoupling between DNA polymerases and helicase activities at replication forks, induced by diverse DNA lesions or replication inhibitors, generate long stretches of primed single-stranded DNA that is implicated in activation of the S-phase checkpoint. It is currently unclear whether nucleation of the essential replication factor RPA onto this substrate stimulates the ATR-dependent checkpoint response independently of its role in DNA synthesis. Using Xenopus egg extracts to investigate the role of RPA recruitment at uncoupled forks in checkpoint activation we have surprisingly found that in conditions in which DNA synthesis occurs, RPA accumulation at forks stalled by either replication stress or UV irradiation is dispensable for Chk1 phosphorylation. In contrast, when both replication fork uncoupling and RPA hyperloading are suppressed, Chk1 phosphorylation is inhibited. Moreover, we show that extracts containing reduced levels of RPA accumulate ssDNA and induce spontaneous, caffeine-sensitive, Chk1 phosphorylation in S-phase. These results strongly suggest that disturbance of enzymatic activities of replication forks, rather than RPA hyperloading at stalled forks, is a critical determinant of ATR activation.     

Functional aspect of a phosphopeptide derived from calf thymus nuclei and DNA

Phosphopeptides (PPs) isolated from highly purified calf thymus DNA (N-DNA) and extracted from calf thymus nuclei were fractionated, and the effect of one PP fraction on DNA replication has been examined. In the absence of inhibitors, the increasing PP concentration caused a linear decrease of 3H-thymidine uptake in L5178Y cells. If PP fraction was mildly hydrolysed with 1NHCl, the decrease in uptake was much steeper. The studies in which the inhibitors were used revealed that by the addition of the unhydrolysed PP fraction the inhibition of 3H-thymidine uptake by alpha-amanitin could be completely overcome, and that the inhibition by puromycin was reduced to 65-77% of the control. With puromycin, there was a gradual decrease of 3H-thymidine uptake with PP concentration above 3 mg/ml. The PPs gave an increase in incorporation of 3H-thymidine even after removal of alpha-amanitin and puromycin; thus, it is suggested that there is no direct interaction of either inhibitor with PP in the cell. Data on the utilization of 3H-cytidine for the synthesise of new DNA suggest that PP fraction might cause an acceleration of DNA replication.     

DNA synthesis following gross alterations of the nucleocytoplasmic ratio in the ciliate Stentor coeruleus

Incorporation of externally supplied and injected ³H-thymidine into DNA was measured autoradiographically. Starved stentors synthesized no DNA, in contrast to well-fed animals, but replication commenced in some cases if they were fed. Grafting starved and well-fed stentors together rapidly induced DNA synthesis in the starved partner. Suppression of synthesis in the well-fed macronucleus was not observed. Well-fed cytoplasm alone induced DNA synthesis in starved stentors, and starved cytoplasm grafted to starved animals also induced synthesis after a lag. Starved animals with the beaded macronucleus reduced to 2 nodes commenced DNA replication after 6 hr; however, initiation was prevented if the normal nuclear complement was restored before the fourth hour.The macronucleus was required to render starved cytoplasm capable of supporting DNA synthesis, but once potentiated the cytoplasm alone could initiate replication in a starved nucleus. Initiation required RNA synthesis, shown by actinomycin sensitivity.This nucleic acid analysis suggests that decreasing the nucleocytoplasmic ratio elicits RNA synthesis in the remaining macronucleus. The RNA codes for proteins involved in DNA synthesis which are synthesized in the cytoplasm and enter the nucleus to initiate DNA replication.