Regulation of AP-1/DNA complex formation in vitro.

The level of AP-1 DNA-binding activity exhibited in vitro by unfractionated extracts of Hela nuclei can be stimulated by a low molecular weight fraction from rabbit reticulocyte lysate. Stimulation also requires a heat labile component of the nuclear extract, probably a protein. Stimulated and unstimulated extracts with high and low AP-1 DNA-binding activities contain the same levels of proteins reactive with antisera against Jun and Fos, proteins which are shown to be involved in the AP-1/DNA complexes detected in vitro. The low molecular weight fraction from reticulocyte lysate can be substituted by the reducing agent dithiothreitol (DTT) in the stimulation reaction and conversely oxidised glutathione greatly reduces formation of AP-1/DNA complexes. The binding activities of transcription factors SP-1, NF-1 and CBP are unaffected by DTT or oxidised glutathione. These observations, taken together, suggest that the efficiency with which pre-existing Fos and Jun proteins can bind an AP-1 target sequence in vitro can be controlled by a nuclear activity which is sensitive to oxidation/reduction and that this control mechanism is specific for AP-1.

Improved method for the measurement of ribonucleotide reductase activity.

An improved method for the measurement of herpes simplex virus type 1 encoded ribonucleotide reductase has been developed. The enzyme which catalyses the conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates was determined by first converting the ribonucleotide substrate and deoxyribonucleotide product to the corresponding nucleosides by treatment with snake venom phosphodiesterase. Then nucleosides were separated by HPLC and measured by flow through scintillation counting and by monitoring their absorbance at 254 nm. Under the conditions used in the experiment cytidine and deoxcytidine, the derivitised substrate and product respectively, eluted from the column at approximately 4 min 33 s and 6 min 24 s. Peak heights and areas were automatically calculated by computer to ascertain the amount of product formed and thus quantitate the assay. Automation of the assay from sample injection to analysis provides a significant saving in time and an improvement in the efficiency of measurement of ribonucleotide reductase activity over other published methods.

The principal hydrogen donor for the herpes simplex virus type 1-encoded ribonucleotide reductase in infected cells is a cellular thioredoxin.

In this study herpes simplex virus type 1-encoded ribonucleotide reductase was shown to be able to utilize thioredoxin purified from the cyanobacterium Anabaena variabilis as a hydrogen donor for the enzyme. An assay has been developed to search for proteins which can function as a hydrogen donor for the viral ribonucleotide reductase. A protein has been identified and purified to homogeneity from infected cell extracts by a combination of fast protein liquid chromatography and gel filtration. This protein, which is also present in mock-infected cells, has been identified as a host cell thioredoxin by similarities in its physical characteristics with other thioredoxins. No evidence for the existence of a major virus-induced thioredoxin was obtained, suggesting that the host cell thioredoxin functions as the hydrogen donor for the herpes simplex virus type 1 ribonucleotide reductase in the infected cell.

Considerations in performing virus spiking experiments and process validation studies.

Virus safety of blood and plasma products produced on an industrial scale depends on several procedures including the ability of the production process to remove or inactivate potential virus contaminants. Because of the highly variable nature of the starting material it is essential to measure the viral clearance capacity of the production process in order to give a high degree of assurance that a particular product will not be contaminated with infectious virus.

The herpes simplex virus type 1 temperature-sensitive mutant ts1222 has a single base pair deletion in the small subunit of ribonucleotide reductase.

The herpes simplex virus type 1 (HSV-1) temperature-sensitive (ts) mutant, ts1222, has a defect within the gene specifying the small subunit of ribonucleotide reductase. Sequence determination of the lesion revealed that the mutant DNA had a single base pair deletion at the 3' end of the gene. The mutation altered the translational reading frame such that the codons of all but one of the last 15 amino acids of the protein were changed and the termination codon removed. Although ts1222 did not induce detectable amounts of enzyme activity at both 31 degrees and 39.5 degrees, it replicated as well as wild-type virus at 31 degrees in exponentially growing tissue culture cells under one step growth conditions. At 39.5 degrees, however, ts1222 behaved as a ts mutant. These findings suggest that at low temperatures the virus-coded enzyme is dispensable for virus growth in actively dividing tissue culture cells but at high temperatures the enzyme is essential for virus replication. Under these conditions altered properties of the host cell contribute to the ts phenotype of the mutant. In the presence of hydroxyurea, which inactivates both the cellular and virus ribonucleotide reductases, growth of the mutant at 31 degrees was inhibited more than wild-type virus replication. Growth of the mutant at the permissive temperature was also sensitive to high concentrations of thymidine whereas wild-type virus multiplication was resistant to the nucleoside. It is therefore likely that ts1222 is dependent on the cellular ribonucleotide reductase for growth at this temperature. In serum-starved cells, growth of the mutant virus at 31 degrees was severely impaired. Thus, like thymidine kinase, the HSV-coded ribonucleotide reductase is required for virus multiplication in resting tissue culture cells.

Virus assay methods: accuracy and validation.

Assay validation is the characterization of assay performance that allows the significance of the values obtained in an assay to be evaluated. Biological assays have several inherent sources of variation and this has been used as an excuse for years to explain unexpected or unanticipated results. However, with careful control of reagents and strictly controlled procedures, biological assays can be successfully validated to allow accurate quantitation and interpretation of the result obtained.

Herpes simplex virus-encoded ribonucleotide reductase: evidence for the dissociation/reassociation of the holoenzyme.

35S-labeled cells infected with herpes simplex virus type 1 (HSV-1), temperature-sensitive (ts) mutant ts 1222 were used as a source of the large subunit of the viral ribonucleotide reductase (RR) to investigate the binding of the large (RR1) and small (RR2) subunits in the active enzyme. Mixing 35S-labeled RR1 from ts 1222 with unlabeled RR1/RR2 complex from wild type (wt) infected cells resulted in the formation of a complex between 35S-labeled RR1 and unlabeled RR2, indicating that the complex between the RR1 and RR2 subunits is dynamic and subunit dissociation/reassociation occurs during enzyme function. Similar results were obtained when unlabeled HSV-2 RR was substituted for HSV-1 RR, demonstrating that the holoenzyme can be formed the large subunit of HSV-1 RR and the small subunit of HSV-2.

Reconstitution of herpes simplex virus type 1 ribonucleotide reductase activity from the large and small subunits.

An assay for the presence of functional large (RR1) and small (RR2) subunits of the herpes simplex virus type 1 (HSV-1) ribonucleotide reductase has been developed. The system utilizes two temperature-sensitive mutants, ts1207, which has a lesion in RR1, and ts1222, which has a lesion in RR2. In cells infected with ts1207 at 39.5 degrees C, the defective RR1 is unable to associate with RR2 to form an active enzyme, and, as a result, a pool of functional RR2 and defective RR1 accumulates. Evidence presented in this paper suggest that cells infected with ts1222 at either 31 degrees C or 39.5 degrees C accumulate a pool of functional RR1, but do not contain detectable RR2. Virus-specific ribonucleotide reductase activity was produced in cells coinfected with both mutants at 39.5 degrees C, each virus contributing one functional subunit to the holoenzyme. No enzyme activity was detected in cells infected with each mutant alone at this temperature. When partially purified extracts of cells infected with ts1207 at the nonpermissive temperature were mixed with those from ts1222-infected cells, a fully functional enzyme was also formed. These results demonstrate that HSV-1 ribonucleotide reductase activity can be reconstituted both in vivo and in vitro from the nondefective subunits produced by ts1222 and ts1207.

A single amino acid substitution in the large subunit of herpes simplex virus type 1 ribonucleotide reductase which prevents subunit association.

The herpes simplex virus type 1 temperature-sensitive (ts) mutant ts1207 does not induce detectable levels of ribonucleotide reductase activity at the non-permissive temperature (NPT, 39.5 degrees C). The ts lesion prevents the association of the enzyme's large (RR1) and small (RR2) subunits to give an active holoenzyme and maps within the gene specifying RR1. Here, it is shown that the ts mutant phenotype is due to the substitution of an asparagine for the wild-type (wt) serine at RR1 position 961, which is located within a region highly conserved between herpesviral and cellular RR1 subunit polypeptides. This ts1207 asparagine is predicted to alter a wt alpha-helix to a beta-strand. We have used synthetic oligopeptides, corresponding to the wt amino acid sequence of the mutation site, and antisera raised against them to determine whether this region is involved in subunit association. Neither the oligopeptides nor the antisera inhibit the enzyme activity, or the reconstituted activity formed by mixing intact RR2 and RR1 subunits present in partially purified extracts of cells infected at the NPT with ts1207 or ts1222 (an HSV-1 mutant with a lesion in the RR2 subunit), respectively. We infer from these results that the site of the mutation is unlikely to be positioned at the surface of RR1 and hence is probably not directly involved in subunit association. We suggest that the mutation site identifies an important RR1 region whose alteration in ts1207 changes the structure of a contact region(s) positioned at the RR1/RR2 interface.