Replication Of Adenovirus Type Research Articles

The influence of extracts from Peucedanum salinum on the replication of adenovirus type 5

IntroductionThe aim of the study was to examine the cytotoxicity and evaluate the antiviral and virucidal activity of methanol and methanol/H2O extracts from the herb of Peucedanum salinum. Plants belonging to the genus Peucedanum (Apiaceae family) have been used in traditional medicine for a long time.Material and methodsThe examination of the cytotoxicity of the extracts in the concentrations of 0.1, 0.2, 0.5, 1, 2, 4 mg/ml was carried out on the cell culture line HEK-293. Cytotoxicity of the examined extracts was measured by the colorimetric MTT (tetrazolium) method. After determination of the highest non-toxic concentration of examined extracts, antiviral and virucidal activity against adenovirus type 5 (Adenoviridae) was established.ResultsThe non-toxic doses were as follows: 1 mg/ml of methanol extract and 2 mg/ml of methanol/H2O extract (1 : 1 v/v). Antiviral activity was observed for the methanol extract of Peucedanum salinum in concentrations of 0.5 and 1 mg/ml. The extract caused the decrease of titre of the virus by 2 log and 1.33 log, respectively. The methanol/H2O extract (1 : 1 v/v) decreased the titre of the virus by 1.33 log and 1.5 log in concentrations of 1 and 2 mg/ml, respectively. The examined extracts had no virucidal activity against adenovirus type 5.ConclusionsThe examined extract is a new, potentially active source of active substances possessing antiviral activity and further studies are needed.

YB-1 als potenzielles Ziel für die Tumortherapie

The 42-kDa multifunctional cellular protein Y-box protein 1 (YB-1) is expressed in various cancers. It is localized in the cytoplasm as well as in the nucleus. In particular, YB-1 is localized in the nuclear compartment following cellular stress, such as radiation, drug treatment, hyperthermia, or viral infection. Within the nucleus, YB-1 can act as a transcription factor, and it is involved in the regulation of important cancer-associated genes. For example, YB-1 triggers the expression of Her-2 and estrogen receptor alpha (ERalpha) in breast cancer. Thus, nuclear YB-1 appears to be a potential target for the inhibition of Her-2- and ERalpha-dependent proliferation signals, particularly with regard to resistance to Her-2-targeting drugs such as trastuzumab. In some cancers, YB-1 may be involved in regulating MDR1/P-glycoprotein, mediating classical multidrug resistance (MDR). Furthermore, YB-1 is involved in the replication of adenovirus type 5, a commonly used vector in gene therapy. Thus, YB-1 can trigger an "oncolytic" effect in YB-1 nuclear positive cancer cells treated with adenoviruses. Besides its impact as a prognostic factor, in the future the diagnostics of cellular YB-1 status may provide the basis for a virotherapy or a gene therapy incorporating adenoviruses.

Midkine promoter-based conditionally replicative adenovirus therapy for midkine-expressing human pancreatic cancer

BackgroundTo develop a novel therapeutic strategy for human pancreatic cancer using a midkine promoter-based conditionally replicating adenovirus.MethodsWe examined midkine mRNA expression and midkine protein expression by seven human pancreatic cancer cell lines (AsPC-1, BxPC-3, CFPAC-1, HPAC, MIAPaCa-2, PANC-1, and Suit-2), as well as by non-cancerous pancreatic tissue and pancreatic cancers. Midkine promoter activity was measured in cancer cell lines by the dual luciferase reporter assay. Adenoviral transduction efficiency was assessed by fluorescent staining of cancer cell lines using adenovirus type 5 containing the green fluorescent protein gene (Ad5GFP). Replication of adenovirus type 5 containing the 0.6 kb midkne promoter (Ad5MK) was assessed by the detection of E1 protein in cancer cell lines. The cytotoxicity of Ad5MK for cancer cells was evaluated from the extent of growth inhibition after viral infection. Infection and replication were also assessed in nude mice with subcutaneous Suit-2 tumors by intratumoral injection of Ad5MK, Ad5GFP, or vehicle. E1a mRNA expression in the treated tumors and expression of the replication-specific adenoviral hexon protein were evaluated. Finally, the anti-tumor activity of Ad5MK against intraperitoneal xenografts of Suit-2 pancreatic cancer cells was examined after intraperitoneal injection of the virus.ResultsBoth midkine mRNA expression and midkine protein expression were strong in AsPC-1 and CFPAC-1 cell liens, moderate in BxPC-3, HPAC, and Suit-2 cell lines, and weak in PANC-1 and MIAPaCa-2 cell lines. Expression of midkine mRNA was significantly stronger in pancreatic cancers than in non-cancerous pancreatic tissues. The relative luciferase activity mediated by the 0.6 kb midkne fragment in AsPC-1, PANC-1, and Suit-2 cell lines was approximately 6 to 20 times greater than that in midkne-negative MIAPaCa-2 cell lines. Pancreatic cancer cell lines exhibited a heterogeneous adenoviral transduction profile. E1A expression was higher in cell lines with strong midkine expression than in cell lines with weak midkine expression. Ad5MK showed much greater cytotoxicity for midkine-expressing Suit-2 and PANC-1 cell lines than for midkine-negative MIAPaCa-2 cell lines. In the Suit-2 subcutaneous xenograft model, expression of E1A was detected in Ad5MK-treated tumors, but not in untreated and Ad5GFP-treated tumors. In the Suit-2 intraperitoneal xenograft model, the Ad5MK group survived for significantly longer than the Ad5GFP, PBS, and untreated groups.ConclusionAd5MK has an anti-tumor effect against human pancreatic cancer cell lines that express midkine mRNA. Midkine promoter-based conditionally replicative adenovirus might be a promising new gene therapy for pancreatic cancer.

Identification of proteins binding to interferon-inducible transcriptional enhancers in hematopoietic cells.

The binding of nuclear proteins of hematopoietic cells to transcriptional enhancers of interferon-inducible genes has been studied before and after exposure to alpha-interferon. Mobility shift assays show that a complex formed with interferon-inducible transcriptional enhancers before interferon induction contains a 73- and 84-kDa protein. Amino acid sequencing of the oligoaffinity column purified 73- and 84-kDa proteins showed that they belonged to a family of DNA-binding proteins which have been previously identified to exhibit binding in a sequence nonspecific manner to the ends of fragmented DNA or the origin of replication of adenovirus Type 2 DNA and sequence-specific binding to the distal regions of the U1 small nuclear RNA promoter, the promoter of the transferrin receptor gene, and the transcriptional regulatory regions of HLA genes. Following exposure to alpha-interferon, more slowly migrating complexes appeared which contained a 48-kDa protein, a 95-kDa protein, and a 105-kDa protein which bound to the 9-27 transcriptional enhancer in a sequence-specific manner.

Effect of linoleic acid hydroperoxide on replication of adenovirus DNA in endothelial cells of bovine aorta

The effect of linoleic acid hydroperoxide on replication of adenovirus type 2 DNA in cultured endothelial cells of bovine aorta was studied to elucidate the deleterious effect of lipid peroxides on replication of DNA in the arterial endothelial cells. When, just after the infection, the cells were incubated with 1 nmol (measured by the thiobarbituric acid reaction and expressed in terms of malondialdehyde of the hydroperoxide, the replication of adenovirus type 2 DNA was markedly reduced. The level of the messenger RNA for adenovirus type 2 DNA polymerase was also reduced. In accordance with the reduction of the messenger RNA, the synthesis of the polymerase in the cells was reduced. The inhibition of DNA replication in the arterial endothelial cells by lipid peroxides was discussed in relation to atherogenesis.

Replication of adenovirus type 4 DNA by a Purified fraction from infected Cells

An extract from Adenovirus type 4 infected HeLa cells was fractionated by ion-exchange and DNA affinity chromatography. One fraction, which bound tightly to single stranded DNA, contained predominantly a protein of apparent molecular weight 65,000 and three less abundant proteins. Immunological cross-reactivity with adenovirus type 2 proteins confirmed the presence of preterminal protein and indicated that the abundant species was the virus coded DNA binding protein. This fraction contained an aphidicolin resistant DNA polymerase activity and in the presence of a linearised plasmid containing the adenovirus type 4 origin of DNA replication efficient transfer of dCMP onto preterminal protein, indicative of initiation, was observed. Furthermore, addition of all four deoxyribonucleotide triphosphates and an ATP regenerating system resulted in the elongation of initiated molecules to generate plasmid molecules covalently attached to preterminal protein. Adenovirus type 4 DNA binding protein was extensively purified from crude adenovirus-4 infected HeLa extract by immunoaffinity chromatography using a monoclonal antibody raised against adenovirus type 2 DNA binding protein. A low level of initiation of DNA replication was detected in the fraction depleted of DNA binding protein but activity was restored by addition of purified DNA binding protein. DNA binding protein therefore plays an important role in the initiation of Ad4 DNA replication.

In vitro exposure of adenovirus types 5 and 7 in oropharyngeal secretions to cigarette smoke

Adenovirus types 5 and 7 were suspended in clarified oropharyngeal secretions. After 1 ml of suspension was dispersed in a thin layer over a 25-cm2 surface in a flask, the suspension was exposed at 37 degrees C to eight 25-ml puffs of smoke from one cigarette. A mechanical smoking apparatus was used. Nonfilter cigarettes used had 23 mg of tar and 1.4 mg of nicotine, and filter cigarettes used had 19 mg of tar and 1.2 mg of nicotine. Smoke was flushed from the flask with normal filtered air. At 0, 0.25, and 1 h after exposure to smoke, untreated and smoke-treated viruses were titrated with monolayer cultures of human epithelioid (HEp-2) cells. Normal air was in contact with the suspensions between puffs and between smoke treatments and virus titrations. Smoke from filter or nonfilter cigarettes had no effect on the infectivity and replication of adenovirus types 5 and 7. Smoke from four cigarettes administered over a 4-h period caused a 2- to 3-log10 drop in the titers of both viruses. Smoke from four cigarettes was also highly toxic to HEp-2 host cells of the viruses.

Further studies on the replication of adenovirus type 2 DNA.

Monolayer cultures of KB cells infected with adenovirus type 2 (Ad2) were subjected to short pulses of tritiated thymidine, at the time of maximal viral DNA synthesis. Nascent viral DNA was selectively extracted from the cells, purified, and fractionated into partly single-stranded (or replicating) molecules and double-stranded (or completed) molecules. Completed molecules were cleaved by the restriction endonucleases from either Escherichia coli (EcoRI), Haemophilus influenzae (Hind III), Haemophilus parainfluenzae (Hpa I), or by the latter two enzymes in succession, and the resulting fragments were separated by electrophoresis through agarose–acrylamide slab gels. In the case of replicating molecules, the double-stranded fragments generated after treatment with the same enzymes were first separated from the partly single-stranded fragments by chromatography on benzoylated–naphthoylated DEAE (BND) cellulose before being subjected to electrophoresis. The relative yields of tritium of the various fragments resolved by the gels were then determined. As already described in several reports, the analysis of the completed molecules obtained after the shorter pulses revealed a strong preferential labeling of the fragments derived from the molecular ends, as expected if both of these ends contained termini for replication. The analysis of double-stranded fragments from replicating molecules revealed a predominant labeling of the central portion of the genome which could be reconciled with initiation occurring either centrally or at both molecular ends. In addition, two gradients of labeling, ascending toward each molecular end and encompassing the outer quarters of the molecule, were noted which slowly receded when the pulse length was increased. The latter finding, which is in agreement with our earlier results, could reflect an overrepresentation of molecules in the late stages of replication in the pool of nascent DNA, such as one would expect if replication slows down when it nears the termination site(s). This interpretation is consistent with the kinetics of labeling of the various portions of the genome observed in the pool of completed molecules. Because of this complication, however, the distribution of label in replicating molecules cannot be used to ascertain unambiguously the location of the origin(s) of replication on the Ad2 DNA molecule.

The mechanism of replication of adenovirus DNA VI. Localization of the origins of the displacement synthesis

Previous investigations have shown that replication of adenovirus type 5 (Ad5) proceeds via a displacement mechanism (see Levine et al., Curr. Topics Microbiol. Immunol. 6, for a review). This communication describes the localization of the origins of the displacement synthesis on the genome by restriction enzyme analysis of replicative intermediates synthesized during the first round of DNA replication. Replicative intermediates were isolated from KB cells infected with the temperature-sensitive mutant H5ts125, in which initiation of DNA replication is impaired. Infection was performed for 16 hr at the nonpermissive temperature (40°) during which no viral DNA synthesis occurred. Then the temperature was shifted to the permissive temperature (32°) and the cells were further incubated at this temperature. Replicative intermediates, labeled during various periods of time after shift-down, were isolated and subjected to restriction enzyme analysis. The highest specific radioactivity was found in restriction enzyme fragments which represent the two molecular ends. Preferentially only one of the two complementary strands at each molecular end was labeled. These results indicate that in the first round of replication the displacement synthesis may start at both molecular termini. The origin of the displacement synthesis of the viral r-strand is located at the molecular right-hand-end and synthesis of this strand proceeds from right to left, while the displacement origin of the viral l-strand is located at the molecular left-hand-end and synthesis of this strand proceeds in the opposite direction.

Characterization of a temperature-sensitive mutant of human adenovirus type 7.

The properties of a naturally occurring temperature-sensitive (ts) mutant of human adenovirus type 7 (Ad7) were studied. Mutant Ad7 (19), or E46-, was the nonhybrid adenovirus component derived from the defective simian virus 40 (SV40)-Ad7 hybrid (PARA). Growth of the mutant was restricted at 40.5 degrees C, and the ratios of virus yields in KB cells at 40.5 and 33 degrees C were 10(-2) to 10(-3). Viral DNA synthesis and the synthesis of adenovirus-specific antigens (tumor, capsid, hexon, and penton antigens) appeared normal at the restrictive temperature. The assembly of virus particles was aberrant, as determined by thin-section of infected cells. The infectivity of mutant virions was heat labile at 50 degrees C, suggesting a ts defect in a structural component of the viron. Analysis by polyacrylamide gel electrophoresis of [35S]methionine-labeled polypeptides synthesized in mutant-infected cells suggested that at least the major virion polypeptides were synthesized at the restrictive temperature. A lack of inhibition of host protein synthesis late in mutant infections, as compared with wild-type (WT) infections at both the permissive and nonpermissive temperatures, made quantitation of infected-cell polypeptides difficult. Analysis of the assembly of capsomeres from cytoplasmic extracts of infected cells on sucrose gradients and by non-dissociating polyacrylamide gel electrophoresis suggested that hexon capsomeres were made at 40.5 degrees C. The hexon capsomeres made by the mutant at either 33 or 40.5 degrees C displayed a decreased migration in the non-dissociating gels compared with the WT hexon capsomeres. The molecular weights of the mutant and WT hexon polypeptides were identical. These results suggest that the ts lesion of this group B human Ad7 mutant may be reflected in altered hexons. The mutant Ad7 interfered with the replication of adenovirus types 2 and 21 at the elevated temperature.

Characterization of single-stranded viral DNA sequences present during replication of adenovirus types 2 and 5

Replication intermediates of adenovirus DNA apparently contain extensive stretches of single-stranded DNA. Such single-stranded viral DNA sequences homologous to different regions of the viral genome present in adenovirus-infected cells during viral DNA replication have therefore been characterized by hybridization to the separated strands of restriction endonuclease fragments of 32P-labeled adenovirus types 2 and 5 DNA. Saturation hybridization experiments with infected cell DNA extracted at late times suggest that all regions of the adenovirus genome are represented in the single-stranded fraction, but at unequal frequencies. This nonuniform representation has been characterized in more detail with self-annealed, total cell DNA extracted 18 hr after adenovirus type 2 infection: the concentration of single-stranded sequences homologous to different regions of the viral genome was determined by comparing the rates of hybridization of 32P-labeled, single-stranded DNA probes with such self-annealed 18 hr DNA to the rates of hybridization of the same probes with equal concentrations of their complements. This approach allows the concentration of single-stranded viral DNA sequences in excess of their complements to be determined. Such sequences can be represented by two concentration gradients across the viral genome: those homologous to the r strand increase in concentration from 27.8–40.9 units toward the right end, whereas sequences homologous to the I strand increase from an area 27.8–40.9 units toward the left end. The time course of synthesis of single-stranded viral DNA sequences relative to accumulation of total viral DNA during the productive cycle and their behavior following a shift of H5ts125-infected cells in which viral DNA replication has begun from a permissive to a nonpermissive temperature support the contention that these sequences are indeed generated as adenovirus DNA is replicated. These results are therefore discussed in terms of current models of adenovirus DNA replication.

Replication of adenovirus type 5 DNA in KB cells: localization and fate of parental DNA during replication.

Abstract The localization and fate of parental adenovirus type 5 (Ad5) DNA in KB cells at the time viral DNA replication occurs has been investigated by electron-microscope autoradiography and biochemical methods. At 18 hr after infection the majority of the parental Ad5 DNA molecules is present in the nucleus. About 60% of these molecules are involved in viral DNA replication by this time, but about 40% have not yet replicated at all. Electron-microscope autoradiography of cells infected with 3H-labeled Ad5 reveals that replicating parental Ad5 DNA molecules are randomly distributed throughout the nucleus, while nonreplicating viral DNA has a preference to locate in the periphery of the nucleus. Based on the experiments reported in this communication, the intranuclear fate of parental Ad5 DNA is discussed.

Localization of adenovirus DNA replication in KB cells

The localization of adenovirus type 5 DNA replication has been investigated by both fractionation of isolated nuclei and electron-microscope autoradiography. Nuclear fractionation by means of the M-band-technique of Tremblay et al. (Tremblay, G. Y., Daniels, M. J., and Schaechter, M. (1969). J. Mol. Biol. 40, 65–76.) reveals that pulse-labeled adenovirus type 5 DNA cosediments with the nuclear membrane. On the basis of this finding it can be supposed that the site of viral DNA replication is at the nuclear membrane. However, electron-microscope autoradiography of infected cells shows that the pulse-labeled viral DNA is not located at the nuclear membrane but randomly distributed over the nucleus. It is concluded that the replication of adenovirus type 5 DNA occurs throughout the cell nucleus; the association of replicating viral DNA with a nuclear membrane complex should be considered as an artifact.

Studies on the mechanism of replication of adenovirus DNA: IV. Discontinuous DNA chain propagation

The replication of adenovirus type 5 DNA occurs by discontinuous chain propagation via short pieces of DNA. These pieces accumulate if the infected cells are treated with hydroxyurea. They have a sedimentation coefficient of 11 S corresponding to a molecular weight of about 700,000, and they contain sequences derived from all parts of the adenovirus genome. Annealing and hybridization studies show that the short pieces represent sequences complementary to both strands of adenovirus type 5 DNA.

Studies on the mechanism of replication of adenovirus DNA. II. The nature of single-stranded DNA in replicative intermediates.

Replicative intermediates of adenovirus type 5 DNA contain large stretches of single-stranded DNA. We have shown that this single-stranded DNA is mainly of parental origin, whereas all new DNA synthesized during one round of replication has a double-stranded structure. Hybridization experiments of the single-stranded DNA with isolated complementary strands of adenovirus type 5 DNA showed that this DNA hybridized only with the viral L-strand (the strand with the lower equilibrium density in alkaline CsCl) indicating that it represents the viral H-strand. This observation implies that replication always starts from one and the same molecular end. Electron microscopy of partially denatured Y-shaped intermediates confirmed this and showed that replication started from the molecular right end (the end richest in A-T base pairs). In conclusion, we have shown that replication of adenovirus type 5 DNA starts at the molecular right end, displacing the parental H-strand.

Effects of dibutyryl-cAMP on adenovirus replication in different cell lines

Replication of adenovirus type 2 (Ad2) was inhibited by dibutyryl-cAMP (db-cAMP) in three fibroblastic cell lines, BHK21, HEK, and WI-38, and in Ad12 transformed BHK21 cells with high intracellular level of cAMP. Yield of Ad12 in WI-38 cells was also reduced. The inhibitory effects were undiminished when db-cAMP was added up to 9 h.p.i. but decreased on later additions. In contrast, replication of either Ad2 or Ad12 was not affected by db-cAMP in two tumor-derived human cell lines, HeLa and KB.

Differential release of cellular lactate dehydrogenase during replication of adenovirus types 5 and 12.

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Thermosensitive events in the replication of adenovirus type 2 at 42°

The production of infectious adenovirus type 2 is reduced more than 99% when infected KB suspension cultures are maintained at 42°. The abortive event(s) at 42° occur late after infection, for cultures held at 42° for 12 hr and then shifted to 37° yield normal amounts of infectious virus. Although 31 S viral DNA and virion polypeptides are synthesized at 42°, no 800 S viral particles are formed. The failure to produce virus at the elevated temperature can be attributed to lack of capsomere assembly. Although infected cultures incubated at 42° accumulate large amounts of structural viral polypeptides, formation of hexon capsomeres is reduced 99% and penton and fiber assembly are diminished more than 90%.

Origin of thymidine kinase in adenovirus-infected human cell lines.

Human adenovirus type 5 enhances the thymidine kinase activity of KB cells but does not induce the enzyme in kinase-deficient HeLa (BU25) cells. Vaccinia induces thymidine kinase activity in both KB and HeLa (BU25) cells. Human adenovirus types 2, 4, 7, and 12 also fail to induce the enzyme in HeLa (BU25) cells. Vaccinia replicates equally well in the presence or absence of HATG (hypoxanthine-aminopterin-thymidine-glycine) in KB and HeLa (BU25) cells. Adenovirus type 5 replicates in KB and in HeLa (BU25) cells in the absence of HATG, and adenovirus type 5 replicates in kinase-positive KB cells in the presence of HATG. However, replication of adenovirus type 5 is grossly inhibited in HeLa (BU25) cells in the presence of HATG. These results suggest that human adenoviruses do not code for a new virus-specific thymidine kinase.

Replication of adenovirus type 7 in monkey cells: a new determinant and its transfer to adenovirus type 2.

A strain of human adenovirus type 7, adapted to replication in green-monkey kidney cells, requires the interaction of two particles to initiate plaque formation in the simian cells. One particle is a true adenovirion. The second, apparently defective, consists of a genome carrying amonkey-adapting component in an adenovirus capsid; this genome does not express known SV40 determinants. The addition of human adenovirus type 7 that is not adapted enhances the titer and changesconditions for plaque formation by the adapted virus to a one-particle requirement. Addition of nonadapted human adenovirus type 2 as helper virus results in the transfer of the monkey-adaptingcomponent from adenovirus type 7 to adenovirus type 2. The population containing the adenovirus 2 transcapsidant then has the ability to replicate in simian cells.