Physiological and Genetic Aspects of Abortive Infection of a Shigella sonnei Strain by Coliphage T7

Phage T7 adsorbed to and lysed cells of Shigella sonnei D(2) 371-48, although the average burst size was only 0.1 phage per cell (abortive infection). No mechanism of host-controlled modification was involved. Upon infection, T7 rapidly degraded host deoxyribonucleic acid (DNA) to acid-soluble material. Phage-directed DNA synthesis was initiated normally, but after a few minutes the pool of phage DNA, including the parental DNA, was degraded. Addition of chloramphenicol, at the time of phage infection, prevented both the initiation of phage-directed DNA synthesis and the degradation of parental phage DNA. Addition of chloramphenicol 4.5 min after phage was added permitted the onset of phage-directed DNA synthesis but prevented breakdown of phage DNA. Mutants of T7 (ss(-) mutants) have been isolated which show normal growth in strain D(2) 371-48. Upon mixed infection of this strain with T7 wild type and an ss(-) mutant, infection was abortive; no complementation occurred. The DNA of the ss(-) mutants was degraded in mixed infection like that of the wild type. Revertant mutants which have lost their ability to grow on D(2) 371-48 were isolated from ss(-) mutants; they are, in essence, phenotypically like T7 wild type. Independently isolated revertants of ss(-) mutants did not produce ss(-) recombinants when they were crossed among themselves. When independently isolated ss(-) mutants were crossed with each other, wild-type recombinants were found; ss(-) mutants could then be mapped in a cluster compatible with the length of one cistron. We concluded that T7 codes for an active, chloramphenicol-sensitive function [ss(+) function (for suicide in Shigella)] which leads to the breakdown of phage DNA in the Shigella host.     

Role of gene 2 in bacteriophage T7 DNA synthesis.

Studies have been carried out to elucidate the in vivo function of gene 2 in T7 DNA synthesis. In gene 2-infected cells the rate of incorporation of (3-H)thymidine into acid-insoluble material is about 60% that of cells infected with T7 wild type. Gene 2 mutants do not however produce viable phage after infection of the nonpermissive host. In T7 wild type-infected cells, a major portion of the newly alkaline sucrose gradients. The concatemers serve as precursors for the formation of mature T7 DNA as demonstrated in pulse-chase experiments. In similar studies carried out with gene 2-infected cells, concatemers are not detected when the intracellular DNA is analyzed at several different times during the infection process. The DNA made during a gene 2 infection is present as duplex structures with a sedimentation rate close to mature T7 DNA.     

Production of IL 2 and IFN-gamma by T cells from malaria patients in response to Plasmodium falciparum or erythrocyte antigens in vitro

T cells from patients acutely infected with malaria exhibit a disease-related stimulation of DNA synthesis in response to Plasmodium falciparum antigen in vitro. This response is weak and short-lived, suggestive of induction of suppressor mechanisms. Exogenous T cell growth factor (IL 2) that was added to antigen-stimulated T cell cultures enhanced proliferation in antigen-responsive cultures, indicating that the lymphocytes expressed IL 2 receptors. In contrast, the addition of IL 2 to cultures that did not respond to antigen had no effect. Antigen-responsive cultures contained endogenous IL 2 as well, and the antigen-induced lymphocyte proliferation was correlated with IL 2 production. However, the results suggested that IL 2 production by the patients' T cells was insufficient or actively shut off, and that this was responsible for the premature cessation of their DNA synthesis. Supernatants from 60% of the T cell cultures treated with malaria antigen and from 30% treated with RBC ghost antigen contained interferon-gamma (IFN-gamma), as determined by a cytopathic effect inhibition assay combined with acid treatment and antibody neutralization or by an IFN-gamma-specific ELISA. There was no obvious correlation between antigen-induced lymphocyte proliferation and the presence of IFN-gamma in the culture supernatants. A high IFN-gamma activity was also seen in antigen-treated cultures from P. falciparum-immune donors living in highly endemic malaria areas. In contrast, no IFN-gamma was found in supernatants of antigen-treated T cells from healthy donors or patients with Plasmodium vivax malaria. Thus, the IFN-gamma activity of these cultures appears to reflect the presence of antigen-reactive T cells and may be useful as a sensitive indicator of cellular immunity in P. falciparum malaria.     

Synthesis of Messenger Ribonucleic Acid After Bacteriophage T1 Infection

Synthesis of host-specific and phage-specific messenger ribonucleic acid (mRNA) was studied in bacteria infected by unmodified (T1 . B) or modified [T1 . B(P1)] bacteriophage T1. In a "standard" infection of Escherichia coli B by T1 . B (no host-controlled modification involved), the rate and amount of T1 mRNA synthesis was intermediate between those values reported for infections by a virulent phage such as T4 or a temperate phage such as lambda. The initial rate of mRNA synthesis was slightly increased after T1 . B(P1) infection of E. coli B in comparison with T1 . B infection of the same host. Little or no phage mRNA synthesis could be detected in T1 . B infection of E. coli B(P1). Phage mRNA synthesis in T1 . B(P1)-infected E. coli B(P1) cells was approximately the same in amount as that seen in T1 . B(P1) infection of E. coli B. Synthesis of host-specific mRNA continued throughout the latent period in all infections studied. However, the enzyme beta-galactosidase could not be induced, except after T1 . B infection of E. coli B(P1). In an attempt to understand the apparent differences in mRNA synthesis after infection of E. coli B by phages T1 . B or T1 . B(P1), the effect of altered T1 deoxyribonucleic acid (DNA) methylation on mRNA synthesis was studied. Methyl-deficient T1 DNA, made in cells infected with ultraviolet-irradiated phage T3, inhibited (14)C-uridine incorporation more strongly than normal T1. One passage of methyl-deficient T1 through E. coli B restored uracil incorporation rates to those seen with ordinary T1. This suggests that methylation of T1 DNA can influence the rate of phage mRNA synthesis. However, attempts to relate the difference in mRNA synthesis seen between T1 . B and T1 . B(P1) in E. coli B to the activity of the P1 modification gene were not conclusive.     

Effect of Prophage W on the Propagation of Bacteriophages T2 and T4

Studies have been undertaken to determine whether the temperate phage ω present in Escherichia coli strain W is responsible for the inability of this strain to act as a host for T2 and T4. E. coli WS, cured of phage ω, was sensitive to T2 and T4. Lysogenation of E. coli C and WS with phage ω resulted in loss of ability to plate T2 and T4. However, E. coli K-12 lysogens still served as hosts for the T -even phage. Two of three WS lysogens studied resembled strain W at the biochemical level. They converted about 30% of infecting T2 deoxyribonucleic acid (DNA) to acid-soluble fragments and limited macromolecular synthesis to a few minutes after infection. The third lysogen did not degrade phage DNA, and nucleic acid and protein synthesis continued for some time, although no phage production occurred. It is concluded that phage ω plays a role in the restriction of virulent phage but that it is not the only factor involved. Since acid solubilization was not observed in all cases of phage ω-mediated restriction of T -even phage, a hypothesis for the restriction has been proposed which is based on an alteration in the cell envelope after lysogenation with phage ω.     

Nonpermissivity of human peripheral blood lymphocytes to adenovirus type 2 infection.

The capacity of freshly explanted human peripheral blood lymphocytes (PBL) to support the replication of human adenovirus type 2 (Ad2) was investigated. Unlike other types of human cells, PBL were found to be highly nonpermissive. Ad2 adsorbed 30 to 40% of both T and non-T cells. Virus uncoating was very slow and inefficient, resulting in a 40-fold reduction compared with HEp-2 cells. On a population basis, viral DNA synthesis was reduced 460-fold and infectious virus production was reduced 10(6)-fold. Only 0.35% of PBL produced infectious centers, yielding 0.8 PFU per infected cell. Phytohemagglutinin stimulation increased DNA synthesis 23-fold, infectious centers 11-fold, and virus yield 14-fold. We conclude that resting human PBL are highly nonpermissive to Ad2 infection and that phytohemagglutinin can only marginally lift this nonpermissiveness.     

下调细胞P21WAF1/CIP1基因表达可抑制单纯疱疹病毒Ⅱ型复制

为了揭示细胞P21蛋白在单纯疱疹病毒Ⅱ型（herpes simplex virus type 2, HSV-2）复制中的作用,通过用HSV-2感染和感染前用特异性小干扰RNA (small interfering RNA,siRNA) 抑制P21基因表达,应用Western 印迹方法检测宿主细胞和病毒蛋白水平,用终点滴定法测定病毒半数组织培养感染量（50% tissue culture infectious dose, TCID50）,以及观察感染细胞的细胞病变效应（cytopathic effect, CPE）等3个方面,揭示细胞P21蛋白水平的变化对病毒复制的影响.结果表明,HSV-2在细胞内复制时可引起P21蛋白水平增高;而用特异性siRNA下调细胞P21基因表达时,可显著地抑制HSV-2 gB蛋白水平,减少培养细胞上清液中病毒TCID50.提示P21蛋白对HSV-2的复制具有重要的作用.     

Identification of the gene controlling the synthesis of the major bacteriophage T5 membrane protein.

After infection of Escherichia coli B by bacteriophage T5, a major new protein species, as indicated by polyacrylamide gel electrophoresis, appears in the cells' membranes. Phage mutants with amber mutations in the first-step-transfer portion of their DNA have been tested for their ability to induce membrane protein synthesis after they infect E. coli B. We have found that phage with mutations in the Al gene of T5 do not induce the synthesis of the T5-specific major membrane protein, whereas phage that are mutant in the A2 gene do induce its synthesis. We conclude that gene Al must function normally for T5-specific membrane protein biosynthesis to occur and that only the first 8% (first-step-transfer piece) of the DNA need be present in the cell for synthesis to occur.     

Deoxyuridine triphosphate pools after polyoma virus infection

The synthesis of polyoma DNA in virus-infected 3T6 mouse fibroblasts is discontinuous with the intermediate formation of short Okazaki fragments. Hydroxyurea, an inhibitor of the enzyme ribonucleotide reductase, inhibits polyoma DNA synthesis, as measured by incorporation of radioactive thymidine. In the inhibited state, almost all incorporation occurs into short fragments. We investigated to what extent formation of short DNA fragments might be the result of incorporation of deoxyuridine triphosphate (dUTP) into DNA, followed by excision and repair reactions. We devised a sensitive enzymatic method for measuring dUTP in cell extracts which allows the determination of the dUTP pool when this pool amounts to between 0.1 and 2% of the dTTP pool. No dUTP was detected in growing mouse fibroblasts. After infection with polyoma virus cell extracts contained 0.4% dUTP (of dTTP) at the peak of DNA synthesis. Addition of hydroxyurea at this point led to a disappearance of dUTP. We conclude that dUTP incorporation can contribute only minimally to the generation of short fragments during polyoma DNA synthesis.     

Induction of human cell DNA synthesis by herpes simplex virus type 2.

The effect of herpes simplex virus type 2 (HSV-2) infection on the synthesis of DNA in human embryonic fibroblast cells was determined at temperatures permissive (37 C) and nonpermissive (42 C) for virus multiplication. During incubation of HSV-2 infected cultures at 42 C for 2 to 4 days or after shift-down from 42 to 37 C, incorporation of (3H)TdR into total DNA was increased 2-to 30-fold as compared with mock-infected cultures. Analysis of the (3H)DNA suggested that host cell DNA synthesis was induced by HSV-2 infection. Induction of host cell DNA synthesis by HSV-2 also occurred in cells arrested in DNA replication by low serum concentration. The three strains of HSV-2 tested were capable of stimulating cellular DNA synthesis. Virus inactivated by UV irradiation, heat, or neutral red dye and light did not induce cellular DNA synthesis, suggesting that an active viral genome is necessary for induction.     

Requirement for protein synthesis for survival of unmodified bacteriophage T1 in a restricting host.

At high multiplication of infection, a substantial fraction of restricting cells (P1 lysogens) could be productively infected by unmodified coliphage T1 (T1.0) provided that protein synthesis was uninhibited during the first 5 min of infection. Successful infection under restricting conditions was accompanied by more genetic recombination than was seen under nonrestricting host, the recombination frequency declined for markers on T1.0 genomes; no effect was seen on recombination between markers on modified (T1.P) genomes. This suggested that recombination between unmodified genomes may be essential for their survival under conditions of host restriction. In a restricting host, genetic markers on T1.0 could recombine with T1.P even when the rescuing phage was added 6 min after T1.0 infection. However, even marker rescue recombination was diminished when protein synthesis was inhibited during early infection. Since DNA restriction is an early event, protein synthesis may be required soon after infection of a restricting host by T1.0 in order to preserve restriction-damaged DNA in a form that can participate in recombination. Experiments are also described that rule out some possibilities for the role of such a protein(s).     

Mutants of bacteriophage T4 deficient in the ability to induce nuclear disruption: shutoff of host DNA and protein synthesis gene dosage experiments, identification of a restrictive host, and possible biological significance.

The shutoff of host DNA synthesis is delayed until about 8 to 10 min after infection when Escherichia coli B/5 cells were infected with bacteriophage T4 mutants deficient in the ability to induce nuclear disruption (ndd mutants). The host DNA synthesized after infection with ndd mutants is stable in the absence of T4 endonucleases II and IV, but is unstable in the presence of these nucleases. Host protein synthesis, as indicated by the inducibility of beta-galactosidase and sodium dodecyl sulfate-polyacrylamide gel patterns of isoptopically labeled proteins synthesize after infection, is shut off normally in ndd-infected cells, even in the absence of host DNA degradation. The Cal Tech wild-type strain of E. coli CT447 was found to restrict growth of the ndd mutants. Since T4D+ also has a very low efficiency of plating on CT447, we have isolated a nitrosoguanidine-induced derivative of CT447 which yields a high T4D+ efficiency of plating while still restricting the ndd mutants. Using this derivative, CT447 T4 plq+ (for T4 plaque+), we have shown that hos DNA degradation and shutoff of host DNA synthesis occur after infection with either ndd98 X 5 (shutoff delayed) or T4D+ (shutoff normal) with approximately the same kinetics as in E. coli strain B/5. Nuclear disruption occurs after infection of CT447 with ndd+ phage, but not after infection with ndd- phage. The rate of DNA synthesis after infection of CT447 T4 plq+ with ndd98 X 5 is about 75% of the rate observed after infection with T4D+ while the burst size of ndd98 X 5 is only 3.5% of that of T4D+. The results of gene dosage experiments using the ndd restrictive host C5447 suggest that the ndd gene product is required in stoichiometric amounts. The observation by thin-section electron microscopy of two distinct pools of DNA, one apparently phage DNA and the other host DNA, in cells infected with nuclear disruption may be a compartmentalization mechanism which separates the pathways of host DNA degradation and phage DNA biosynthesis.     

Interleukin-2 mRNA expression, lymphokine production and DNA synthesis in glutathione-depleted T cells

The stimulation of DNA synthesis in lymphocyte populations was previously shown to depend strongly on the intracellular glutathione (GSH) level. Since T cell growth is known to depend on interleukin 2 (IL-2), the experiments in this report were designed to determine whether intracellular GSH depletion may inhibit IL-2 production or the IL-2 dependent DNA synthesis. Our experiments revealed that IL-2 production and DNA synthesis of mitogenically stimulated splenic T cells have indeed different requirements for GSH. The addition of relatively high concentrations of GSH (5 mM) to cultures of concanavalin A (Con A)-stimulated splenic T cells was found to augment strongly the DNA synthesis but inhibited the production of IL-2. Moderate intracellular GSH levels, however, are apparently not inhibitory for IL-2 production, since intracellular GSH depletion by cysteine starvation or by graded concentrations of DL-buthionine sulfoximine (BSO) had virtually no effect on IL-2-specific mRNA expression and the production of T cell growth factor (TCGF). The DNA synthesis activity, in contrast, was strongly suppressed after GSH depletion with either method. As in cultures of splenic T cells, GSH depletion had no substantial effect on the induction of IL-2 mRNA and TCGF production in several mitogenically stimulated T cell clones. Taken together, our experiments suggest that complex immune response may operate best at intermediate GSH levels that are not too high to inhibit IL-2 production but sufficient to support DNA synthesis.     

Simian virus 40 gene A regulation of cellular DNA synthesis. II. In nonpermissive cells.

The stimulation of host macromolecular synthesis and induction into the cell cycle of serum-deprived G0-G1-arrested mouse embryo fibroblasts were examined after infection of resting cells with wild-type simian virus 40 or with viral mutants affecting T antigen (tsA58) or small t antigen (dl884). At various times after virus infection, cell cultures were analyzed for DNA synthesis by autoradiography and flow microfluorimetry. Whereas mock-infected cultured remained quiescent and displayed either a 2N DNA content (80%) or a 4N DNA content (15%), mouse cells infected with wild-type simian virus 40, tsA58 at 33 degrees C, or dl884 were induced into active cell cycling at approximately 18 h postinfection. Although dl884-infected mouse cells were induced to cycle initially at the same rate as wild type-infected cells, they became arrested earlier after infection and also failed to reach the saturation densities of wild-type simian virus 40-infected cells. Infection with dl884 also failed to induce loss of cytoplasmic actin cables in the majority of the infected cell population. Mouse cells infected with tsA58 and maintained at 39.5 degrees C showed a transient burst of DNA synthesis as reflected by changes in cell DNA content and an increase in the number of labeled nuclei during the first 24 h postinfection; however, after the abortive stimulation of DNA synthesis at 39.5 degrees C shift experiments demonstrated that host DNA replication was regulated by a functional A gene product. It is concluded that both products of the early region of simian virus 40 DNA play a complementary role in recruiting and maintaining simian virus 40-infected cells in the cell cycle.     

Kinetics of Nucleic Acid Synthesis in Human Embryonic Kidney Cultures Infected with Adenovirus 2 or 12: Inhibition of Cellular Deoxyribonucleic Acid Synthesis

Infection of human embryonic kidney (HEK) cell cultures with adenovirus types 2 or 12 resulted in an initial drop in the rate of incorporation of (3)H-thymidine into deoxyribonucleic acid (DNA) during the early latent period of virus growth, followed by a marked rise in label uptake. It was shown by cesium chloride isopycnic centrifugation that, after adenovirus 2 infection, there was a decrease in the rate of incorporation of thymidine into cellular DNA. Moreover, DNA-DNA hybridization experiments revealed that, by 28 to 32 hr after infection with either adenovirus 2 or 12, the amount of isolated pulse-labeled DNA capable of hybridizing with HEK cell DNA was reduced by approximately 60 to 70%. Autoradiographic measurements showed that the inhibition of cellular DNA synthesis was due to a decrease in the ability of an infected cell to synthesize DNA. The adenovirus-induced inhibition of host cell DNA synthesis was not due to degradation of cellular DNA. (3)H-thymidine incorporated into cellular DNA at the time of infection remained acid-precipitable, and labeled material was not incorporated into viral DNA. Furthermore, when zone sedimentation through neutral or alkaline sucrose density gradients was employed, no detectable change was observed in the sedimentation rate of this cellular DNA at various times after infection with adenovirus 2 or 12. In addition, there was no increase in deoxyribonuclease activity in cells infected with either virus. Cultures infected for 38 hr with adenovirus 2 or 12 incorporated three to four times as much (3)H-uridine into ribonucleic acid (RNA) as did non-infected cultures. Furthermore, the net RNA synthesized by infected cultures substantially exceeded that of control cultures. The activity of thymidine kinase was induced, but there was no stimulation of uridine kinase.     

Temperature-sensitive mutants of Balb/3T3 cells: description of a mutant affected in cellular and polyoma virus DNA synthesis.

A temperature-sensitive Dna- mutant (ts-2) of the mouse cell Balb/3T3 is characterized. Studies with synchronized cells indicate that the defect is in DNA synthesis itself, rather than in progress toward its initiation. ts-2 supports polyoma DNA synthesis after infection at 33degreesC but not at 38degreesC. Viral DNA synthesis begun at 33degreesC is inhibited upon shift to 38degreesC. A procedure is proposed by which viral DNA synthesis can be used to distinguish different classes of cell Dna- mutants.     

Multiplication of Polyoma Virus in Mouse-Hamster Somatic Hybrids: a Hybrid Cell Line Which Produces Viral Particles Containing Predominantly Host Deoxyribonucleic Acid

The multiplication of polyoma virus in a mouse-hamster (3T3 x BHK) somatic hybrid line (10A), which, although permissive for viral multiplication, produces very low amounts of virus, has been studied. In this cell line, the efficiency of productive infection is high, but the yield of infectious virus is on the order of 0.5% of that of 3T3 cells. The amount of viral deoxyribonucleic acid (DNA) synthesized by these cells upon infection is about 5% of that of 3T3 cells. An examination of the virus produced in hybrid 10A revealed that it was only one-tenth as infectious as the virus grown in 3T3. Although the viral DNA synthesized in the infected 10A cells is normal, the DNA extracted from purified virus grown in 10A consists of approximately 10% of normal, supercoiled polyoma DNA molecules and of approximately 90% linear DNA molecules with a sedimentation coefficient of 14 to 16S. These DNA molecules appear to be of cellular origin but contain a limited amount of viral DNA sequences. The host DNA-containing particles are not infectious but appear to possess some biological activity; they give rise to a weak complementation effect, and part of them are able to induce T-antigen synthesis. In addition, the host DNA present in these particles is predominantly that which has been synthesized after infection. The correlation between the block in viral DNA synthesis in this cell line and the abnormal encapsidation of host DNA is discussed.     

Nonproductive Infection and Induction of Cellular Deoxyribonucleic Acid Synthesis by Bovine Adenovirus Type 3 in a Contact-Inhibited Mouse Cell Line

Bovine adenovirus type 3 (BAV-3), which has been reported to produce tumors in newborn hamsters, induced cellular deoxyribonucleic acid (DNA) synthesis in a contact-inhibited mouse kidney cell line (C3H2K). In this system, the virus did not multiply, whereas virus-specific tumor antigen (T antigen) was detected in nearly all cells. Replication of viral DNA could not be detected by DNA-DNA hybridization on membrane filters. The cellular DNA synthesis induced by BAV-3 did occur in the absence of added serum. Extent of induction of cellular DNA synthesis was closely correlated with the multiplicity of infection. Cells activated to synthesize DNA in the serum-free medium by the virus infection progressed to cell division without noticeable cell killing.