Purification of a bacterially expressed herpes simplex virus type 1 origin binding protein for use in posttranslational processing studies.

The origin binding protein (OBP) encoded by the UL9 open reading frame of herpes simplex virus type 1 (HSV-1) plays an essential role in productive infection by promoting the initiation of viral DNA synthesis. In this study, OBP was inducibly expressed in Escherichia coli and purified to homogeneity using a two-step chromatographic separation procedure. The properties of this recombinant OBP (rOBP) were found to be indistinguishable from those of the virus-encoded protein. Since rOBP was synthesized in bacterial cells, it lacked the posttranslational processing which normally occurs in OBP produced in HSV-1-infected mammalian cells and could therefore be exploited in experiments which addressed the effects of protein modification on OBP function. As an initial study, the impact of phosphorylation on enzymatic activity was examined using rOBP which had been treated with a panel of purified cellular kinases. rOBP was found to act as a substrate for nearly all of the kinases tested in (32)P-labeled phosphate transfer assays. However, only phosphorylation by protein kinase A (PKA, or cAMP-dependent protein kinase) was shown to significantly alter the enzymatic properties of rOBP, as it increased by five- to eightfold the ATPase activity associated with this protein. Activation of this critical viral DNA replication enzyme by a cAMP-dependent kinase such as PKA may be of some relevance in the natural course of HSV-1 infections, since reactivation of latent virus is thought to involve both signal transduction events and the induction of viral DNA synthesis. Thus, the expression and purification strategy outlined in this work provides an economical source of unmodified HSV-1 OBP that should prove useful in future in vitro studies.

Identification and characterization of a benzothiophene inhibitor of herpes simplex virus type 1 replication which acts at the immediate early stage of infection.

Analysis of a large compound library in a high throughput virus infection assay screen identified the benzothiophene PD146626 as a potent and specific inhibitor of herpes simplex virus type 1 (HSV-1) replication. PD146626 possessed an EC(50) and EC(90) against HSV-1 of 0.1 and 1 microM, respectively, and mediated no detectable cytotoxicity in cells at concentrations up to 1 microM. Western blot analyses and time of addition experiments demonstrated that in the presence of PD146626 HSV-1 underwent a specific block in viral gene expression at the immediate early stage. However, several observations indicated that a cellular function rather than a viral immediate early transactivator protein represented the molecular target for PD146626, including the lack of resistance of VP16 and ICP0 mutant viruses to the compound, the inability to select resistant strains of HSV-1 following exhaustive serial passaging of virus in the presence of the compound, and the sensitivity of human cytomegalovirus, which lacks VP16 and ICP0 homologs, to the compound. Moreover, kinetic studies suggested an unusual pattern of responsiveness of the host cell to PD146626, in that the compound could induce an extended antiviral state in cells after only a brief exposure. Together these results suggest that PD146626 targets a novel cellular function that is critical for the expression of HSV-1 immediate early genes but not host cell genes.

A colorimetric assay for high-throughput screening of inhibitors of herpes simplex virus type 1 alkaline nuclease.

Herpes simplex virus type 1 (HSV-1) encodes a deoxyribonuclease that is frequently referred to as alkaline nuclease (AN) because of its elevated pH optimum. Studies with recombinant viruses which contain deletions in the HSV-1 gene encoding AN have indicated that this enzyme is required for efficient virus replication and therefore represents a potential target for novel antiviral therapies. A simple colorimetric assay for deoxyribonuclease activity employing a DNA-methyl green substrate was adapted for use in a high-throughput screen to identify small molecule inhibitors of this enzyme. This screen identified 1,2-benzoisothiazolin-3-one as a specific inhibitor of AN, since it exhibited activity against AN but was completely inactive against bovine pancreatic DNaseI. Subsequent studies revealed that this compound most likely inhibited AN by forming disulfide linkages with one or more exposed cysteine residues on the surface of the enzyme and that AN was sensitive to sulfhydryl-group-modifying reagents in general. These results demonstrated the utility of this DNA-methyl green substrate-based assay in both the rapid identification and the characterization of novel small molecule inhibitors of the AN encoded by HSV-1 and other herpesviruses.

Substrate specificity and characterization of partially purified rat liver 13-hydroxyoctadecadienoic acid (13-HODE) dehydrogenase.

Oxidation products of linoleic acid, such as 13-hydroxyoctadecadienoic acid (13-HODE), exhibit biological activity in a number of systems. One major metabolic fate of 13-HODE is oxidation to the 2,4-dienone, 13-oxooctadecadienoic acid by an NAD(+)-dependent dehydrogenase (13-HODE dehydrogenase). The present work describes the partial purification and characterization of 13-HODE dehydrogenase from rat liver cytosol. The enzyme was purified using a combination of ammonium sulfate precipitation, as well as hydroxylapatite, gel permeation, and hydrophobic interaction chromatography. Analysis of the most purified preparation by SDS-polyacrylamide gel electrophoresis indicates two subunits of approximately 55 kDa, suggesting the possibility of a heterodimeric enzyme. However, due to aggregation in the purified preparation, an accurate molecular mass for the native enzyme has not yet been obtained. Using 13-HODE as a substrate, the purified enzyme has a Km of 6.3 microM and a Vmax of 5.7 nmol/min/mg. More importantly, the enzyme has a narrow substrate specificity with 13-HODE being the preferred substrate. From a series of 17 potential substrates, only 9-HODE (53% the activity of 13-HODE) and 15-hydroxyeicosatetraenoic acid (64% the activity of 13-HODE) showed significant activity as substrates. A number of other unsaturated hydroxy fatty acids, including several eicosanoids, are not substrates. The narrow substrate specificity displayed by the enzyme suggests that it could play a key role in modulating the effects of oxidized derivatives of linoleic acid.

The product of the UL12.5 gene of herpes simplex virus type 1 is a capsid-associated nuclease.

The UL12 open reading frame of herpes simplex virus type 1 (HSV-1) encodes a deoxyribonuclease that is frequently referred to as alkaline nuclease (AN) because of its high pH optimum. Recently, an alternate open reading frame designated UL12.5 was identified within the UL12 gene. UL12.5 and UL12 have the same translational stop codon, but the former utilizes an internal methionine codon of the latter gene to initiate translation of a 60-kDa amino-terminal truncated form of AN. Since the role of the UL12.5 protein in the HSV-1 life cycle has not yet been determined, its properties were investigated in this study. Unlike AN, which can be readily solubilized from infected cell lysates, the UL12.5 protein was found to be a highly insoluble species, even when isolated by high-salt detergent lysis. Since many of the structural polypeptides which constitute the HSV-1 virion are similarly insoluble, a potential association of UL12.5 protein with virus particles was examined. By using Western blot analysis, the UL12.5 protein could be readily detected in preparations of intact virions, isolated capsid classes, and even capsids that had been extracted with 2 M guanidine-HCl. In contrast, AN was either missing or present at only low levels in each of these structures. Since the inherent insolubility of the UL12.5 protein prevented its potential deoxyribonuclease activity from being assayed in infected-cell lysates, partially purified fractions of soluble UL12.5 protein were generated by selectively solubilizing either insoluble infected-cell proteins or isolated capsid proteins with urea and renaturing them by stepwise dialysis. Initial analysis of these preparations revealed that they did contain an enzymatic activity that was not present in comparable fractions from cells infected with a UL12.5 null mutant of HSV-1. Additional biochemical characterization revealed that UL12.5 protein was similar to AN with respect to pH optimum, ionic strength, and divalent cation requirements and possessed both exonucleolytic and endonucleolytic functions. The finding that the UL12.5 protein represents a capsid-associated form of AN which exhibits nucleolytic activity suggests that it may play some role in the processing of genomic DNA during encapsidation.

Purification and characterization of herpes simplex virus type 1 alkaline exonuclease expressed in Escherichia coli.

The alkaline exonuclease (AE) encoded by the herpes simplex virus type 1 (HSV-1) UL12 open reading frame was inducibly expressed in Escherichia coli and purified without the use of chromatographic separation. This recombinant AE was found to exhibit the same biochemical properties as the virus-encoded protein and was used to confirm the existence of a weak endonucleolytic activity in the enzyme. Antisera raised against the recombinant protein recognized several forms of the AE in HSV-1-infected cells. This expression and purification strategy will provide an economical and easily accessible alternative source of HSV-1 AE for future in vitro studies.

Decreased levels of 13-hydroxyoctadecadienoic acid (13-HODE) dehydrogenase in neoplastic tissue of human colon biopsies.

Recent studies have identified a role for the oxidation product of linoleic acid, 13-hydroxyoctadecadienoic acid (13-HODE) in cell proliferation. The enzyme 13-HODE dehydrogenase catalyzes the conversion of 13-HODE to 13-oxooctadecadienoic acid. This enzyme has been shown to correlate with the degree of differentiation of intestinal cells in both in vitro and in vivo models. Higher enzyme levels are found in more differentiated cell types. The present study was done to determine if enzyme levels of 13-HODE dehydrogenase are predictive of the differentiation status of biopsies from human colonic mucosa. Twenty-eight patients who underwent diagnostic colonoscopy (10 patients with adenocarcinoma and 18 with adenomatous polyps) had biopsies taken from both normal rectal mucosa and neoplastic mucosa. The determination of 13-HODE dehydrogenase activity was conducted by high-performance liquid chromatography analysis of all biopsy samples. Sixteen of the 18 patients with polyps had lower 13-HODE dehydrogenase activity in the adenoma than in the uninvolved rectal mucosa (P = 0.001). The colon adenocarcinomas also had less 13-HODE dehydrogenase activity in the cancer biopsy tissue than in uninvolved rectal mucosa (P = 0.041) These data are consistent with a role for 13-HODE dehydrogenase in intestinal cell differentiation. Understanding the precise role of this enzymatic reaction could be important potentially in the therapy and biology of colon cancer. In addition, measurements of 13-HODE dehydrogenase may be a useful parameter by which to ascertain the differentiation status of intestinal cells in vitro.

Formation of adducts between 13-oxooctadecadienoic acid (13-OXO) and protein-derived thiols, in vivo and in vitro.

Linoleic acid is metabolized by numerous tissues to oxidized derivatives possessing biological activity. In the current experiments, we have investigated the reaction of 13-oxooctadecadienoic acid (13-OXO) and the metabolic precursor 13-hydroxyoctadecadienoic acid (13-HODE) with cellular macromolecules and model cellular nucleophiles. Colonic mucosal explants from Sprague-Dawley rats were incubated in the presence of [1-14C]-13-OXO or [1-14C]-13-HODE. The binding of radiolabel to the protein and nucleic acid fractions was analyzed by isopycnic centrifugation in Cs2SO4. Cellular homogenates incubated with either 13-OXO or 13-HODE resulted in the binding of radiolabel to cellular protein. No significant amounts of reaction with cellular RNA or DNA were observed. To assess possible modes of reaction with cellular constituents, the oxidized fatty acids were incubated in vitro with oxygen, sulfur, or nitrogen, nucleophiles including, serine, cysteine, glutathione, methionine, lysine, adenosine, and guanosine. Under physiologic conditions, in the absence of cellular homogenates, only 13-OXO was reactive. In addition, only the sulfur-containing compounds cysteine and glutathione showed significant rates of reaction. Furthermore, treatment of colonic homogenates with N-ethlymaleimide reduced the binding of [1-14C]-13-OXO to cellular protein. These data support the suggestion that 13-HODE requires metabolic activation, by dehydrogenation to 13-OXO, prior to binding to cellular protein and that protein-derived thiol groups are involved in the binding reactions.

The product of a 1.9-kb mRNA which overlaps the HSV-1 alkaline nuclease gene (UL12) cannot relieve the growth defects of a null mutant.

Alkaline nuclease, a relatively abundant viral phosphoprotein in herpes simplex virus type 1 (HSV-1)- or HSV-2-infected cells, is encoded by a 2.3-kb mRNA (R. H. Costa, K. G. Draper, L. Banks, K. L. Powell, G. Cohen, R. Eisenberg, and E. K. Wagner, 1983. J. Virol. 48, 591-603). This mRNA is a member of a family of five unspliced 3'-coterminal messages. Costa et al. proposed that another member of this family of mRNAs (1.9-kb) may encode an N-terminally truncated protein which shares its carboxy-terminus with the alkaline nuclease protein. We previously described the isolation of AN-1, a deletion/insertion mutant of the alkaline nuclease gene (S. K. Weller, R. M. Seghatoleslami, L. Shao, D. Rowse, and E. P. Carmichael, 1990. J. Gen. Virol. 71, 2941-2952). The deletion in AN-1 would be predicted to abolish gene products of both the 2.3- and the 1.9-kb mRNAs. To investigate whether the putative truncated version of alkaline nuclease encoded by the 1.9-kb mRNA has enzymatic activity and plays a role in the viral life cycle, a viral mutant (AN-F1) was constructed which is predicted to abolish the gene product of the 2.3-kb mRNA (full-length alkaline nuclease) but leave intact the putative product of the 1.9-kb mRNA. Using a highly sensitive polyclonal antiserum raised against a bacterially expressed full-length alkaline nuclease, we observed a 60-kDa protein in KOS- and AN-F1-infected cells but not in AN-1-infected cells. This suggests that the 60-kDa protein is likely to be expressed from the 1.9-kb mRNA; the open reading frame is now designated UL12.5. Despite the presence of the 60-kDa band, AN-F1 failed to exhibit any alkaline exonuclease activity. This result suggests that the truncated polypeptide (UL12.5) is not enzymatically active, has low levels of activity, or possesses enzymatic activity which is not detected because of the low abundance of the polypeptide. AN-1 and AN-F1 are both severely restricted with respect to growth in Vero cells, as viral yields are 100- to 1000-fold lower than those of wild-type virus. We previously reported that the major defect in AN-1 is in the ability of DNA-containing capsids which form in the nucleus to mature into the cytoplasm (L. Shao, L. M. Rapp, and S. K. Weller, 1993. Virology 196, 146-162); AN-F1 exhibits the same defect. These results indicate that although the 1.9-kb mRNA encodes a 60-kDa protein presumably from the UL12.5 open reading frame, this polypeptide cannot substitute for the full-length UL12 product.

The correlation between 13-hydroxyoctadecadienoate dehydrogenase (13-HODE dehydrogenase) and intestinal cell differentiation.

The enzyme 13-hydroxyoctadecadienoate dehydrogenase (13-HODE dehydrogenase) catalyzes the NAD(+)-dependent oxidation of 13-hydroxyoctadecadienoic acid to 13-oxooctadecadienoic acid. In that the oxygenation of linoleic acid is increasingly being shown to be involved in the regulation of cellular function, this enzyme is poised to play a key role in the expression of the biological activity of these compounds. We have measured the activity of 13-HODE dehydrogenase in rat intestinal cells at various stages of differentiation. The specific activity of 13-HODE dehydrogenase shows a strong positive correlation with the degree of differentiation of intestinal mucosal cells from both the small and large intestines. In the small intestine the gradient of activity parallels that of alkaline phosphatase, while in the colon the incorporation of 3H-deoxythymidine and 13-HODE dehydrogenase are inversely related. Since the expression of 13-HODE dehydrogenase is most likely not associated with the nutritive function of the intestinal tract, these data raise the possibility the enzyme plays a role in the process of cellular differentiation.

Increases in 13-hydroxyoctadecadienoic acid dehydrogenase activity during differentiation of cultured cells.

Recently, oxidation products of linoleic acid such as 13-hydroxyoctadecadienoic acid (HODE) have been implicated in the regulation of cellular physiology including the proliferative response to growth factor treatment. In addition, an NAD(+)-dependent 13-HODE dehydrogenase was recently described. To evaluate the contribution of this enzyme to cellular processes we have examined the behavior of the enzyme under different conditions. In the present report, changes in the activity of 13-hydroxyoctadecadienoic acid dehydrogenase during in vitro differentiation of two different cell lines were examined. The cell line HT-29 undergoes induced differentiation via manipulation of the medium while the Caco-2 line undergoes spontaneous differentiation upon attainment of confluence. In both cell lines, longer culture times were accompanied by increases in 13-HODE dehydrogenase activity. The increase in enzyme activity continued even after cell proliferation had ceased. Cellular differentiation was verified by the observation of increases in sucrase and alkaline phosphatase activities. In addition, the activity of 13-HODE dehydrogenase was measured in growing, early confluent and late confluent cultures of undifferentiating Swiss mouse 3T3 fibroblasts. In the fibroblast line, no significant changes in 13-HODE dehydrogenase activity were observed during the course of the experiment. The specific activity of 13-HODE dehydrogenase was also significantly different between the three cell lines, consistent with the extent of differentiation. Highest levels of activity were found in Caco-2 cells (200-400 pmol/min/mg) and barely detectable levels in the fibroblasts (0.6-2 pmol/min/mg). The correlation between 13-HODE dehydrogenase and cell differentiation suggests the enzyme may have a role to play in the partitioning of cells between proliferation and differentiation pathways.

Failure of rectal ornithine decarboxylase to identify adenomatous polyp status.

Rectal mucosal ornithine decarboxylase (ODC) activity has been reported to distinguish between patients with and without adenomatous polyps (AP). In the present investigation, ODC activity has been measured in 28 patients with AP and 34 patients without AP. To assess the intraindividual variation in ODC activity, repeat biopsies were performed on 11 patients. In addition, the effect of postbiopsy sample handling was investigated by storage of samples on either dry or wet ice during transport to the laboratory. The mean rectal mucosal ODC activity in patients with AP was 196.0 +/- 195.5, whereas that in AP negative patients was 182.2 +/- 320.5. The rectal mucosal ODC activity in patients with colorectal cancer was 388.2 +/- 581. Repeat samples in individuals were generally within the same range as the original samples. The method of sample transport did not significantly affect the level of ODC measured in a particular biopsy. Because of high variability in rectal mucosal ODC activity within the population, there was wide overlap in ODC values between those patients with and without AP in an unselected general population. Thus, the measurement of flat rectal mucosal ODC activity is not a good predictor of the presence or absence of AP. Additional studies of the factors affecting mucosal ODC activity are necessary before the potential clinical utility of the method can be realized in the general population.

Metabolism of oxidized linoleic acid: characterization of 13-hydroxyoctadecadienoic acid dehydrogenase activity from rat colonic tissue.

An oxidized derivative of linoleic acid, 13-hydroxyoctadecadienoic acid (13-HODE), is dehydrogenated by an NAD+ dependent dehydrogenase present in rat colon mucosa. The product of the reaction is the 2,4-dienone, 13-oxooctadecadienoic acid. Enzyme activity was determined by HPLC analysis of incubation mixtures as well as by measuring the increase in absorbance at 285 nm, which represents formation of the 2,4-dienone chromophore. Characteristics of the reaction with respect to protein concentration, time of incubation and substrate dependence were investigated. Several inhibitors of known dehydrogenases had no effect on the 13-HODE dehydrogenase. These include, ethanol, indomethacin, 6-methyl-17-hydroxyprogesterone acetate, 4-(diethylamino)-benzaldehyde, and aspirin. The enzyme was mildly inhibited by pyrazole, 4-methylpyrazole and ibuprofen. Disulfiram was found to be a potent inhibitor of enzyme activity with an IC50 of 200 microM. Inhibitor specificity, and other characteristics of the reaction suggest the enzyme is neither alcohol dehydrogenase, diol dehydrogenase, nor a prostaglandin dehydrogenase. It is possible this enzyme plays an important role in the response of the colonic mucosa to the mitogenic effect of oxidized fatty acids.

Metabolism of oxidized linoleic acid: distribution of activity for the enzymatic oxidation of 13-hydroxyoctadecadienoic acid to 13-oxooctadecadienoic acid in rat tissues.

Oxidation products of linoleic acid, including hydroperoxy- and hydroxyoctadecadienoic acids have been shown to possess biological activities in a number of different systems. In this work we describe an enzymatic activity which catalyzes the conversion of 13-hydroxyoctadecadienoic acid to a 2,4-dienone product, 13-oxooctadecadienoic acid. The enzyme activity is widely distributed, with the highest activity in the colon and the liver. The distribution of activity among various tissues is distinct from other dehydrogenases known to use oxygenated unsaturated fatty acids as substrates. This enzyme may play a key role in the metabolism of 13-hydroxyoctadecadienoic acid in epithelial tissues.

Production of unsaturated carbonyl compounds during metabolism of hydroperoxy fatty acids by colonic homogenates.

Recent evidence has suggested that unsaturated carbonyl compounds may be the ultimate mitogens produced from the primary auto-oxidation products of unsaturated fatty acids. The present study has investigated the metabolism of 13-hydroperoxyoctadecandienoic acid (13-ROOH) by rat colon homogenates. This hydroperoxide is one of the primary products formed from the oxygenation of linoleic acid, the most abundant dietary polyunsaturated fatty acid. Incubation mixtures contained [1-14C]13-ROOH and colonic homogenates prepared from male Sprague-Dawley rats. After 30 min of incubation the reaction was quenched and the products extracted for analysis by HPLC. The identity of the eluted products were verified by UV, MS and NMR spectroscopy. The major products include a mixture of isomers of 2,4-dienone C18 fatty acids and 13-hydroxyoctadecadienoic acid. Direct comparison of homogenate metabolism to the hematin-catalyzed, alkoxyl radical-mediated decomposition of 13-ROOH shows some significant differences. In particular, no epoxy products are detected in the presence of tissue homogenates whereas these are the major products observed during the decomposition of 13-ROOH by hematin and a number of other agents. These experiments demonstrate the production of relatively large amounts of unsaturated carbonyl-containing fatty acids during the metabolism of hydroperoxy fatty acids by colonic tissue. The major product, 13-oxo-9Z,11E-octadecadienoic acid, when instilled intrarectally stimulates the incorporation of [3H]deoxythymidine into colonic mucosal DNA, and induces colonic mucosal ornithine decarboxylase activity in vivo. These findings have important implications for the mechanism by which dietary fat promotes colon tumorigenesis as the formation of relatively reactive 2,4-dienones may be a key to the in vivo mitogenic activity of oxidized fatty acids.

The essential fatty acid requirement for azoxymethane-induced intestinal carcinogenesis in rats.

The essential fatty acid requirement for the development of intestinal carcinogenesis was determined and compared to the overall essential fatty acid status of the animals as measured by the triene/tetraene ratio in the plasma, liver and colon. To induce tumors, male Sprague-Dawley rats were given two weekly injections (20 mg/kg body wt) of azoxymethane. Two weeks after the last injection, the rats were divided into groups of 25 and given one of six diets containing various levels of essential fatty acids (as linoleate). The diets contained 5% total fat and were prepared by mixing safflower oil (high essential fatty acids, beef fat (low essential fatty acids), and medium chain triglyceride oil (no essential fatty acids). One group of rats was fed a 20% beef fat diet. The range of essential fatty acids was from less than 0.03% to 1.28% (w/w). Twenty-six weeks after the first azoxymethane injection, the animals were killed and intestinal tumor incidence and multiplicity were determined. Samples of plasma, liver and colon were also taken for measurement of the triene/tetraene ratio by gas chromatography. Large bowel tumor incidence showed a dependence on the essential fatty acid content of the diet. The results were as follows: (percent essential fatty acids: percent tumor incidence) Group A (1.28: 72.4), Group B (0.60: 73.3), Group C (0.11: 55.2), Group D (0.08: 39.3), Group E (less than 0.03: 37.9) and Group F, which was fed 20% beef fat, (0.34: 88.5).(ABSTRACT TRUNCATED AT 250 WORDS)