Heterologous expression, purification, and characterization of adenovirus DNA polymerase and preterminal protein.
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Adenoviridae↗
Biomedical subjects
R Padmanabhan
Publications and source records attributed to R Padmanabhan.
Synthesis and characterization of an infectious dengue virus type-2 RNA genome (New Guinea C strain).
Dengue virus type 2 (DEN-2), a member of the Flaviviridae family, has a positive-strand RNA genome, 10,723 nucleotides (nt) in length and encoding a single polyprotein precursor consisting of 3391 amino acids (aa). In order to construct a full-length cDNA clone, the viral genome was cloned into 5' (nt 1-2203 under the control of the T7 promoter (pT7)) and 3' (nt 2203-10,723) constructs. A full-length DEN-2 cDNA under pT7 control was assembled in vitro after excising the two cDNA inserts from the 5' and 3' constructs, and joining them with T4 DNA ligase. The RNA produced by in vitro transcription of the cDNA using T7 RNA polymerase was infectious, as shown by transfection of permissive BHK-21 and Vero cells, and propagation of the virus particles released into the culture media. The virus particles stably maintained the conservative mutation introduced into the 5' construct, and the cells infected with the infectious RNA-derived virus synthesized virus-specific DEN-2 antigens, as shown by immunofluorescence and immunoprecipitations. The full-length infectious clone for DEN-2 should be useful for the study of molecular mechanisms involved in viral RNA replication and virus assembly.
Antigens, Viral↗
Association between NS3 and NS5 proteins of dengue virus type 2 in the putative RNA replicase is linked to differential phosphorylation of NS5.
Dengue virus type 2, a member of the family Flaviviridae, encodes a single polyprotein precursor consisting of 3391 amino acids residues that is processed to at least 10 mature proteins by host and viral proteases. The NS3 protein contains a domain commonly found in cellular serine proteinases that in cooperation with NS2B is involved in polyprotein processing. In addition, NS3 and NS5 proteins contain conserved motifs found in several RNA helicases and RNA-dependent RNA polymerases, respectively. Both enzymatic activities have been suggested to be involved in viral RNA replication. In this report, we demonstrate that the NS3 and NS5 proteins interact in vivo in dengue virus type 2-infected monkey kidney (CV-1) cells and in HeLa cells coinfected with recombinant vaccinia viruses encoding these proteins as shown by coimmunoprecipitations and immunoblotting methods. We also show by immunofluorescence, metabolic labeling, and two-dimensional peptide mapping that NS5 is a nuclear phosphoprotein and that phosphorylation occurs on serine residues at multiple sites. Furthermore, NS5 exists in differentially phosphorylated states in the nuclear and the cytoplasmic fractions, and only the cytoplasmic form of NS5 is found to coimmunoprecipitate with NS3, suggesting that differential phosphorylation may control the interaction between these proteins and its function in the viral RNA replicase.
Amino Acid Sequence↗
Aspirin-alcohol interaction in the production of cleft palate and limb malformations in the TO mouse.
Our objective in the present study was to determine the effects of alcohol on stages when the limb buds and renal primordia develop in the TO mouse and to see if aspirin pretreatment would prevent these organ systems from being malformed as was shown by Randall et al. ('91) in the C57 mice. On one of days 9-12 of gestation, groups of TO mice were injected intraperitoneally (IP) with a single dose of 200 mg/kg of aspirin, or a proportionate volume of physiological saline. An hour later, half of the aspirin-treated animals received a single dose of 0.03 ml/g of freshly prepared 25% (v/v) solution of absolute alcohol and the other half received a proportionate volume of saline. Half of the saline-treated animals received a single dose of 0.03 ml/g of saline or a proportionate volume of alcohol solution. All animals were killed on day 18 of gestation. Alcohol significantly increased embryonic resorption and caused remarkable intrauterine growth retardation (IUGR). It also induced arched palate, cleft palate and deformities of the digits with haematomas in a modest number of embryos. Aspirin alone did not have any teratogenic effects. Pretreatment with aspirin significantly augmented alcohol-induced resorption, IUGR, cleft palate and digital malformations associated with haematomas. Chronological observations on the development of the treated limbs showed the occurrence of vascular stasis, haematomas, edema and cell death at early stages. Subsequently, digital rays were either destroyed (ectrodactyly) or remained hypoplastic (brachydactyly). It appears that limb development in the aspirin- and alcohol-treated TO mouse embryos is largely affected by vascular disruption. These data provide further evidence to our earlier observation that alcohol and aspirin interact in the production of malformations and that the teratogenic effects of alcohol in the TO mouse are possibly not mediated via treatment related prostaglandin elevation.
Abnormalities, Drug-Induced↗
Expression, nuclear transport, and phosphorylation of adenovirus DNA replication proteins.
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Adenoviridae↗
Modeling and optimising dextrose fermentation using a fluorosensor.
Dextrose has been fermented in a Tokyo Rikakikai Fermentor at 32 degree celsius using seven different concentrations of yeast as seeding. The progress of the reaction was followed by measuring the fluorescent signal due to NADH with a Dr. Ingold (Switzerland) fluorosensor which has an excitation wavelength of 360 nm and measurement wavelength of 450 nm. The optimum concentration for this fermentation reaction is 30 percent seeding. At this concentration the biomass growth rate and final biomass concentration are a maximum. The fluorescent voltage vs time data fitted a first order model with an error of less than one percent. Further work on optimising the temperature is in progress.
Biomedical Engineering↗
Biochemical characterization of a temperature-sensitive adenovirus DNA polymerase.
Involvement of adenovirus DNA polymerase (AdPol) in viral DNA replication was initially demonstrated using two temperature-sensitive (ts) adenoviruses, Ad5ts36 and Ad5ts149. Ad5ts36 is particularly interesting because a single mutation mapping to Leu391-->Phe in the AdPol gene confers ts phenotype not only for DNA replication in human cells but also for transformation in rat cells. In order to elucidate the mechanism(s) involved in the ts phenotype, we introduced the ts36 mutation in AdPol of adenovirus type 2 and overexpressed the protein using recombinant vaccinia virus expression system in order to study its biochemical properties. The results show that the ts36 AdPol is defective in the in vitro DNA replication initiation and DNA polymerase elongation assays if the protein is expressed at the nonpermissive temperature (37 degrees) independent of whether the assays are performed at 32 or 37 degrees. In contrast, the ts36 AdPol expressed at 32 degrees had activities similar to that of the wild-type protein when assayed at either temperature. Furthermore, unlike the wild-type protein, ts36 AdPol expressed at 37 degrees failed to recognize the viral DNA replication origin, but bound to a single-stranded DNA cellulose column with greater affinity, suggesting that the defect in the ts36 AdPol for DNA replication can be attributed to its altered DNA-binding properties.
Adenoviridae↗
Phosphorylation-dependent interaction of adenovirus preterminal protein with the viral origin of DNA replication.
Adenovirus preterminal protein (pTP) exists as a heterodimer with the viral DNA polymerase (AdPol) and becomes covalently linked to a dCMP residue during initiation of DNA replication. The in vivo phosphorylation of pTP could be demonstrated when pTP is overproduced using recombinant vaccinia viruses, or by a large scale metabolic labeling of adenovirus 2 (Ad2)-infected HeLa cells. Phosphoserine was the only phosphoamino acid obtained by acid hydrolysis of 32P-labeled pTP immunoprecipitated from metabolically labeled HeLa cells infected with either Ad2 or recombinant vaccinia virus. Tryptic peptide maps of pTP expressed using recombinant vaccinia virus system in HeLa cells revealed that phosphorylation of pTP occurred on multiple sites. Dephosphorylation of pTP with calf intestinal alkaline phosphatase resulted in a significant decrease in its activity in the in vitro DNA replication initiation assays. Further characterization of the phosphatase-treated pTP indicated that although dephosphorylation did not affect its interaction with AdPol, the specific recognition of the DNA replication origin by pTP was significantly reduced as determined by gel electrophoresis-based DNA mobility shift assays.
Adenoviruses, Human↗
Constitutive over-expression of transforming growth factor-alpha in rat liver epithelial cells leads to increased cell cycling without transformation.
Over-expression of transforming growth factor-alpha (TGF-alpha) is consistently seen in spontaneous transformants of rat liver derived epithelial cells (RLE phi 13) and has been implicated in the transformation of other cultured cells. We have constitutively over-expressed TGF-alpha in RLE phi 13 cells, which are known to express epidermal growth factor receptors, to determine if TGF-alpha over-expression plays a role in transformation or differentiation, or both, of these cells. Early passage RLE phi 13 cells were infected with a replication-defective murine retrovirus that expresses both the full length coding sequence for human TGF-alpha and the neomycin-resistance gene. Integration of the transcriptionally active provirus and expression of TGF-alpha mRNA were confirmed. Neither morphologic transformation nor molecular evidence for differentiation was noted in TGF-alpha-producing clones. However, these clones did exhibit an accelerated growth rate, increased expression of several cell cycle related genes including mitotic cyclic B1, proliferating cell nuclear antigen, c-myc, and p53 as well as increased expression of the preneoplastic marker enzyme, glutathione-S-transferase. This suggests that over-expression of TGF-alpha results in increased cell cycling, and that subsequent events must be necessary for cellular transformation or differentiation or both.
Animals↗
Effect of pre-treatment with aspirin on alcohol-induced neural tube defects in the TO mouse fetuses.
This study was planned to investigate the effects of ethanol on the genetic susceptibility of the TO mouse to neural tube malformations and to determine the ameliorative effects, if any, of aspirin a potent prostaglandin inhibitor. The TO mouse exhibits a spontaneous incidence of 3.6% exencephaly. The mice were exposed to single (i.p.) dose of 0.03 ml/gm body weight of a solution of (25%v/v) of absolute alcohol in physiological saline on day 7 or 8 of gestation. Subteratogenic doses (150 or 200 mg/kg) of aspirin were administered (i.p.) an hour before ethanol exposure. Fetuses were collected on day 18 and compared with those of the untreated, and saline treated pair-fed pair-watered controls as well as with those of the aspirin alone treatment group. A total of 175 litters were studied. Alcohol caused a three-fold increase against the background incidence of exencephaly. Several craniofacial anomalies and growth retardation were also observed. Alizarin red-S stained skeletal preparations revealed extensive malformations of the craniofacial skeleton in the exencephalic fetuses. Both doses of aspirin administered prior to alcohol treatment significantly accentuated the alcohol-induced prenatal mortality. The rescue effect of aspirin on alcohol-induced intrauterine growth retardation was also significant although fetal weight was not restored to levels comparable to those of the controls. Pre-treatment with aspirin (both 150 and 200 mg/kg) on day 8 of gestation resulted in a numerical, though not statistically significant increase in alcohol-induced exencephaly. On the other hand pre-administration of the lower dose on day 7 exencephaly. On the other hand pre-administration of the lower dose on day 7 of gestation caused a significant reduction while the higher dose gave rise to a significant increase in the incidence of this malformation. Aspirin also reduced the frequency of alcohol-induced arched palate and the baseline exencephaly. These data provide evidence for the possible interaction of alcohol with the genetic susceptibility to exencephaly in this strain of mice. The lack of a clear dose-dependent antagonistic effect of aspirin on alcohol-induced exencephaly suggests that the production of this malformation is probably not mediated by prostaglandin as it was shown for limb and renal abnormalities (Randall, C.L., Anton, R.F. and Becker, H.C. (1991). Aspirin dose dependently reduces alcohol induced birth defects and prostaglandin E levels in mice. Teratology 44, 521-529).
Anencephaly↗
A modified method for isolation of recombinant vaccinia virus.
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DNA-Directed DNA Polymerase↗
Expression of the extracellular domain of the rat liver prolactin receptor and its interaction with ovine prolactin.
A clone of the extracellular domain of the rat liver prolactin receptor was generated by the RNA-based polymerase chain reaction, and the NH2-terminal 210 amino acids were expressed in HeLa cells using a vaccinia virus/T7 hybrid expression system. The protein was isolated from serum-free culture medium directly by chromatography on an ovine prolactin affinity column and yielded approximately 1.5 mg of protein/liter of suspension culture. The extracellular domain of the rat prolactin receptor inhibited the ovine prolactin-dependent mitogenesis of rat lymphoma Nb2 cells with an IC50 of 7.1 pM and bound 125I-labeled ovine prolactin with a Kd of 1.21 +/- 0.19 nM. In contrast, the binding of the 125I-labeled extracellular domain to ovine prolactin exhibited positive cooperativity with a Hill coefficient of 1.73. High pressure gel filtration chromatography was used to demonstrate the formation of a complex consisting of one molecule of ovine prolactin and two molecules of the extracellular domain of the rat prolactin receptor. Complex formation occurred with human growth hormone, but not with ovine growth hormone, a non-lactogen.
Animals↗
Adenovirus DNA polymerase is phosphorylated by a stably associated histone H1 kinase.
Adenovirus DNA polymerase (AdPol) exists as a complex with the preterminal protein (pTP) and is essential for both initiation and elongation stages of viral DNA replication. Recent evidence from our laboratory indicates that AdPol is a phosphoprotein and that the major in vivo phosphorylation site, serine 67, occurs within the consensus substrate recognition sequence for cdc2 kinases. In this study, we found that a protein kinase which also exhibits histone H1 phosphorylation activity is stably associated with AdPol. AdPol forms a multimeric complex with this histone H1 kinase and pTP in HeLa cells infected with adenovirus or coinfected with recombinant vaccinia viruses encoding AdPol and pTP. The associated protein kinase and the p34cdc2 kinase phosphorylate AdPol at the same sites which are utilized in vivo, suggesting that the p34cdc2 kinase or a related kinase may be involved in the in vivo phosphorylation of AdPol. Serine 67 is also one of the major in vitro phosphorylation sites, and the substitution of alanine for serine at this position abolishes DNA replication initiation activity of AdPol.
Adenoviruses, Human↗
Role of protein conformation in the processing of dengue virus type 2 nonstructural polyprotein precursor.
The dengue virus type-2 (DEN-2) genome is a positive-strand RNA encoding a single polyprotein precursor, C-prM(M)-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B- NS5, consisting of 3391 amino acids (aa). The N-terminal region of the polyprotein precursor, C-prM(M)-E, encodes the structural proteins and is processed cotranslationally by the host signal peptidase. The nonstructural region NS1-->NS5 is processed by the viral protease(s), as well as by the signal peptidase. A two-component viral protease consisting of NS2B and the serine protease domain of NS3 has been shown to be required for cleavages having the consensus sequence of dibasic aa (K-R, R-R, R-K, or Q-R). In this study, the region encoding all the nonstructural proteins, NS1-->NS5, was expressed using a recombinant vaccinia virus system. Cleavages at the consensus viral protease recognition sites, 2B-3 at the N terminus and 3-4A at the C terminus, are prerequisites to the release of mature NS3 protease. Although the 2B-3 site was cleaved readily in a variety of polyprotein precursors containing the intact NS2B and the NS3 protease domain, the 3-4A site was most efficiently cleaved, similar to that found in DEN-2-infected cells, only in the polyprotein precursor encoding the entire nonstructural region. Removal of NS1 at the N terminus or of NS5 coding sequences at the C terminus affected the cleavage at the 3-4A site to produce the processing intermediate, NS3-NS4A. These results indicate that the conformation of the nonstructural polyprotein precursor, NS1-->NS5, plays a major role in the efficient cleavage at the 3-4A site.
Animals↗
Adenovirus DNA polymerase is a phosphoprotein.
Biological activities of many of the eukaryotic DNA replication proteins are modulated by protein phosphorylation. Investigations of the phosphorylation of adenovirus DNA polymerase (AdPol) have been difficult mainly because of its low level of synthesis in adenovirus-infected HeLa cells. However, when AdPol was overproduced using the recombinant vaccinia virus (RV-AdPol) and the baculovirus expression systems, or by a large scale metabolic labeling of adenovirus 2-infected HeLa cells (native AdPol), in vivo phosphorylation of AdPol could be demonstrated. Phosphoamino acid analysis of [32P]AdPol indicated the presence of phosphoserine independent of the source of AdPol. Comparison of tryptic peptide maps of native AdPol and RV-AdPol revealed that the majority of phosphopeptides were common. Fractionation by high performance liquid chromatography and sequencing of one of the major phosphopeptides revealed serine 67 as a site of phosphorylation. Interestingly, this site is located close to the nuclear localization signal of AdPol and has a consensus substrate recognition sequence for histone H1 (cdc2-related) kinases and mitogen-activated protein kinases. Dephosphorylation of AdPol with calf intestinal alkaline phosphatase resulted in significant decrease in its activity in the in vitro DNA replication initiation assay, suggesting that phosphorylation is important for its biological activity.
Adenoviruses, Human↗
Characterization of an unusual mutant of human melanoma cells resistant to anticancer drugs that inhibit topoisomerase II.
The topoisomerase II inhibitor, VP-16 (etoposide), is an important component in many chemotherapeutic regimens. To characterize resistance to this drug, the human melanoma cell line, FEM-X, was selected in multiple steps with VP-16. To prevent the development of typical multidrug resistance, an inhibitor of P-glycoprotein, the tiapamil analog, RO-11-2933, was added to the selections. The resultant clone FVP3 is 56-fold resistant to VP-16 and cross-resistant to doxorubicin (Adriamycin) (9-fold) and VM-26 (27-fold). These cells are also two- to four-fold resistant to m-AMSA, daunorubicin, and mitoxantrone. FVP3 is not resistant to the P-glycoprotein substrates vinblastine, does not express the MDR1 gene at detectable levels, and does not show reduced 3H-VP-16 accumulation. Unlike other cell lines that exhibit resistance to inhibitors of topoisomerase II, FVP3 has the same level of topoisomerase II expression and activity as FEM-X. Using live cells treated with VP-16, band depletion assays and KCI/SDS precipitation assays show that topoisomerase II from FVP3 is much less susceptible to drug-induced cleavable complex formation than is that from FEM-X. This difference in sensitivity to VP-16 is also detected using lysates from disrupted cells, but not with isolated nuclei devoid of cytoplasmic and membrane components. In addition, the topoisomerase II present in nuclear extracts from FVP3 is not resistant to the effects of VP-16 as measured by: (1) inhibition of strand passing activity during decatenation of kinetoplast DNA, (2) drug-induced linearization of plasmid DNA, and (3) immunodepletion by VP-16. These results suggest that some component of the cytoplasm or cellular membranes, or a factor depleted from nuclei during their isolation, is responsible for the resistance to VP-16 in FVP3.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Modified Doppler flowmetry in the splanchnic circulation.
BACKGROUND: Absolute measurements of blood flow by Doppler ultrasonography are subject to substantial error. A simplified method of Doppler flowmetry using conventional duplex Doppler equipment was studied to establish its validity in determining short-term changes in flow in the splanchnic circulation in portal hypertension. METHODS: Two simple indices were measured: peak velocity averaged over two cardiac cycles and vessel diameter. The relationship between peak and mean velocity was studied using a Doppler phantom. The feasibility of studying relevant vessels and the constancy of regional flow was determined by performing Doppler flowmetry in a basal state twice within 2 hours. RESULTS: Constancy of relationship between peak and mean velocities was confirmed (r > 0.98). Examination was successful in the majority of 28 consecutive patients with portal hypertension for superior mesenteric, main portal, right portal, and paraumbilical (when patent) veins, but was successful in only 54% for the hepatic artery. Constancy of basal flow was found in 11 selected patients when group data were considered, but in individuals, substantial and unpredictable variance was found. CONCLUSIONS: Modified Doppler flowmetry appears valid in the assessment of short-term changes in splanchnic blood flow of patients with portal hypertension. Individual variance in baseline flow is identified as a potential problem and is further reason for caution in the interpretation of measurements of Doppler flow.
Hepatic Artery↗
Polymerase chain reaction-based point mutagenesis protocol.
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Adenoviridae↗