Clb6-Cdc28 Promotes Ribonucleotide Reductase Subcellular Redistribution during S Phase.

A tightly controlled cellular deoxyribonucleotide (deoxynucleoside triphosphate [dNTP]) pool is critical for maintenance of genome integrity. One mode of dNTP pool regulation is through subcellular localization of ribonucleotide reductase (RNR), the enzyme that catalyzes the rate-limiting step of dNTP biosynthesis. In Saccharomyces cerevisiae, the RNR small subunit, Rnr2-Rnr4, is localized to the nucleus, whereas the large subunit, Rnr1, is cytoplasmic. As cells enter S phase or encounter DNA damage, Rnr2-Rnr4 relocalizes to the cytoplasm to form an active holoenzyme complex with Rnr1. Although the DNA damage-induced relocalization requires the checkpoint kinases Mec1-Rad53-Dun1, the S-phase-specific redistribution does not. Here, we report that the S-phase cyclin-cyclin-dependent kinase (CDK) complex Clb6-Cdc28 controls Rnr2-Rnr4 relocalization in S phase. Rnr2 contains a consensus CDK site and exhibits Clb6-dependent phosphorylation in S phase. Deletion of CLB6 or removal of the CDK site results in an increased association of Rnr2 with its nuclear anchor Wtm1, nuclear retention of Rnr2-Rnr4, and an enhanced sensitivity to the RNR inhibitor hydroxyurea. Thus, we propose that Rnr2-Rnr4 redistribution in S phase is triggered by Clb6-Cdc28-mediated phosphorylation of Rnr2, which disrupts the Rnr2-Wtm1 interaction and promotes the release of Rnr2-Rnr4 from the nucleus.

p53R2 as a novel prognostic biomarker in nasopharyngeal carcinoma.

BACKGROUND: p53R2 is a target of p53 gene, which is essential for DNA repair, mitochondrial DNA synthesis, protection against oxidative stress, chromosomal instability, chronic inflammation and tumorigenesis. This study is aimed to investigate the expression of ribonucleotide reductase (RR) subunit p53R2 in nasopharyngeal carcinoma and its significance in the prognosis. METHODS: The expression levels of p53R2 in 201 patients with NPC were examined by immunohistochemical assay. The correlations of p53R2 expression and clinicopathological features of nasopharyngeal carcinoma patient were analysed by chi-square test. The Kaplan-Meier survival analysis and Cox multivariate regression model were used to analyze the prognostic significance of the patients with NPC. RESULTS: Immunohistochemical results showed that p53R2 was positively expressed in 92.5% (186/201) of nasopharyngeal carcinoma and the high expression rate was 38.3% (77/201). Further analysis observed that the negative correlation between expression of p53R2 and pT status had statistical significance (P < 0.05). Kaplan-Meier survival analysis found that the mean survival time of patients with high expression of p53R2 was 143.32 months, while the patients with low expression level of p53R2 was 121.63 months (P < 0.05). Cox regression analysis suggested that p53R2 protein expression could be used as an independent prognostic factor for nasopharyngeal carcinoma (P < 0.05). CONCLUSIONS: This study drew a conclusion that p53R2 could be used as a prognostic biomarker indicative of the favorable outcome for patients with nasopharyngeal carcinoma.

A phase I-II evaluation of veliparib (NSC #737664), topotecan, and filgrastim or pegfilgrastim in the treatment of persistent or recurrent carcinoma of the uterine cervix: an NRG Oncology/Gynecologic Oncology Group study.

PURPOSE: The aim of this study was to evaluate the tolerability and efficacy of poly(ADP-ribose) polymerase (PARP) inhibition by veliparib during cytotoxic topotecan administration with filgrastim or pegfilgrastim neutrophil support in women with persistent or recurrent uterine cervix cancer. EXPERIMENTAL DESIGN: This phase I-II trial examined twice-daily oral veliparib (10 mg) given during once-daily intravenous topotecan (0.6 mg/m²) on days 1 to 5 of each treatment cycle. Cycles were repeated every 21 days until disease progression or until toxicity prohibited further therapy. Toxicity and objective response rate were primary endpoints. RESULTS: Twenty-seven women were enrolled. Frequently reported grade 3 or higher treatment-related toxicities were anemia (59%), thrombocytopenia (44%), leukopenia (22%), and neutropenia (19%). There were 2 partial responses (7% [90% confidence interval, 1%-22%]). Four patients had a disease progression date more than 6 months after the start of veliparib-topotecan therapy. Patients with low immunohistochemical expression (0-1+) of PARP-1 in their primary uterine cervix cancer were more likely to have a longer progression-free interval (hazard ratio, 0.25; P = 0.02) and survival (hazard ratio, 0.12; P = 0.005) after veliparib-topotecan therapy. CONCLUSIONS: Clinical activity of a veliparib-topotecan combination was minimal in women with persistent or recurrent uterine cervix cancer. Women whose uterine cervix cancers express PARP-1 at low levels may benefit preferentially from PARP inhibitors combined with cytotoxic therapies, suggesting further study of PARP expression as an integral triage biomarker.

Hug1 is an intrinsically disordered protein that inhibits ribonucleotide reductase activity by directly binding Rnr2 subunit.

Rad53 is a conserved protein kinase with a central role in DNA damage response and nucleotide metabolism. We observed that the expression of a dominant-lethal form of RAD53 leads to significant expression changes for at least 16 genes, including the RNR3 and the HUG1 genes, both of which are involved in the control of nucleotide metabolism. We established by multiple biophysical and biochemical approaches that Hug1 is an intrinsically disordered protein that directly binds to the small RNR subunit Rnr2. We characterized the surface of interaction involved in Hug1 binding to Rnr2, and we thus defined a new binding region to Rnr2. Moreover, we show that Hug1 is deleterious to cell growth in the context of reduced RNR activity. This inhibitory effect of Hug1 on RNR activity depends on the binding of Hug1 to Rnr2. We propose a model in which Hug1 modulates Rnr2-Rnr1 association by binding Rnr2. We show that Hug1 accumulates under various physiological conditions of high RNR induction. Hence, both the regulation and the mode of action of Hug1 are different from those of the small protein inhibitors Dif1 and Sml1, and Hug1 can be considered as a regulator for fine-tuning of RNR activity.

Biomarkers for predicting the response of esophageal squamous cell carcinoma to neoadjuvant chemoradiation therapy.

This review summarizes and evaluates the literature regarding the biomarkers for predicting the response and/or prognosis of esophageal squamous cell carcinoma (ESCC) patients treated with neoadjuvant chemoradiation therapy (CRT). There are seven categories of molecules known to correlate with the response and/or prognosis: tumor suppressors (p53, p21), cell cycle regulators (Cyclin D1, CDC25B, 14-3-3sigma), DNA repair molecules (p53R2, ERCC1), drug resistance proteins [metallothionein (MT)], angiogenic factors (VEGF), molecules involved in cell proliferation/invasion/metastasis (Ki-67, COX-2) and hedgehog signaling molecules (Gli-1). Of the above molecules, the tumor suppressor p53 is expected to be a representative biomarker for predicting the response and prognosis. The cell cycle markers CDC25B and 14-3-3sigma have potential as response biomarkers independent of the p53 status. The DNA repair markers, p53R2 or ERCC1, angiogenic molecule (VEGF), and hedgehog signaling pathway factor Gli-1 also have potential to predict the response and prognosis of ESCC. However, there are still many unanswered questions with regard to predicting the clinical effects of neoadjuvant CRT.

Single-cell analysis of ribonucleotide reductase transcriptional and translational response to DNA damage.

The ribonucleotide reductase (RNR) enzyme catalyzes an essential step in the production of deoxyribonucleotide triphosphates (dNTPs) in cells. Bulk biochemical measurements in synchronized Saccharomyces cerevisiae cells suggest that RNR mRNA production is maximal in late G(1) and S phases; however, damaged DNA induces RNR transcription throughout the cell cycle. But such en masse measurements reveal neither cell-to-cell heterogeneity in responses nor direct correlations between transcript and protein expression or localization in single cells which may be central to function. We overcame these limitations by simultaneous detection of single RNR transcripts and also Rnr proteins in the same individual asynchronous S. cerevisiae cells, with and without DNA damage by methyl methanesulfonate (MMS). Surprisingly, RNR subunit mRNA levels were comparably low in both damaged and undamaged G(1) cells and highly induced in damaged S/G(2) cells. Transcript numbers became correlated with both protein levels and localization only upon DNA damage in a cell cycle-dependent manner. Further, we showed that the differential RNR response to DNA damage correlated with variable Mec1 kinase activity in the cell cycle in single cells. The transcription of RNR genes was found to be noisy and non-Poissonian in nature. Our results provide vital insight into cell cycle-dependent RNR regulation under conditions of genotoxic stress.

COP9 signalosome subunit 7 from Arabidopsis interacts with and regulates the small subunit of ribonucleotide reductase (RNR2).

The COP9 Signalosome protein complex (CSN) is a pleiotropic regulator of plant development and contains eight-subunits. Six of these subunits contain the PCI motif which mediates specific protein interactions necessary for the integrity of the complex. COP9 complex subunit 7 (CSN7) contains an N-terminal PCI motif followed by a C-terminal extension which is also necessary for CSN function. A yeast-interaction trap assay identified the small subunit of ribonucelotide reductase (RNR2) from Arabidopsis as interacting with the C-terminal section of CSN7. This interaction was confirmed in planta by both bimolecular fluorescence complementation and immuoprecipitation assays with endogenous proteins. The subcellular localization of RNR2 was primarily nuclear in meristematic regions, and cytoplasmic in adult cells. RNR2 was constitutively nuclear in csn7 mutant seedlings, and was also primarily nuclear in wild type seedlings following exposure to UV-C. These two results correlate with constitutive expression of several DNA-damage response genes in csn7 mutants, and to increased tolerance of csn7 seedlings to UV-C treatment. We propose that the CSN is a negative regulator of RNR activity in Arabidopsis.

Upregulation of the p53R2 ribonucleotide reductase subunit by nitric oxide.

The p53R2 ribonucleotide reductase subunit is a p53-inducible protein involved in DNA repair and mitochondrial DNA replication. It has been shown that p53 is activated by nitric oxide, which can damage DNA at high concentrations. This suggests that NO may regulate p53R2 expression through p53 activation. We show here that NO increases p53 protein expression in p53-wt cell lines and upregulates p53R2 at the protein and mRNA levels in a p53-dependent manner. Other p53 target genes, such as DDB2, WAF1 and PCNA, are also induced by NO. Surprisingly, p53R2 is similarly upregulated by NO in two p53-deficient cell lines, showing the existence of p53-independent regulatory mechanisms. Delta Np73, which is overexpressed in many cancers, inhibits the transcriptional activity of p53 and p53 homologs. In p53-wt cells, the Delta Np73alpha isoform inhibits basal and NO-induced p53R2 protein expression. In p53-null cells, it also strongly inhibits p53R2 expression, and represses the enhancer activity of the p53-responsive element present in the p53R2-encoding gene. These results demonstrate that p53R2 expression can be controlled by p53 homologs in the absence of p53, and is downregulated by oncogenic Delta Np73 isoforms. Knocking down p53R2 in p53-wt cells dramatically enhances NO-induced DNA damages, indicating a protective function of the p53R2 ribonucleotide reductase subunit in prevention or repair of NO-mediated genotoxic injury.

Upregulation of enzymes metabolizing 5-fluorouracil in colorectal cancer.

BACKGROUND: The purpose of this study was to compare the activities of various enzymes, participating in the metabolism of 5-fluorouracil, between colorectal cancer and nontumor tissues and to investigate the association of the enzyme activities with clinicopathological backgrounds. METHODS: Activities of seven enzymes involved in nucleic acid metabolism--orotate phosphoribosyltransferase (OPRT), ribonucleotide reductase (RNR), thymidylate synthase, dihydropyrimidine dehydrogenase, thymidine phosphorylase, uridine phosphorylase and thymidine kinase (TK)--were measured in tumor and nontumor tissues from 28 patients who were operated on for colorectal cancers. RESULTS: OPRT, thymidylate synthase, RNR, thymidine phosphorylase, uridine phosphorylase and thymidine kinase activities were significantly higher in tumor areas than in nontumor areas. OPRT showed the highest T/N ratio (the ratio of each enzyme activity in tumor areas to that in nontumor areas). The T/N ratio of RNR activity showed a tendency to be associated with lymph node metastasis and Dukes classification. CONCLUSION: The results suggest that OPRT is a main enzyme participating in the phosphorylation of 5-fluorouracil and has an important role in tumor growth. The T/N ratio of RNR may be predictive of tumor progression.

The ribonucleotide reductase subunit M2B subcellular localization and functional importance for DNA replication in physiological growth of KB cells.

Ribonucleoside diphosphate reductase (EC 1.17.4.1) (RR) is a potential target for antineoplastic agents due to its crucial role in DNA replication and repair. The expression and activity of RR subunits are highly regulated to maintain an optimal dNTP pool, which is required to maintain genetic fidelity. The human RR small subunit M2B (p53R2) is thought to contribute to DNA repair in response to DNA damage. However, it is not clear whether M2B is involved in providing dNTPs for DNA replication under physiological growth conditions. Serum starvation synchronized studies showed that a rapid increase of M2B was associated with cyclin E, which is responsible for regulation of G(1)/S-phase transition. A living cell sorting study that used KB cells in normal growth, further confirmed that M2B increased to maximum levels at the G(1)/S-phase transition, and decreased with DNA synthesis. Confocal studies revealed that M2B redistributed from the cytoplasm to the nucleus earlier than hRRM2 in response to DNA replication. Nuclear accumulation of M2B is associated with dynamic changes in dNTP at early periods of serum addition. By using M2B-shRNA expression vectors, inhibition of M2B may result in growth retardation in KB cells. We conclude that M2B may translocate from the cytoplasm into the nucleus and allow dNTPs to initiate DNA synthesis in KB cells under physiological conditions. Thus, our findings suggested that M2B might play an important role for initiating DNA replication of KB cells in normal growth.

Nature of the peroxo intermediate of the W48F/D84E ribonucleotide reductase variant: implications for O2 activation by binuclear non-heme iron enzymes.

Analysis of the spectroscopic signatures of the R2-W48F/D84E biferric peroxo intermediate identifies a cis mu-1,2 peroxo coordination geometry. DFT geometry optimizations on both R2-W48F/D84E and R2-wild-type peroxo intermediate models including constraints imposed by the protein also identify the cis mu-1,2 peroxo geometry as the most stable peroxo intermediate structure. This study provides significant insight into the electronic structure and reactivity of the R2-W48F/D84E peroxo intermediate, structurally related cis mu-1,2 peroxo model complexes, and other enzymatic biferric peroxo intermediates.

UV-C response of the ribonucleotide reductase large subunit involves both E2F-mediated gene transcriptional regulation and protein subcellular relocalization in tobacco cells.

E2F factors are implicated in various cellular processes including specific gene induction at the G1/S transition of the cell cycle. We present in this study a novel regulatory aspect for the tobacco large subunit of ribonucleotide reductase (R1a) and its encoding gene (RNR1a) in the UV-C response. By structural analyses, two E2F sites were identified on the promoter of this gene. Functional analysis showed that, in addition to their role in the specific G1/S induction of the RNR1a gene, both E2F sites were important for regulating specific RNR1a gene expression in response to UV-C irradiation in non-synchronized and synchronized cells. Concomitantly, western blot and cellular analyses showed an increase of a 60 kDa E2F factor and a transient translocation of a GFP-R1a protein fusion from cytoplasm to nucleus in response to UV irradiation.

Ribonucleotide reductase of shrimp white spot syndrome virus (WSSV): expression and enzymatic activity in a baculovirus/insect cell system and WSSV-infected shrimp.

Infection of shrimp cells with white spot syndrome virus (WSSV) results in an increase in ribonucleotide reductase (RR) expression at the RNA level. In this article we further express and characterize the induction of a novel ribonucleotide reductase after WSSV infection of shrimp cells. A baculovirus/insect system was used to express the two recombinant protein subunits RR1 and RR2, and a DNA polymerase coupled RR activity assay showed a marked increase in ribonucleotide reductase activity when cell extracts containing recombinant RR1 and RR2 were combined. The same assay revealed that RR activity increased as infection advanced in the gills of experimentally infected shrimp. An increase in RR expression was also detected at the protein level in WSSV-infected shrimp cells. An immunocytochemistry assay by confocal laser scanning microscopy showed that in hemocytes collected from WSSV-infected shrimp, both of the subunit proteins (RR1 and RR2) were concentrated mainly around the nucleus, but only RR1 was detected inside it. All of these results suggest that WSSV RR is functionally involved during WSSV infection.

Molecular markers and targeted therapy with novel agents: prospects in the treatment of non-small cell lung cancer.

Detection of genomic differences predictive of drug response or resistance in individual patients may allow therapy to be customized to the characteristics of particular tumors. Preliminary findings are that non-small cell lung cancer patients overexpressing ERCC1 mRNA have lower response to cisplatin chemotherapy, while those overexpressing ribonucleotide reductase mRNA have limited benefit from gemcitabine. In addition, overexpression of beta-tubulin III and stathmin can influence the sensitivity to microtubule interacting drugs, like vinorelbine and paclitaxel. The introduction of biological agents which target highly specific intracellular pathways offers the promise of enhancing the efficacy of cytotoxic chemotherapy. Among many promising biological agents is the monoclonal antibody C225, which blocks the EGFR receptor. The addition of C225 appears to induce responses in a proportion of colon cancer patients refractory to 5-FU or irinotecan, supporting pre-clinical evidence of synergistic activity. It also appears from xenograft data that C225 enhances the sensitivity of tumors to radiation and docetaxel or the combination.

The use of high field/frequency EPR in studies of radical and metal sites in proteins and small inorganic models.

Low temperature electron paramagnetic resonance (EPR) spectroscopy with frequencies between 95 and 345 GHz and magnetic fields up to 12 T have been used to study radicals and metal sites in proteins and small inorganic model complexes. We have studied radicals, Fe, Cu and Mn containing proteins. For S = 1/2 systems, the high frequency method can resolve the g-value anisotropy. It was used in mouse ribonucleotide reductase (RNR) to show the presence of a hydrogen bond to the tyrosyl radical oxygen. At 285 GHz the type 2 Cu(II) signal in the complex enzyme laccase is clearly resolved from the Hg(II) containing laccase peroxide adduct. For simple metal sites, the systems over S = 1/2 can be described by the spin Hamiltonian: H(S) = BgS + D[Sz2 - S(S + 1)/3 + E/D (Sx2 - Sy2)]. From the high frequency EPR the D-value can be determined directly by, (I) shifts of g(eff) for half-integer spin systems with large D-values as observed at 345 GHz on an Fe(II)-NO-EDTA complex, which is best described as S = 3/2 system with D = 11.5 cm(-1), E = 0.1 cm(-1) and gx = gy = gz = 2.0; (II) measuring the outermost signal, for systems with small D values, distant of (2S - 1) x absolute value(D) from the center of the spectrum as observed in S= 5/2 Fe(III)-EDTA. In Mn(II) substituted mouse RNR R2 protein the weakly interacting Mn(II) at X-band could be observed as decoupled Mn(II) at 285 GHz.

Analysis of proliferating cell fraction determined by monoclonal antibody to M1-subunit ribonucleotide reductase and Ki-67 in relation to p53 protein expression in fine-needle aspirates from non-Hodgkin's lymphomas.

The purpose of this study was to analyse the proliferative fraction with the monoclonal antibody M1-R-R to M1-subunit ribonucleotide reductase and with MIB-1 to Ki-67 antigen in relation to p53 protein expression in fine needle aspirates from B-cell non-Hodgkin's lymphomas. One hundred and thirty-seven cases, previously diagnosed and sub-typed according to the Kiel classification and characterized by immunophenotyping, were included in the study. The M-1 subunit ribonucleotide reductase (M1-R-R), Ki-67 and p53 antigens were detected using monoclonal antibodies on stored cytospin preparations. There was a good correlation (r = 0.72) between Ki-67 and M1-R-R positive cell fraction in both high and low grade lymphomas. High-grade lymphomas had a median percentage of M1-R-R/MIB-1 positive cells of 53.0/73.0 for lymphoblastic, 61.0/52.0 for immunoblastic and 33.5/41.0 for centroblastic lymphomas, respectively. In low grade lymphomas figures of median percentage of M1-R-R/MIB-1 were 9.0/15.0 for centroblastic/centrocytic, 11.0/9.5 for chronic lymphocytic leukaemia, 16.0/27.0 for centrocytic and 12.0/9.0 for immunocytomas, respectively. The median percentages of M1-R-R/MIB-1 for high and low grade lymphomas were 37.0/50.5 and 11.0/12.0, respectively. In the p53 positive cases the proliferation rate as measured by staining for M1-R-R and MIB-1 was higher than in p53 negative cases, but the difference was not statistically significant. The results show that cytospin material obtained by fine needle aspiration and stored at -70 degrees C for years can be used reliably for both peroxidase-avidin-biotin and three-step alkaline phosphatase immunocytochemical staining. In addition, proliferation fraction determined by M1-R-R monoclonal antibody staining correlates well with that measured by an established marker for cell proliferation, the Ki-67 antibody. However, the proliferation fraction as measured by the two antibodies differs in the various subtypes of non-Hodgkin's lymphoma which indicates that they may contribute different prognostic information.

A simple and sensitive ribonucleotide reductase assay.

Ribonucleotide reductase (RR) is a key regulatory enzyme in the DNA synthesis pathway and is the target of the cancer chemotherapeutic agent hydroxyurea. The study of RR is significantly hindered by the tedious and labor-intensive nature of enzymatic assay. In this report, we present a novel RR assay in which detection of the deoxyribonucleotides produced by RR occurs via coupling to the DNA polymerase reaction, and is enhanced by using RNase to degrade endogenous RNA. Cell extracts from various cell lines were treated with RNase and then reacted with ATP and radioactive ribonucleotide diphosphate as the substrate. Incorporation of the radioactive substrate [14C]CDP into DNA was linear over 30 min and was linear with the amount of extract, which provided RR activity. The reaction was inhibited by hydroxyurea and required Mg2+ and ATP, suggesting that the assay is specific to RR activity. While RR activities determined by our method and by a conventional method were comparable, this novel method proved to be simpler, faster, more sensitive and less expensive. In addition, assay of the RR activity for multiple samples can easily be performed simultaneously. It is superior to other RR assays in all aspects.

Bivariate flow cytometric analysis of DNA content versus immunopositivity for ribonucleotide reductase M1 subunit in the cell cycle.

Ribonucleotide reductase (RR) is a cytoplasmatic enzyme catalyzing the reduction of all four ribonucleotides to their corresponding deoxyribonucleotides. Its activity strongly correlates to the rate of DNA synthesis. By using a specific monoclonal antibody against the large M1 subunit of RR, we assessed the expression of M1-RR versus DNA content by dual-parameter flow cytometry. The aim of this paper was to compare the variations in the immunopositivity for M1-RR during the cell cycle to the positivity for other cell cycle markers identifying either proliferating cells (Ki-67 and PCNA) or quiescent cells (statin). To do this, normal human embryonic fibroblasts in different growth conditions as well as several other mammalian cell lines (rat C6 glioma cells; mouse 3T3 fibroblasts and B16 melanoma cells; human epithelial EUE cells and mammary carcinoma MCF-7 cells) were used. The expression of M1-RR antigen was found to correlate positively with the expression of Ki-67 and PCNA, and negatively with the expression of statin. During early G1 phase, M1-RR becomes detectable by specific antibodies relatively later compared to PCNA and Ki-67; therefore, the lack of immunopositivity for M1-RR cannot be taken as an absolute indication of cell quiescence in G0.

Identification of RNR4, encoding a second essential small subunit of ribonucleotide reductase in Saccharomyces cerevisiae.

Ribonucleotide reductase (RNR), which catalyzes the rate-limiting step for deoxyribonucleotide production required for DNA synthesis, is an alpha2beta2 tetramer consisting of two large and two small subunits. RNR2 encodes a small subunit and is essential for mitotic viability in Saccharomyces cerevisiae. We have cloned a second essential gene encoding a homologous small subunit, RNR4. RNR4 and RNR2 appear to have nonoverlapping functions and cannot substitute for each other even when overproduced. The lethality of RNR4 deletion mutations can be suppressed by overexpression of RNR1 and RNR3, two genes encoding the large subunit of the RNR enzyme, indicating genetic interactions among the RNR genes. RNR2 and RNR4 may be present in the same reductase complex in vivo, since they coimmunoprecipitate from cell extracts. Like the other RNR genes, RNR4 is inducible by DNA-damaging agents through the same signal transduction pathway involving MEC1, RAD53, and DUN1 kinase genes. Analysis of DNA damage inducibility of RNR2 and RNR4 revealed partial inducibility in dun1 mutants, indicating a DUN1-independent branch of the transcriptional response to DNA damage.

The herpes simplex virus type 1 ribonucleotide reductase is required for acute retinal disease.

We have used a herpes simplex virus type 1 (HSV-1) ribonucleotide reductase (RR) null mutant (ICP6 delta) to determine if the HSV-1 RR is required for acute retinal disease. Injection of the ICP6 delta mutant into the vitreous induced mild transient signs of infection (vitreal infiltrate, retinal inflammation, and changes in retinal cytology). In contrast, the parental KOS and a revertant virus (ICP6 delta + 3.1) in which the RR gene had been restored, caused severe retinitis. Injection of media alone also induced mild transient signs of disease. Two months after infection, ICP6 delta injected eyes could not be distinguished from normal eyes. Repeated injection of ICP6 delta (3 times, 2 weeks apart) resulted in vitreal infiltrate near the site of injection but the retina did not appear damaged. The mutant, ICP6 delta, grew to peak titers 1 x 10(3) to 1 x 10(5)-fold lower and cleared faster than KOS or ICP6 delta + 3.1 in the injected eyes suggesting that the reduced virulence was due to reduced ability of the virus to grow. These results show that the viral RR is required for acute retinal disease.