Viral Deoxyribonucleic Acid Synthesis Research Articles

Insect cells and their potential as stabilization barriers for DNA of multiple and single nucleopolyhedroviruses against ultraviolet-B-simulated sunlight inactivation.

A cell line from Trichoplusia ni (TN-CL1) infected with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV-HPP) and a cell line from Helicoverpa zea (BCIRL-HZ-AM1) infected with the Helicoverpa zea single nucleopolyhedrovirus (HzSNPV/BrCL2) were subjected to ultraviolet-B (UV-B) irradiation at a predetermined level of exposure that would inactivate greater than 95% of the virus suspended in the liquid. The working hypothesis was that the homologous insect cells would utilize their inherent deoxyribonucleic acid (DNA) repair mechanism(s) to prevent, repair, or at least mitigate the damaging effects of UV-B light on viral DNA synthesis. We attempted to determine this by using infected cells that were subjected to UV-B irradiation at different postinoculation periods under two experimental conditions of exposure: (1) shielded, and (2) nonshielded. Of the two cell lines infected with their respective homologous viruses, the virus from TN-CL1 cells was the least sensitive to UV-B light because the extracellular virus (ECV) and occlusion body (OB) levels of virus-infected TN-CL1 cells were higher than those of the virus-infected BCIRL-HZ-AM1 cells. Production of ECV and OB from both cell lines was lower in the exposed, nonshielded treatment than in the exposed, shielded treatment. However, AcMNPV-HPP was produced in enough quantity to indicate that TN-CL1 might impart a level of protection to the virus against UV light.

Adenovirus chromatin structure at different stages of infection.

We investigated the structure of adenovirus deoxyribonucleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococcal nuclease. Parental (infecting) DNA was digested into multimers which had a unit fragment size that was indistinguishable from the size of the nucleosomal repeat of cellular chromatin. This pattern was maintained in parenteral DNA throughout infection. Similar repeating units were detected in hamster cells that were nonpermissive for human adenovirus and in cells pretreated with n-butyrate. Late in infection, the pattern of digestion of viral DNA was determined by two different experimental approaches. Nuclear DNA was electrophoresed, blotted, and hybridized with labeled viral sequences; in this procedure all virus-specific DNA was detected. This technique revealed a diffuse protected band of viral DNA that was smaller than 160 base pairs, but no discrete multimers. All regions of the genome were represented in the protected DNA. To examine the nuclease protection of newly replicated viral DNA, infected cells were labeled with [3H]thymidine after blocking of cellular DNA synthesis but not viral DNA synthesis. With this procedure we identified a repeating unit which was distinctly different from the cellular nucleosomal repeat. We found broad bands with midpoints at 200, 400, and 600 base pairs, as well as the limit digest material revealed by blotting. High-resolution acrylamide gel electrophoresis revealed that the viral species comprised a series of closely spaced bands ranging in size from less than 30 to 250 base pairs.

Adenovirus chromatin structure at different stages of infection.

We investigated the structure of adenovirus deoxyribonucleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococcal nuclease. Parental (infecting) DNA was digested into multimers which had a unit fragment size that was indistinguishable from the size of the nucleosomal repeat of cellular chromatin. This pattern was maintained in parenteral DNA throughout infection. Similar repeating units were detected in hamster cells that were nonpermissive for human adenovirus and in cells pretreated with n-butyrate. Late in infection, the pattern of digestion of viral DNA was determined by two different experimental approaches. Nuclear DNA was electrophoresed, blotted, and hybridized with labeled viral sequences; in this procedure all virus-specific DNA was detected. This technique revealed a diffuse protected band of viral DNA that was smaller than 160 base pairs, but no discrete multimers. All regions of the genome were represented in the protected DNA. To examine the nuclease protection of newly replicated viral DNA, infected cells were labeled with [3H]thymidine after blocking of cellular DNA synthesis but not viral DNA synthesis. With this procedure we identified a repeating unit which was distinctly different from the cellular nucleosomal repeat. We found broad bands with midpoints at 200, 400, and 600 base pairs, as well as the limit digest material revealed by blotting. High-resolution acrylamide gel electrophoresis revealed that the viral species comprised a series of closely spaced bands ranging in size from less than 30 to 250 base pairs.

Tumor promoters alter the temporal program of adenovirus replication in human cells.

In this study we evaluated the effect of phorbol ester tumor promoters on the kinetics of adenovirus type 5 (Ad5) replication in human cells. When added at the time of infection, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) accelerated the appearance of an early virus antigen (72,000-molecular-weight [72K] deoxyribonucleic acid-binding protein), the onset of viral deoxyribonucleic acid synthesis, and the production of infectious virus. The appearance of an Ad5-specific cytopathic effect (CPE) was also accelerated in infected cultures exposed to TPA, whereas phorbol, 4 alpha-phorbol-12,13-didecanoate and 4-OmeTPA, which are inactive as tumor promoters, were ineffective in inducing this morphological change. The acceleration of the CPE seen in TPA-treated Ad5-infected cells was not caused by TPA induction of the protease plasminogen activator, since the protease inhibitors leupeptin and antipain do not inhibit the earlier onset of this CPE and, in contrast, epidermal growth factor, which induces plasminogen activator in HeLa cells, does not induce an earlier CPE. Evidence for a direct effect of TPA on viral gene expression was obtained by analyzing viral messenger ribonucleic acid (mRNA) synthesis. TPA accelerated the appearance of mRNA from all major early regions of Ad5, transiently stimulated the accumulation of region III mRNA, and accelerated the appearance of late Ad5 mRNA. Thus, TPA altered the temporal program of Ad5 mRNA production and accelerated the appearance of at least some Ad5-specific polypeptides during lytic infection of human cells. These effects presumably explain the earlier onset of the Ad5-specific CPE in TPA-treated cells and may have relevance to the effects of TPA on viral gene expression in nonpermissive cells carrying integrated viral deoxyribonucleic acid sequences.

Tumor promoters alter the temporal program of adenovirus replication in human cells.

In this study we evaluated the effect of phorbol ester tumor promoters on the kinetics of adenovirus type 5 (Ad5) replication in human cells. When added at the time of infection, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) accelerated the appearance of an early virus antigen (72,000-molecular-weight [72K] deoxyribonucleic acid-binding protein), the onset of viral deoxyribonucleic acid synthesis, and the production of infectious virus. The appearance of an Ad5-specific cytopathic effect (CPE) was also accelerated in infected cultures exposed to TPA, whereas phorbol, 4 alpha-phorbol-12,13-didecanoate and 4-OmeTPA, which are inactive as tumor promoters, were ineffective in inducing this morphological change. The acceleration of the CPE seen in TPA-treated Ad5-infected cells was not caused by TPA induction of the protease plasminogen activator, since the protease inhibitors leupeptin and antipain do not inhibit the earlier onset of this CPE and, in contrast, epidermal growth factor, which induces plasminogen activator in HeLa cells, does not induce an earlier CPE. Evidence for a direct effect of TPA on viral gene expression was obtained by analyzing viral messenger ribonucleic acid (mRNA) synthesis. TPA accelerated the appearance of mRNA from all major early regions of Ad5, transiently stimulated the accumulation of region III mRNA, and accelerated the appearance of late Ad5 mRNA. Thus, TPA altered the temporal program of Ad5 mRNA production and accelerated the appearance of at least some Ad5-specific polypeptides during lytic infection of human cells. These effects presumably explain the earlier onset of the Ad5-specific CPE in TPA-treated cells and may have relevance to the effects of TPA on viral gene expression in nonpermissive cells carrying integrated viral deoxyribonucleic acid sequences.

Chinese hamster ovary cells replicate adenovirus deoxyribonucleic acid.

Chinese hamster ovary (CHO) cells infected with adenovirus type 2 (Ad2) produced amounts of viral deoxyribonucleic acid (DNA) equal to that synthesized in permissively infected HeLa cells. However, there was 6,000-fold less virion produced in CHO cells. Since the structural viral polypeptides were not detected by pulse-labeling CHO cells at various times postinfection, the block in virion formation is located between the synthesis of viral DNA and late proteins. Extracts of CHO cells could also function in a recently reported in vitro Ad2 DNA synthesis system which is dependent upon the addition of exogenous Ad2 DNA covalently linked to a 5'-terminal protein (Ikeda et al., Proc. Natl. Acad. Sci. U.S.A. 77:5827-5831, 1980). Extracts of infected CHO cytoplasm were able to complement uninfected CHO nuclear extracts to synthesize viral DNA on Ad2 templates. This in vitro replication system has the potential to probe host DNA synthesis requirements as well as viral factors.

Chinese hamster ovary cells replicate adenovirus deoxyribonucleic acid.

Chinese hamster ovary (CHO) cells infected with adenovirus type 2 (Ad2) produced amounts of viral deoxyribonucleic acid (DNA) equal to that synthesized in permissively infected HeLa cells. However, there was 6,000-fold less virion produced in CHO cells. Since the structural viral polypeptides were not detected by pulse-labeling CHO cells at various times postinfection, the block in virion formation is located between the synthesis of viral DNA and late proteins. Extracts of CHO cells could also function in a recently reported in vitro Ad2 DNA synthesis system which is dependent upon the addition of exogenous Ad2 DNA covalently linked to a 5'-terminal protein (Ikeda et al., Proc. Natl. Acad. Sci. U.S.A. 77:5827-5831, 1980). Extracts of infected CHO cytoplasm were able to complement uninfected CHO nuclear extracts to synthesize viral DNA on Ad2 templates. This in vitro replication system has the potential to probe host DNA synthesis requirements as well as viral factors.

Virus-Induced Specific Cell Surface Antigen(s) on Mouse Adenovirus-Infected Cells

A virus-specific cell surface antigen(s) was detected on mouse adenovirus-infected cells by the immunofluorescent antibody technique. It was demonstrated in infected cells regardless of the mouse strain or animal species used. It became detectable on the cell surface about 6 h postinfection and was demonstrated in 90% of infected cells at 24 to 36 h postinfection. On the other hand, intracellular capsid antigens became detectable by the immunofluorescent antibody technique at about 24 h postinfection. Since it was also detected on virus-infected cells treated with 5'-fluorodeoxyuridine to block viral deoxyribonucleic acid synthesis, it was synthesized at the early stage of infection and may not be a virus structural component. The relationship between this serologically detectable cell surface antigen(s) and cell-mediated immune response to virus infections is discussed.

Rhesus monkeys kidney cells persistently infected with Simian Virus 40: production of defective interfering virus and acquisition of the transformed phenotype

Monolayer cultures of LLC-MK2 rhesus monkey kidney cells became persistently infected with simian virus 40 (SV40) when infected at a multiplicity of infection of 100 plaque-forming units/cell. A stable carrier state developed characterized by extensive viral proliferation without obvious cytopathic effect other than the slow growth of these cultures. By 11 weeks all cells produced the SV40 T antigen. In contrast, less than 5% of the cells produced V antigen. Virus-free clonal isolates were obtained by cloning in SV40 antiserum. Continuous cultivation in antiserum resulted in a temporary cure of unclone cultures. When virus did eventually reappear in the "cured" cultures the titers remained low. The virus produced by the carrier culture was defective at both 31 and 37% c, and it interfered with the growth of standard s40 during mixed infection of CV-1 green monkey kidney cells. All of the interfering activity in carrier culture homogenates could be sedimented by centrifugation at 109,000 x g for 3 h. These cultures were completely susceptible to vesicular stomatitis virus. Extensive viral deoxyribonucleic acid synthesis occurred in CV-1 cells infected with carrier culture virus. Carrier culture homogenates are only slightly less cytopathic to CV-1 cells than standard SV40. The carrier culture express several properties of SV40 transformation.

Antiviral Activity of Arabinosyladenine and Arabinosylhypoxanthine in Herpes Simplex Virus-Infected KB Cells: Selective Inhibition of Viral Deoxyribonucleic Acid Synthesis in the Presence of an Adenosine Deaminase Inhibitor

The antiviral activity of the fraudulent nucleoside arabinosyladenine (ara-A) against herpes simplex virus (HSV) type 1 was increased nearly 20-fold by the adenosine deaminase inhibitor, coformycin. The combination of ara-A plus coformycin was 90 times more potent in blocking HSV replication than was arabinosylhypoxanthine (ara-H). In suspension culture both drugs were more active than they were in monolayer culture. Deoxyribonucleic acid (DNA) synthesis also was inhibited by the nucleosides. Depending upon the species of DNA examined, ara-A was 8 to 15 times more active in the presence of coformycin, and the combination was 35 to 70 times more potent than ara-H. Both drugs inhibited total DNA synthesis to the same extent in uninfected and HSV-infected KB cells. In contrast, viral DNA synthesis was three to six times more susceptible to inhibition than was cellular DNA synthesis. Inhibition of viral DNA synthesis was more pronounced in suspension culture than in monolayer culture. However, the method of cell propagation did not alter the degree to which the drugs inhibited DNA synthesis in uninfected KB cells. An index has been derived to quantitate the extent of the selective inhibition of viral or cellular DNA synthesis. Fifty percent inhibitory concentrations of a drug were calculated for uninfected KB DNA synthesis and viral DNA synthesis and expressed as a ratio. The logarithm of this ratio was termed the selective index and was positive if viral DNA synthesis was inhibited preferentially or negative if uninfected KB DNA synthesis was more strongly inhibited. Data from experiments performed in monolayer culture gave positive selective index values of 0.3, 0.5, and 0.4 for ara-A plus coformycin, ara-A, and ara-H, respectively. Values of 0.7 and 0.6 were obtained from suspension culture data for ara-A plus coformycin and ara-H, respectively. Considered collectively, the data presented in this communication establish that coformycin increased the potency of ara-A but did not increase its selectivity.

Quantitative cytophotometry evaluated as a method for analyses of herpes simplex viral deoxyribonucleic acid synthesis

Feulgen deoxyribonucleic acid (F-DNA) microspectrophotometry was evaluated as a potential tool for quantification of herpes simplex virus type 1 (HSV-1) and 2(HSV-2) DNA synthesis in single cells. Since HSV DNA synthesis has been extensively studied using incorporation of radioactive precursors into viral and cellular DNA, microspectrophotometric measures were correlated with biochemical data obtained using tritium-labeled thymidine ([3H]TdR). It was established that: (i) viral-induced increaae in F-DNA can be cytophotometrically detected between 1 and 6 h postinjection (p.i.), which corresponds to the initial incorporation of [3H) TdR into viral DNA; (ii) peak F-DNA levels occurred 8 h p.i., which supported cytological observations of a more rapid development of an inclusion body in HSV-2-infected nuclei. Despite the fact that F-DNA cytophotometry is unable to distinguish between cell and viral DNA, the overall study supports the existence of a good correlation between data obtained using microspectrophotometric and conventional isotope methods. Furthermore, cytophotometry complements biochemical evaluations in that it permits analyses of DNA changes on a single-cell basis or the detection of infection in very small numbers of cells.

Antiviral Activity of Arabinosyladenine and Arabinosylhypoxanthine in Herpes Simplex Virus-Infected KB Cells: Selective Inhibition of Viral Deoxyribonucleic Acid Synthesis in Synchronized Suspension Cultures

The drug 9-beta-d-arabinofuranosyladenine (ara-A) significantly suppressed the formation of herpes simplex virus type 1-induced syncytia in BHK-21/4 cells at concentrations as low as 0.1 mug/ml. Optimal activity was noted when the drug was added before initiation of viral deoxyribonucleic acid (DNA) synthesis (3.5 h postinfection). The deaminated derivative of ara-A, 9-beta-d-arabinofuranosylhypoxanthine (ara-H), was at least 10 times less effective in suppressing the development of herpes simplex virus-induced syncytia. The replication of herpes simplex virus was measured by assaying fluids and cells from infected drug-treated cultures by using a plaque production technique. Ara-A at drug levels of >10 < 32 mug/ml completely blocked the replication of infectious virus particles. Ara-H was less effective than ara-A in reducing the replication of virions. Rates of host and viral DNA synthesis were monitored by pulse labeling herpes simplex virus-infected synchronized KB cells with [(3)H]thymidine and subsequently separating viral from cellular DNA in CsCl density gradients. During synthetic (S) phase, ara-A or ara-H at concentrations ranging from 3.2 to 32 mug/ml selectively inhibited viral DNA synthesis. At 3.2 mug of ara-A per ml, viral DNA synthesis was reduced 74% although total cellular DNA synthesis was unaffected. Increasing concentrations of ara-A produced increasing temporal delays in the maximal rate of host DNA synthesis. This time shift was not observed in cells treated with ara-H.

Adenovirus deoxyribonucleic acid replication. II. Synthesis of viral deoxyribonucleic acid in vitro by a nuclear membrane fraction from infected KB cells.

A cell-free system for the study of viral DNA replications was developed by the isolation of a nuclear membrane fraction "DNA replication complex" from adenovirus 2-infected human KB cells late after infection. This complex which possesses both DNA polymerase activity and a virus-specific endonuclease synthesizes exclusively virus-specific DNA sequences in vitro by a semiconservative mechanism. Analysis by rate zonal sedimentation in alkaline sucrose gradients showed that the products of the reaction are small DNA chains approximately 6 to 9 S, presumably "Okazaki intermediates," that are not sealed under our in vitro conditions. Analysis by rate zonal sedimentation in neutral sucrose gradients showed that labeled viral DNA fragments are hydrogen bonded to viral 18 S DNA segments, 0.25 the size of the linear, viral 31 S DNA genome. The 18 S DNA is probably a specific cleavage product of the viral endonuclease found in the replication complex and could represent intermediates in viral DNA replication or degradation products.

Mechanism of induction of cell-mediated immunity to virus infections: in vitro inhibition of intracellular multiplication of mouse adenovirus by immune spleen cells

Mice were protected from lethal infection with mouse adenovirus (M-Ad) by adoptive transfer of immune spleen cells (ISC) that were prepared from mice immunized with M-Ad and not protected by sonicated ISC. However, a similar extent of protection was also observed by passive immunization with anti-M-Ad serum. In contrast, by in vitro experiments ISC were shown to be able to interrupt intracellular multiplication of M-Ad, whereas sonicated ISC, unimmunized mouse spleen cells, or anti-M-Ad serum were unable to do so. ISC were inhibitory in vitro when added within 12 h postinfection but not when added later. The inhibitory activity of ISC was regarded as due to cell killing by ISC, since the trypan blue exclusion test showed that above 80% of infected cells were killed by ISC even when 5'-fluorodeoxyuridine was added to the cells to block viral deoxyribonucleic acid synthesis, under which conditions control infected cells, to which ISC were not added or normal spleen cells were added, were kept alive at least for a few days. Kinetics studies in M-Ad-infected mice showed that the inhibitory activity of ISC became highest at 1 to 2 weeks postinfection and faded away thereafter in a few weeks, whereas serum antibody titer assayed by passive hemagglutination reached its peak level at about 4 weeks postinfection and declined gradually therafter.

Adenovirus-transformed cells restrict Herpes simplex virus replication

A cell line that normally supports the replication of herpes simplex virus types 1 and 2 became resistant to these viruses after transformation by simian adenovirus 7. Kinetic studies of the mechanism of resistance demonstrated that both herpesviruses were able to attach to the transformed cells and express some early genomic functions, as demonstrated by the presence of low levels of viral thymidine kinase. However, isopycnic centrifugation studies of the abortive system failed to detect viral deoxyribonucleic acid synthesis, whereas indirect immunofluorescent studies of viral proteins revealed that less than 10 per cent of the cells contained these viral macromolecules at any given time. Collectively the data suggest that after transformation by simian adenovirus 7 these cells are altered so as to render them resistant or incapable of supporting the growth of herpes simplex virus types 1 and 2. The results further suggest that the block occurs after viral absorption and prior to viral deoxyribonucleic acid synthesis.

Synthesis of Vaccinia Virus Polypeptides in the Presence of Hydroxyurea

Hydroxyurea (5 x 10(-3) M) inhibits the development of vaccinia virus in HeLa cells early after infection and prevents the synthesis of viral deoxyribonucleic acid. In the presence of hydroxyurea, late viral polypeptides are not formed, as observed by electrophoresis on polyacrylamide gels.

Mechanism of Shope fibroma virus-induced suppression of host deoxyribonucleic acid synthesis.

The effects of treatment with live or inactivated Shope fibroma virus on host cell deoxyribonucleic acid (DNA) synthesis were determined. The incorporation of (3)H-thymidine into nuclear DNA was suppressed by both active and inactivated virus, although live virus was more effective. During the early phase of infection, stimulation of host nuclear DNA synthesis of up to 240% of control value was observed in cells infected with active virus. Inhibition of DNA synthesis began at about the 8th h and was maximal by 12 h postinfection. Virus inactivated by ultraviolet-irradiation or heat treatment did not induce viral DNA synthesis but was, nevertheless, able to suppress host DNA synthesis.

Replication of simian virus 40 deoxyribonucleic acid: analysis of the one-step growth cycle.

The time course of replication of simian virus 40 deoxyribonucleic acid (DNA) was investigated in growing monolayer cultures of subcloned CV1 cells. At multiplicities of infection of 30 to 60 plaque-forming units (PFU)/cell, first progeny DNA molecules (component 1) were detected by 10 hr after infection. During the following 10 to 12 hr, accumulation of virus DNA proceeded at ever increasing rates, albeit in a non-exponential fashion. The rate of synthesis then remained constant, until approximately the 40th hour postinfection, when DNA replication stopped. Under these conditions, the duration of the virus growth cycle was approximately 50 hr. The time needed for the synthesis of one DNA molecule was found to be approximately 15 min. At multiplicities of infection of 1 or less than 1 PFU/cell, the onset of the linear phase of DNA accumulation was delayed, but the final rate of DNA synthesis was the same, independent of the input multiplicity. This was taken as a proof that templates for the synthesis of viral DNA multiply in the cell during the early phase of replication. However, the probability for every replicated DNA molecule to become in turn replicative decreased constantly during that phase. This could be accounted for by assuming a limited number of replication sites in the infected cell.

Gene expression during the development of Bacillus subtilis bacteriophage phi29. II. Resolution of viral-specific ribonucleic acid molecules.

The ribonucleic acid (RNA) specified by bacteriophage phi29 was isolated under conditions which minimized physical and enzymatic degradation, reduced aggregation, and enriched for completed molecules. This RNA was fractionated both by sedimentation through sucrose density gradients and electrophoresis through polyacrylamide gels to measure the size and relative amount of each component. Early RNA consisted of six components of molecular weight 0.75 x 10(6), 0.44 x 10(6), 0.37 x 10(6), 0.25 x 10(6), 0.09 x 10(6), and 0.04 x 10(6), accounting for 35% of the coding capacity of phi29 deoxyribonucleic acid (DNA). All of these components except the one at 0.44 x 10(6) were detected when infection occurred in the presence of chloramphenicol. Synthesis of the major early component (0.75 x 10(6)) ceased shortly after the onset of viral DNA synthesis. The other species of early RNA were synthesized throughout the latent period. Three additional components, 1.75 x 10(6), 0.93 x 10(6), and 0.07 x 10(6), appear at late times. The two large RNAs may be polycistronic messenger RNAs corresponding to the seven viral capsid proteins.

Antiviral action of camptothecin.

At a concentration of 10 mum, camptothecin inhibited vaccinia deoxyribonucleic acid (DNA) synthesis in HeLa cells. Inhibition of viral DNA synthesis was observed when the drug was added before infection or at 1 or 2 hr after infection. Inhibitory effects of camptothecin on vaccinia DNA synthesis could be reversed, even after exposure to the alkaloid for 2 hr. Viral DNA, isolated from vaccinia-infected, camptothecin-treated cells, displayed an altered sedimentation constant after alkaline sucrose density gradient centrifugation. Incorporation of uridine into vaccinia messenger ribonucleic acid was inhibited by camptothecin, but the activity of ribonucleic acid polymerase, as tested in isolated vaccinia cores, was not affected by the drug. Camptothecin had essentially no effect on replication of poliovirus in HeLa cells.