Herpes Simplex Virus Ribonucleotide Reductase Research Articles

Combination of mutated herpes simplex virus type 1 (G207 virus) with radiation for the treatment of squamous cell carcinoma of the head and neck

G207 is an oncolytic herpes simplex virus (HSV) with deletions at both γ 134.5 loci and a LacZ gene insertion inactivating the HSV ribonucleotide reductase gene. Ionising radiation induces the growth arrest-inducible gene, GADD34, and ribonucleotide reductase. GADD34 is a protein that correlates with apoptosis following radiation and has homology with the G207 γ 134.5 gene. We hypothesised that the combination of radiotherapy with G207 may have a potentiating effect on viral replication and anti-tumour efficacy. The purpose of this study was therefore to evaluate the combination of G207 with radiation therapy to treat head and neck tumours. The cytotoxicity of G207 was tested in six head and neck squamous carcinoma cell lines, in the presence or absence of irradiation. For in vivo experiments, flank tumours in C3H/HeJ mice or in nude mice were treated with direct injections of G207, with or without radiation. All head and neck squamous cancer cell lines tested demonstrated significantly increased antitumour effects with the combination of G207 virus and radiation therapy compared with each individual modality ( P < 0.01). Furthermore, the combination treatment effect was better than the expected additive effect of the two therapies in combination. Even the radiation-resistant cell lines (SCC25, MSKQLL2, SCCVII) were susceptible. The combination of direct G207 injection with radiation therapy suppressed human and murine squamous cell carcinoma growth significantly ( P < 0.05 and P < 0.001) compared with controls or single modality therapy. G207 enhanced the effectiveness of radiation therapy and low-dose radiation potentiated the effectiveness of G207 viral therapy in head and neck cancer. These findings suggest a potential clinical application for this combined therapy as treatment for radiation-resistant head and neck cancers.

The R1 subunit of herpes simplex virus ribonucleotide reductase has chaperone-like activity similar to Hsp27

HSV-2 R1, the R1 subunit of herpes simplex virus (HSV) ribonucleotide reductase, protects cells against apoptosis. Here, we report the presence in HSV-2 R1 of a stretch exhibiting similarity to the α-crystallin domain of the small heat shock proteins, a domain known to be important for oligomerization and cytoprotective activities of these proteins. Also, the HSV-2 R1 protein, which forms multimeric structures in the absence of nucleotide, displayed chaperone ability as good as Hsp27 in a thermal denaturation assay using citrate synthase. In contrast, mammalian R1, which does not contain an α-crystallin domain, has neither chaperone nor anti-apoptotic activity. Thus, we propose that the chaperone activity of HSV-2 R1 could play an important role in viral pathogenesis.

The R1 subunit of herpes simplex virus ribonucleotide reductase protects cells against apoptosis at, or upstream of, caspase-8 activation.

The R1 subunit of herpes simplex virus (HSV) ribonucleotide reductase, which in addition to its C-terminal reductase domain possesses a unique N-terminal domain of about 400 amino acids, is thought to have an additional, as yet unknown, function. Here, we report that the full-length HSV-2 R1 has an anti-apoptotic function able to protect cells against death triggered by expression of R1(Delta2-357), an HSV-2 R1 subunit with its first 357 amino acids deleted. We further substantiate the R1 anti-apoptotic activity by showing that its accumulation at low level could completely block apoptosis induced by TNF-receptor family triggering. Activation of caspase-8 induced either by TNF or by Fas ligand expression was prevented by the R1 protein. As HSV R1 did not inhibit cell death mediated by several agents acting via the mitochondrial pathway (Bax overexpression, etoposide, staurosporine and menadione), it is proposed that it functions to interrupt specifically death receptor-mediated signalling at, or upstream of, caspase-8 activation. The N-terminal domain on its own did not exhibit anti-apoptotic activity, suggesting that both domains of R1 or part(s) of them are necessary for this new function. Evidence for the importance of HSV R1 in protecting HSV-infected cells against cytokine-induced apoptosis was obtained with the HSV-1 R1 deletion mutants ICP6Delta and hrR3. These results show that, in addition to its ribonucleotide reductase function, which is essential for virus reactivation, HSV R1 could contribute to virus propagation by preventing apoptosis induced by the immune system.

Radiation Inactivation of Ribonucleotide Reductase, an Enzyme with a Stable Free Radical

Herpes simplex virus ribonucleotide reductase (RR) is a tetrameric enzyme composed of two homodimers of large R1 and small R2 subunits with a tyrosyl free radical located on the small subunit. Irradiation of the holoenzyme yielded simple exponential decay curves and an estimated functional target size of 315 kDa. Western blot analysis of irradiated holoenzyme R1 and R2 yielded target sizes of 281 kDa and 57 kDa (approximately twice their expected size). Irradiation of free R1 and analysis by all methods yielded a single exponential decay with target sizes ranging from 128–153 kDa. For free R2, quantitation by enzyme activity and Western blot analyses yielded simple inactivation curves but considerably different target sizes of 223 kDa and 19 kDa, respectively; competition for radioligand binding in irradiated R2 subunits yielded two species, one with a target size of ∼210 kDa and the other of ∼20 kDa. These results are consistent with a model in which there is radiation energy transfer between the two monomers of both R1 and R2 only in the holoenzyme, a radiation-induced loss of free radical only in the isolated R2, and an alteration of the tertiary structure of R2.

Hydrogen Bonding to Tyrosyl Radical Analyzed by Ab Initio g-Tensor Calculations

Hydrogen bonding to the tyrosyl radical in ribonucleotide reductase (RNR) has been simulated by a complex between the phenoxyl radical and a water molecule. Multiconfigurational self-consistent field linear response theory was used to calculate the g-tensor of the isolated phenoxyl radical and of the phenoxyl−water model. The relevance of the model was motivated by the fact that spin density distributions and electron paramagnetic resonance (EPR) spectra of the phenoxyl and tyrosyl radicals are very similar. The calculated g-tensor anisotropy of the phenoxyl radical was comparable with experimental findings for tyrosyl in those RNRs where the H-bond is absent: gx = 2.0087(2.0087), gy = 2.0050(2.0042), and gz = 2.0025(2.0020), where the tyrosyl radical EPR data from Escherichia coli RNR are given in parentheses. The hydrogen bonding models reproduced a shift toward a lower gx value that was observed experimentally for mouse and herpes simplex virus RNR where the H-bond was detected by electron−nuclear double resonance after deuterium exchange. This decrease could be traced to lower angular momentum and spin-orbit coupling matrix elements between the ground 2B1 and the first excited 2B2 states (oxygen lone-pair n to πSOMO excitation) upon hydrogen bonding in a linear configuration. The gx value was further decreased by hydrogen bonding in bent configurations due to a blue shift of this excitation.

The PK domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is required for immediate-early gene expression and virus growth.

The large subunit of herpes simplex virus (HSV) ribonucleotide reductase (RR), RR1, contains a unique amino-terminal domain which has serine/threonine protein kinase (PK) activity. To examine the role of the PK activity in virus replication, we studied an HSV type 2 (HSV-2) mutant with a deletion in the RR1 PK domain (ICP10DeltaPK). ICP10DeltaPK expressed a 95-kDa RR1 protein (p95) which was PK negative but retained the ability to complex with the small RR subunit, RR2. Its RR activity was similar to that of HSV-2. In dividing cells, onset of virus growth was delayed, with replication initiating at 10 to 15 h postinfection, depending on the multiplicity of infection. In addition to the delayed growth onset, virus replication was significantly impaired (1,000-fold lower titers) in nondividing cells, and plaque-forming ability was severely compromised. The RR1 protein expressed by a revertant virus [HSV-2(R)] was structurally and functionally similar to the wild-type protein, and the virus had wild-type growth and plaque-forming properties. The growth of the ICP10DeltaPK virus and its plaque-forming potential were restored to wild-type levels in cells that constitutively express ICP10. Immediate-early (IE) genes for ICP4, ICP27, and ICP22 were not expressed in Vero cells infected with ICP10DeltaPK early in infection or in the presence of cycloheximide, and the levels of ICP0 and p95 were significantly (three- to sevenfold) lower than those in HSV-2- or HSV-2(R)-infected cells. IE gene expression was similar to that of the wild-type virus in cells that constitutively express ICP10. The data indicate that ICP10 PK is required for early expression of the viral regulatory IE genes and, consequently, for timely initiation of the protein cascade and HSV-2 growth in cultured cells.

The antiviral activity of the ribonucleotide reductase inhibitor BILD 1351 SE in combination with acyclovir against HSV type-1 in cell culture

BILD 1351 SE is a selective peptidomimetic subunit association inhibitor of the herpes simplex virus (HSV) ribonucleotide reductase (RR) with potent antiviral activity both in cell culture assays and animal models of HSV disease. The ability of BILD 1351 SE to inhibit the replication of HSV-1 when used in combination with acyclovir (ACV) for the treatment of HSV infections was investigated in baby hamster kidney cells using a 96-well enzyme-linked immunosorbent assay. The effective concentrations to achieve 50% inhibition (EC50) of virus replication by BILD 1351 SE in serum-starved and non serum-starved cells were 2±0.9 and 4.1±1.6 μM, respectively. The EC50 of ACV under both assay conditions was equal to 2.7±0.9 μM when tested alone. However, upon addition of BILD 1351 SE, the antiviral activity of ACV was potentiated in a synergistic manner as determined by the isobole method. At a concentration of BILD 1351 SE that produced 30% inhibition of HSV-1 replication, the EC50 of ACV decreased by about 15-fold in confluent cells and 17-fold in serum-starved cells. Similar conclusions were reached when evaluating drug interactions by the median dose-effect. Assuming mutually non-exclusive conditions at a drug ratio of ACV/BILD 1351 SE of 1/2, synergy was demonstrated in confluent cells with a drug enhancement index at EC50 of 14 and a combination index of 0.25. None of the drug combinations tested showed increased cytotoxicity in comparison with each drug alone. These results are consistent with the expected mode of action of a selective HSV RR inhibitor and support the strategy of combining these inhibitors with ACV for improved therapy of HSV infections.

Antiviral activity of a selective ribonucleotide reductase inhibitor against acyclovir-resistant herpes simplex virus type 1 in vivo.

The present study reports the activity of BILD 1633 SE against acyclovir (ACV)-resistant herpes simplex virus (HSV) infections in athymic nude (nu/nu) mice. BILD 1633 SE is a novel peptidomimetic inhibitor of HSV ribonucleotide reductase (RR). In vitro, it is more potent than ACV against several strains of wild-type as well as ACV-resistant HSV mutants. Its in vivo activity was tested against cutaneous viral infections in athymic nude mice infected with the ACV-resistant isolates HSV type 1 (HSV-1) dlsptk and PAAr5, which contain mutations in the viral thymidine kinase gene and the polymerase gene, respectively. Following cutaneous infection of athymic nude mice, both HSV-1 dlsptk and PAAr5 induced significant, reproducible, and persistent cutaneous lesions that lasted for more than 2 weeks. A 10-day treatment regimen with ACV given topically four times a day as a 5% cream or orally at up to 5 mg/ml in drinking water was partially effective against HSV-1 PAAr5 infection with a reduction of the area under the concentration-time curve (AUC) of 34 to 48%. The effects of ACV against HSV-1 dlsptk infection were not significant when it was administered topically and were only marginal when it was given in drinking water. Treatment under identical conditions with 5% topical BILD 1633 SE significantly reduced the cutaneous lesions caused by both HSV-1 dlsptk and PAAr5 infections. The effect of BILD 1633 SE against HSV-1 PAAr5 infections was more prominent and was inoculum and dose dependent, with AUC reductions of 96 and 67% against infections with 10(6) and 10(7) PFU per inoculation site, respectively. BILD 1633 SE also significantly decreased the lesions caused by HSV-1 dlsptk infection (28 to 51% AUC reduction). Combination therapy with topical BILD 1633 SE (5%) and ACV in drinking water (5 mg/ml) produced an antiviral effect against HSV-1 dlsptk and PAAr5 infections that was more than the sum of the effects of both drugs. This is the first report that a selective HSV RR subunit association inhibitor can be effective against ACV-resistant HSV infections in vivo.

The R1 Subunit of Herpes Simplex Virus Ribonucleotide Reductase Is a Good Substrate for Host Cell Protein Kinases but Is Not Itself a Protein Kinase

The N terminus of the R1 subunit of herpes simplex virus type 2 ribonucleotide reductase is believed to be a protein kinase domain mainly because the R1 protein was phosphorylated in a protein kinase assay on blot. Using Escherichia coli and adenovirus expression vectors to produce R1, we found that, whereas the reductase activity of both recombinant proteins was similar, efficient phosphorylation of R1 and casein in the presence of Mg2+ was obtained only with the R1 purified from eukaryotic cells. Phosphorylation of this R1, in solution or on blot, results mainly from the activity of casein kinase II (CKII), a co-purifying protein kinase. Labeling on blot occurs from CKII leakage off the membrane and its subsequent high affinity binding to in vivo CKII-phosphorylated R1. CKII target sites were mapped to an acidic serine-rich segment of the R1 N terminus. Improvement in purification of the R1 expressed in eukaryotic cells nearly completely abolished its phosphorylation potential. An extremely low level of phosphorylation observed in the presence of Mn2+ with the R1 produced in E. coli was probably due to an unidentified prokaryotic protein kinase. These results provide evidence that the herpes simplex virus type 2 R1 does not possess an intrinsic protein kinase activity.

Peptidomimetic inhibitors of herpes virus ribonucleotide reductase. Correlation between herpes simplex and varicella zoster virus

Peptidomimetic inhibitors based on the C-terminal sequence of herpes simplex virus (HSV) ribonucleotide reductase (RR) small subunit (R2) also inhibit varicella zoster virus (VZV) RR. There is generally good correlation between the potencies against both HSV and VZV RR. There are, however, two important differences. A urea moiety at the N-terminus improves potency against HSV RR but offers no advantages against VZV RR. γ-Methylleucine at the C-terminus is also beneficial for HSV RR but decreases potency considerably for VZV RR.

The Novel Protein Kinase of the RR1 Subunit of Herpes Simplex Virus Has Autophosphorylation and Transphosphorylation Activity That Differs in Its ATP Requirements for HSV-1 and HSV-2

The large subunit of herpes simplex virus (HSV) ribonucleotide reductase (RR1) designated ICP6 and ICP10 for HSV-1 and HSV-2, respectively, has a novel protein kinase (PK) enzymatic activity. ICP10 is localized on the cell surface, a localization that depends on an intact transmembrane (TM) segment. We used immunocomplex PK assays to examine the PK activity of ICP10 in stably transfected eukaryotic cells. Activity was distinct from that of casein kinase II (CKII) in that it did not require monovalent ions and was not inhibited by zinc sulfate. PK activity was eliminated by deletion of the conserved PK catalytic motifs or of the TM segment and it was significantly impaired by mutation of the invariant Lys (Lys176). Loss of PK activity by Lys176mutation resulted in the failure to bind ATP. A truncated ICP10 PK expressed in bacteria (pp291a1) retained auto- and transphosphorylating activity (for calmodulin) after purification to apparent homogeneity. PK activity was also absent in cells infected with a recombinant virus (ICP10ΔPK) deleted in the ICP10 PK catalytic motifs. In cells infected with HSV-1 or HSV-2, RR1 had auto- and transphosphorylating activity for the small subunit of HSV ribonucleotide reductase (RR2) and immunoglobulin G (IgG). Comparing the PK activity of ICP6 and ICP10 we found that ICP6 requires five-fold higher concentrations of [γ-32P]ATP than ICP10 and both enzymes are Mn2+dependent, which is also different from CKII that is primarily Mg2+-dependent. Similar results were obtained for various HSV strains and in different cell lines. The data are consistent with the conclusion that the RR1 PK activity is intrinsic.

Resistance of herpes simplex virus type 1 to peptidomimetic ribonucleotide reductase inhibitors: selection and characterization of mutant isolates.

Herpes simplex virus (HSV) encodes its own ribonucleotide reductase (RR), which provides the high levels of deoxynucleoside triphosphates required for viral DNA replication in infected cells. HSV RR is composed of two distinct subunits, R1 and R2, whose association is required for enzymatic activity. Peptidomimetic inhibitors that mimic the C-terminal amino acids of R2 inhibit HSV RR by preventing the association of R1 and R2. These compounds are candidate antiviral therapeutic agents. Here we describe the in vitro selection of HSV type 1 KOS variants with three- to ninefold-decreased sensitivity to the RR inhibitor BILD 733. The resistant isolates have growth properties in vitro similar to those of wild-type KOS but are more sensitive to acyclovir, possibly as a consequence of functional impairment of their RRs. A single amino acid substitution in R1 (Ala-1091 to Ser) was associated with threefold resistance to BILD 733, whereas an additional substitution (Pro-1090 to Leu) was required for higher levels of resistance. These mutations were reintroduced into HSV type 1 KOS and shown to be sufficient to confer the resistance phenotype. Studies in vitro with RRs isolated from cells infected with these mutant viruses demonstrated that these RRs bind BILD 733 more weakly than the wild-type enzyme and are also functionally impaired, exhibiting an elevated dissociation constant (Kd) for R1-R2 subunit association and/or reduced activity (kcat). This work provides evidence that the C-terminal end of HSV R1 (residues 1090 and 1091) is involved in R2 binding interactions and demonstrates that resistance to subunit association inhibitors may be associated with compromised activity of the target enzyme.

Peptidomimetic inhibitors of herpes simplex virus ribonucleotide reductase with improved in vivo antiviral activity.

We have been investigating the potential of a new class of antiviral compounds. These peptidomimetic derivatives prevent association of the two subunits of herpes simplex virus (HSV) ribonucleotide reductase (RR), an enzyme necessary for efficient replication of viral DNA. The compounds disclosed in this paper build on our previously published work. Structure-activity studies reveal beneficial modifications that result in improved antiviral potency in cell culture in a murine ocular model of HSV-induced keratitis. These modifications include a stereochemically defined (2,6-dimethylcyclohexyl)amino N-terminus, two ketomethylene amide bond isosteres, and a (1-ethylneopentyl)amino C-terminus. These three modifications led to the preparation of BILD 1351, our most potent antiherpetic agent containing a ureido N-terminus. Incorporation of the C-terminal modification into our inhibitor series based on a (phenylpropionyl)valine N-terminus provided BILD 1357, a significantly more potent antiviral compound than our previously published best compound, BILD 1263.

Peptidomimetic inhibitors of herpes simplex virus ribonucleotide reductase: a new class of antiviral agents

We have been investigating a new class of antiviral compounds effective against herpes simplex virus (HSV) in vitro and in vivo. Antiviral activity results from inhibition of HSV ribonucleotide reductase (RR). The inhibitors are designed as mimics of the RR small subunit C-terminus, a region essential for RR subunit association and consequently enzymatic activity. Inhibition results from specific binding of the inhibitor to the HSV RR large subunit thereby preventing subunit association. This report details the structure--activity studies that lead to the indentification of BILD 1263, a potent inhibitor of HSV RR subunit association (IC50, 0.2 nM) that also inhibits the replication of HSV types 1 and 2 in cell culture (EC50, 3 and 4 microM) and reduces the severity of HSV-1-induced keratitis in a murine ocular model. The discovery of inhibitors with in vitro antiviral results from a combination of improving inhibitor potency in a RR binding assay and modifying inhibitor physicochemical properties. The importance and possible role of the new structural modifications introduced into this inhibitor series is discussed.

Characterization of the novel protein kinase activity present in the R1 subunit of herpes simplex virus ribonucleotide reductase.

We have compared the protein kinase activities of the R1 subunits from herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) ribonucleotide reductase following expression in Escherichia coli. Autophosphorylation activity was observed when kinase assays were performed with immunoprecipitated R1 or proteins purified to homogeneity, and the activity was stimulated by the basic protein protamine. Transphosphorylation of histones or calmodulin by purified or immunoprecipitated HSV-1 and HSV-2 R1 was not observed, and our results suggest that the activities of these two proteins are similar. We further characterized the protein kinase activity of HSV-1 R1 by producing insertion and deletion mutants constructed with a plasmid expressing R1 amino acids 1 to 449. C-terminal deletion analysis identified the catalytic core of the enzyme as comprising residues 1 to 292, and this polypeptide will be useful for structural determinations by X-ray crystallography. Insertion of a 4-amino-acid sequence at sites within the protein kinase domain identified regions essential for activity; insertions at residues 22 and 112 completely inactivated activity, and an insertion at residue 136 reduced activity sixfold. Similar insertions at residues 257, 262, 292, and 343 had no effect on activity. The ATP analog 5'-fluorosulfonylbenzoyladenosine, which covalently modifies conventional eukaryotic kinases at an essential lysine residue within the active site, did label HSV R1, but this labelling occurred outside the N-terminal domain. These data indicate that the HSV R1 kinase is novel and distinct from other eukaryotic protein kinases.

Improved Adenovirus Vector Provides Herpes Simplex Virus Ribonucleotide Reductase R1 and R2 Subunits Very Efficiently

We have constructed a new adenovirus (Ad) expression vector, pAdBM5, that allows for the production of unprecedented levels of recombinant protein in the human 293 cell line using the Ad expression system. The main feature of this vector is a combination of enhancer sequences that increases the activity of the ectopic major late promoter (MLP) in recombinant Ad. In 293 cells infected with helper-free Ad recombinants generated with the pAdBM5 transfer vector, both herpes simplex virus (HSV) ribonucleotide reductase R1 and R2 subunits represent the most abundant polypeptides, accounting for as much as 15-20% of total cellular proteins. Our data suggest that this level of expression is probably very close to the upper limit of the system. Furthermore, when compared to the widely utilized baculovirus (Bac)/Sf9 expression system, the improved Ad vector showed a better performance for the production and purification of active HSV-2 ribonucleotide reductase R1 and R2 subunits. The R2 subunit was about 5-fold more abundant in recombinant Ad-infected 293 cells than in Bac-infected Sf9 cells while the R1 subunit was produced at roughly similar levels with either system. However, the amount of active soluble R1 obtained from recombinant Ad-infected 293 cells was at least 5 times higher because most of the R1 produced in Sf9 cells was insoluble.

Polymorphism within the herpes simplex virus (HSV) ribonucleotide reductase large subunit (ICP6) confers type specificity for recognition by HSV type 1-specific cytotoxic T lymphocytes.

A panel of herpes simplex virus type 1 (HSV-1)-specific, CD8+, major histocompatibility complex class I (H-2Kb)-restricted cytotoxic T-lymphocyte (CTL) clones was derived from HSV-1-immunized C57BL/6 (H-2b) mice in order to identify the HSV-1 CTL recognition epitope(s) which confers type specificity. HSV-1 x HSV-2 intertypic recombinants were used to narrow the region encoding potential CTL recognition epitopes to within 0.51 to 0.58 map units of the HSV-1 genome. Using an inhibitor of viral DNA synthesis and an ICP6 deletion mutant, the large subunit of ribonucleotide reductase (ICP6, RR1) was identified as a target protein for these type-specific CTL. Potential CTL recognition epitopes within RR1 were located on the basis of the peptide motif predicted to bind to the MHC class I H-2Kb molecule. A peptide corresponding to residues 822 to 829 of RR1 was shown to confer susceptibility on H-2Kb-expressing target cells to lysis by the type 1-specific CTL. On the basis of a comparison of the HSV-1 RR1 epitope (residues 822 to 829) with the homologous sequence of HSV-2 RR1 (residues 828 to 836) and by the use of amino acid substitutions within synthetic peptides, we identified HSV-1 residue 828 as being largely responsible for the type specificity exhibited by HSV-1-specific CTL. This HSV-1 RR1 epitope, when expressed in recombinant simian virus 40 large T antigen in primary C57BL/6 cells, was recognized by the HSV-1 RR1-specific CTL clones. These results indicate that an early HSV protein with enzymatic activity provides a target for HSV-specific CTL and that type specificity is dictated largely by a single amino acid.

Disruption of herpes simplex virus ribonucleotide reductase quaternary structure by peptide inhibitors as a novel approach to antiviral therapy.

Peptides capable of selectively disrupting protein-protein interactions that are required for viral replication represent potential agents for antiviral therapy. The first example of viral product that could be inhibited by the peptide YAGAVVNDL, targeted to the functional interaction between subunits, is the ribonucleotide reductase of herpes simplex virus. However, this peptide alone has no effect on virally infected cells, presumably because it is too large to enter cells unaided. Escherichia coli heat-labile enterotoxin B subunit has been used as a protein carrier for the delivery of YAGAVVNDL into HSV-1 infected cells and been shown to specifically inhibit viral replication. This provides evidence of the usefulness of carrier-mediated delivery of putative antiviral peptides for the evaluation of their efficacy.

Identification of structural domains within the large subunit of herpes simplex virus ribonucleotide reductase.

The large subunit (R1) of herpes simplex virus (HSV) ribonucleotide reductase is a bifunctional protein consisting of a unique N-terminal protein kinase domain and a ribonucleotide reductase domain. Previous studies showed that the two functional domains are linked by a protease sensitive site. Here we provide evidence for two subdomains, of 30K and 53K, within the reductase domain. The two fragments, which were produced by limited proteolysis and were resistant to further degradation, remained tightly associated in a complex containing two molecules of each. They were capable of binding the R2 subunit of HSV ribonucleotide reductase with approximately the same affinity as the intact protein but the complex did not complement the small subunit (R2) to give an active enzyme. At low concentrations (0.4 micrograms/ml) of trypsin or V8 protease, cleavage between the subdomains was prevented by the presence of the N-terminal protein kinase domain. At higher protease concentrations (1 micrograms/ml) the N-terminal domain is extensively proteolysed and the 30K and 53K domains were generated. Identical results were obtained using purified R1 isolated from infected cell extracts or following expression in Escherichia coli. The origin of the two domains was investigated by N-terminal sequencing of the 53K fragment and by examining their reactivity with a panel of R1-specific monoclonal antibodies which we isolated and epitope mapped for that purpose. The trypsin cleavage site was found to lie between arginine 575 and asparagine 576, and proteolysis in this region was not prevented by the presence of R2 or the nonapeptide YAGAVVNDL. We propose that the ribonucleotide reductase region of HSV R1 exists in a two domain structure, and that the interdomain linking region is protected by the unique N terminus.

The critical C‐terminus of the small subunit of herpes simplex virus ribonucleotide reductase is mobile and conformationally similar to C‐terminal peptides

The C-terminus of the small subunit of class I ribonucleotide reductases is essential for subunit association and enzymatic activity. 1H NMR analysis of the small subunit (2 x 38 kDa as a homodimer) of herpes simplex virus ribonucleotide reductase shows that this critical binding site is mobile and exposed in relation to the rest of the protein. Assignments of six C-terminal amino acids are made by comparing the TOCSY and NOESY spectra of the small subunit with the spectra of an identical protein truncated by seven amino acids at the C-terminus and the spectra of an analogous 15 amino acid peptide. The mobility of the C-terminus may be important for subunit recognition and could be general for other ribonucleotide reductases. The spectral comparisons also suggest that the six C-terminal amino acids of the small subunit and peptide are conformationally similar. This observation may be important for the design of inhibitors of ribonucleotide reductase subunit association.