Large Subunit Of Ribonucleotide Reductase Research Articles

Maize LSC encoding a large subunit of ribonucleotide reductase is required for subsidiary cell development and plant growth.

The four-celled stomatal complex consists of a pair of guard cells (GCs) and two subsidiary cells (SCs) in grasses, which benefits fast adjustment of stomatal pore aperture. The formation and development of SCs are thus important for stomatal functionality. Here, we report the maize loss of subsidiary cells (lsc) mutant with a large number of stomata lacking one or two SCs. The loss of SCs is supposed to be resulted from impeded subsidiary mother cells (SMC) polarization and asymmetrical division. Besides the defect in SCs, the lsc mutant also displays a dwarf morphology and pale and stripped newly-grown leaves. LSC encodes a large subunit of ribonucleotide reductase (RNR), an enzyme involved in deoxyribonucleotides (dNTPs) synthesis. Consistently, the dNTPs levels and expression of genes involved in DNA replication, cell cycle progression, and SC development were significantly reduced in the lsc mutant compared with the wild-type B73 inbred line. Conversely, overexpression of maize LSC increase dNTP synthesis and promotes plant growth in both maize and Arabidopsis. Our data indicate that LSC regulates dNTP production and is required for SMC polarization, SC differentiation, and plant growth.

RRM1 is mediated by histone acetylation through gemcitabine resistance and contributes to invasiveness and ECM remodeling in pancreatic cancer.

The invasiveness of pancreatic cancer and its resistance to anticancer drugs define its malignant potential, and are considered to affect the peritumoral microenvironment. Cancer cells with resistance to gemcitabine exposed to external signals induced by anticancer drugs may enhance their malignant transformation. Ribonucleotide reductase large subunit M1 (RRM1), an enzyme in the DNA synthesis pathway, is upregulated during gemcitabine resistance, and its expression is associated with worse prognosis for pancreatic cancer. However, the biological function of RRM1 is unclear. In the present study, it was demonstrated that histone acetylation is involved in the regulatory mechanism related to the acquisition of gemcitabine resistance and subsequent RRM1 upregulation. The current invitro study indicated that RRM1 expression is critical for the migratory and invasive potential of pancreatic cancer cells. Furthermore, a comprehensive RNA sequencing analysis showed that activated RRM1 induced marked changes in the expression levels of extracellular matrix‑related genes, including N‑cadherin, tenascin‑C and COL11A. RRM1 activation also promoted extracellular matrix remodeling and mesenchymal features, which enhanced the migratory invasiveness and malignant potential of pancreatic cancer cells. The present results demonstrated that RRM1 has a critical role in the biological gene program that regulates the extracellular matrix, which promotes the aggressive malignant phenotype of pancreatic cancer.

Proteome-Based Investigation Identified Potential Drug Repurposable Small Molecules Against Monkeypox Disease.

Monkeypox Virus (MPXV), the causative agent of Monkeypox (MPX) disease, is an emerging zoonotic pathogen spreading in different endemic and non-endemic nations and creating outbreaks. MPX treatment mainly includes Cidofovir and Tecovirimat but they have several side effects and solely depending on these drugs may promote the emergence of drug-resistant variants. Hence, new drugs are required to control the spread of the disease. In this study, we explored the MPXV proteome to suggest repurposable drugs. DrugBank screening revealed drugs such as Brinzolamide, Dorzolamide, Methazolamide, Zidovudine, Gemcitabine, Hydroxyurea, Fludarabine, and Tecovirimat as controls. Structural analogs of these compounds were extracted from ChEMBL Database. After Molecular docking and Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET)-based screening, we identified Zidovudine (binding affinity-5.9kcal/mol) and a Harmala alkaloid (2S,4R)-4-(9H-Pyrido[3,4-b]indol-1-yl)-1,2,4-butanetriol (binding affinity -6.6kcal/mol) against L2R receptor (Thymidine Kinase). Moreover, Fludarabine (binding affinity -6.4kcal/mol) and 5'-Dehydroadenosine (binding affinity -6.4kcal/mol) can strongly interact with the I4L receptor (Ribonucleotide reductase large subunit R1). Molecular Dynamics (MD) simulations suggest all of these compounds can change the C-alpha backbone, residue mobility, compactness, and solvent accessible surface area of L2R and I4L. Our results strongly suggest that these drug repurposing small molecules are worth exploring in vivo and in vitro for clinical applications.

Essential Roles of Ribonucleotide Reductases under DNA Damage and Replication Stresses in Cryptococcus neoformans.

ABSTRACTA balance in the deoxyribonucleotide (dNTPs) intracellular concentration is critical for the DNA replication and repair processes. In the model yeast Saccharomyces cerevisiae, the Mec1-Rad53-Dun1 kinase cascade mainly regulates the ribonucleotide reductase (RNR) gene expression during DNA replication and DNA damage stress. However, the RNR regulatory mechanisms in basidiomycete fungi during DNA replication and damage stress remain elusive. Here, we observed that in C. neoformans, RNR1 (large RNR subunit) and RNR21 (one small RNR subunit) were required for cell viability, but not RNR22 (another small RNR subunit). RNR22 overexpression compensated for the lethality of RNR21 suppression. In contrast to the regulatory mechanisms of RNRs in S. cerevisiae, Rad53 and Chk1 kinases cooperatively or divergently controlled RNR1 and RNR21 expression under DNA damage and DNA replication stress. In particular, this study revealed that Chk1 mainly regulated RNR1 expression during DNA replication stress, whereas Rad53, rather than Chk1, played a significant role in controlling the expression of RNR21 during DNA damage stress. Furthermore, the expression of RNR22, not but RNR1 and RNR21, was suppressed by the Ssn6-Tup1 complex during DNA replication stress. Notably, we observed that RNR1 expression was mainly regulated by Mbs1, whereas RNR21 expression was cooperatively controlled by Mbs1 and Bdr1 as downstream factors of Rad53 and Chk1 during DNA replication and damage stress. Collectively, the regulation of RNRs in C. neoformans has both evolutionarily conserved and divergent features in DNA replication and DNA damage stress, compared with other yeasts.IMPORTANCE Upon DNA replication or damage stresses, it is critical to provide proper levels of deoxynucleotide triphosphates (dNTPs) and activate DNA repair machinery. Ribonucleotide reductases (RNRs), which are composed of large and small subunits, are required for synthesizing dNTP. An imbalance in the intracellular concentration of dNTPs caused by the perturbation of RNR results in a reduction in DNA repair fidelity. Despite the importance of their roles, functions and regulations of RNR have not been elucidated in the basidiomycete fungi. In this study, we found that the roles of RNR1, RNR21, and RNR22 genes encoding RNR subunits in the viability of C. neoformans. Furthermore, their expression levels are divergently regulated by the Rad53-Chk1 pathway and the Ssn6-Tup1 complex in response to DNA replication and damage stresses. Therefore, this study provides insight into the regulatory mechanisms of RNR genes to DNA replication and damage stresses in basidiomycete fungi.

Requirement and functional redundancy of two large ribonucleotide reductase subunit genes for cell cycle, chloroplast biogenesis and photosynthesis in tomato.

Ribonucleotide reductase (RNR), functioning in the de novo synthesis of deoxyribonucleoside triphosphates (dNTPs), is crucial for DNA replication and cell cycle progression. In most plants, the large subunits of RNR have more than one homologous gene. However, the different functions of these homologous genes in plant development remain unknown. In this study, we obtained the mutants of two large subunits of RNR in tomato and studied their functions. The mutant ylc1 was obtained by ethyl methyl sulfonate (EMS) treatment. Through map-based cloning, complementation and knock-out experiments, it was confirmed that YLC1 encodes a large subunit of RNR (SlRNRL1). The expression level of the genes related to cell cycle progression, chloroplast biogenesis and photosynthesis was assessed by RNA-sequencing. In addition, we knocked out SlRNRL2 (a SlRNRL1 homologue) using CRISPR-Cas9 technology in the tomato genome, and we down-regulated SlRNRL2 expression in the genetic background of slrnrl1-1 using a tobacco rattle virus-induced gene silencing (VIGS) system. The mutant slrnrl1 exhibited dwarf stature, chlorotic young leaves and smaller fruits. Physiological and transcriptomic analyses indicated that SlRNRL1 plays a crucial role in the regulation of cell cycle progression, chloroplast biogenesis and photosynthesis in tomato. The slrnrl2 mutant did not exhibit any visible phenotype. SlRNRL2 has a redundant function with SlRNRL1, and the double mutant slrnrl1slrnrl2 is lethal. SlRNRL1 is essential for cell cycle progression, chloroplast biogenesis and photosynthesis. In addition, SlRNRL1 and SlRNRL2 possess redundant functions and at least one of these RNRLs is required for tomato survival, growth and development.

De novo deoxyribonucleotide biosynthesis regulates cell growth and tumor progression in small-cell lung carcinoma

Deoxyribonucleotide biosynthesis from ribonucleotides supports the growth of active cancer cells by producing building blocks for DNA. Although ribonucleotide reductase (RNR) is known to catalyze the rate-limiting step of de novo deoxyribonucleotide triphosphate (dNTP) synthesis, the biological function of the RNR large subunit (RRM1) in small-cell lung carcinoma (SCLC) remains unclear. In this study, we established siRNA-transfected SCLC cell lines to investigate the anticancer effect of silencing RRM1 gene expression. We found that RRM1 is required for the full growth of SCLC cells both in vitro and in vivo. In particular, the deletion of RRM1 induced a DNA damage response in SCLC cells and decreased the number of cells with S phase cell cycle arrest. We also elucidated the overall changes in the metabolic profile of SCLC cells caused by RRM1 deletion. Together, our findings reveal a relationship between the deoxyribonucleotide biosynthesis axis and key metabolic changes in SCLC, which may indicate a possible link between tumor growth and the regulation of deoxyribonucleotide metabolism in SCLC.

MTORC2 regulates ribonucleotide reductase to promote DNA replication and gemcitabine resistance in non-small cell lung cancer

Ribonucleotide reductase (RNR) is the key enzyme that catalyzes the production of deoxyribonucleotides (dNTPs) for DNA replication and it is also essential for cancer cell proliferation. As the RNR inhibitor, Gemcitabine is widely used in cancer therapies, however, resistance limits its therapeutic efficacy and curative potential. Here, we identified that mTORC2 is a main driver of gemcitabine resistance in non-small cell lung cancers (NSCLC). Pharmacological or genetic inhibition of mTORC2 greatly enhanced gemcitabine induced cytotoxicity and DNA damage. Mechanistically, mTORC2 directly interacted and phosphorylated RNR large subunit RRM1 at Ser 631. Ser631 phosphorylation of RRM1 enhanced its interaction with small subunit RRM2 to maintain sufficient RNR enzymatic activity for efficient DNA replication. Targeting mTORC2 retarded DNA replication fork progression and improved therapeutic efficacy of gemcitabine in NSCLC xenograft model in vivo. Thus, these results identified a mechanism through mTORC2 regulating RNR activity and DNA replication, conferring gemcitabine resistance to cancer cells.

APOBECs and Herpesviruses.

The apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like (APOBEC) family of DNA cytosine deaminases provides a broad and overlapping defense against viral infections. Successful viral pathogens, by definition, have evolved strategies to escape restriction by the APOBEC enzymes of their hosts. HIV-1 and related retroviruses are thought to be the predominant natural substrates of APOBEC enzymes due to obligate single-stranded (ss)DNA replication intermediates, abundant evidence for cDNA strand C-to-U editing (genomic strand G-to-A hypermutation), and a potent APOBEC degradation mechanism. In contrast, much lower mutation rates are observed in double-stranded DNA herpesviruses and the evidence for APOBEC mutation has been less compelling. However, recent work has revealed that Epstein-Barr virus (EBV), Kaposi’s sarcoma-associated herpesvirus (KSHV), and herpes simplex virus-1 (HSV-1) are potential substrates for cellular APOBEC enzymes. To prevent APOBEC-mediated restriction these viruses have repurposed their ribonucleotide reductase (RNR) large subunits to directly bind, inhibit, and relocalize at least two distinct APOBEC enzymes—APOBEC3B and APOBEC3A. The importance of this interaction is evidenced by genetic inactivation of the EBV RNR (BORF2), which results in lower viral infectivity and higher levels of C/G-to-T/A hypermutation. This RNR-mediated mechanism therefore likely functions to protect lytic phase viral DNA replication intermediates from APOBEC-catalyzed DNA C-to-U deamination. The RNR-APOBEC interaction defines a new pathogen-host conflict that the virus must win in real-time for transmission and pathogenesis. However, partial losses over evolutionary time may also benefit the virus by providing mutational fuel for adaptation.

A Missense Mutation in a Large Subunit of Ribonucleotide Reductase Confers Temperature-Gated Tassel Formation.

Temperature is a major factor regulating plant growth. To reproduce at extreme temperatures, plants must develop normal reproductive organs when exposed to temperature changes. However, little is known about the underlying molecular mechanisms. Here, we identified the maize (Zea mays) mutant thermosensitive vanishing tassel1-R (tvt1-R), which lacks tassels at high (restrictive) temperatures due to shoot apical meristem (SAM) arrest, but forms normal tassels at moderate (permissive) temperatures. The critical stage for phenotypic conversion in tvt1-R mutants is V2 to V6 (Vn, where "n" is the number of leaves with collars visible). Positional cloning and allelism and complementation tests revealed that a G-to-A mutation causing a Arg277-to-His277 substitution in ZmRNRL1, a ribonucleotide reductase (RNR) large subunit (RNRL), confers the tvt1-R mutant phenotype. RNR regulates the rate of deoxyribonucleoside triphosphate (dNTP) production for DNA replication and damage repair. By expression, yeast two-hybrid, RNA sequencing, and flow cytometric analyses, we found that ZmRNRL1-tvt1-R failed to interact with all three RNR small subunits at 34°C due to the Arg277-to-His277 substitution, which could impede RNR holoenzyme (α2β2) formation, thereby decreasing the dNTP supply for DNA replication. Decreased dNTP supply may be especially severe for the SAM that requires a continuous, sufficient dNTP supply for rapid division, as demonstrated by the SAM arrest and tassel absence in tvt1-R mutants at restrictive temperatures. Our study reveals a novel mechanism of temperature-gated tassel formation in maize and provides insight into the role of RNRL in SAM maintenance.

Identification of interaction domains in the pseudorabies virus ribonucleotide reductase large and small subunits

Alphaherpesviral ribonucleotide reductase (RNR) is composed of large (pUL39, RR1) and small (pUL40, RR2) subunits. This enzyme can catalyze conversion of ribonucleotide to deoxynucleotide diphosphates that are further phosphorylated into deoxynucleotide triphosphate (dNTPs). The dNTPs are substrates for de novo viral DNA synthesis in infected host cells. The enzymatic activity of RNR depends on association between RR1 and RR2. However, the molecular basis underlying alphaherpesviral RNR complex formation is still largely unknown. In the current study, we investigated the pseudorabies virus (PRV) RNR interaction domains in pUL39 and pUL40. The interaction of pUL39 and pUL40 was identified by co-immunoprecipitation (co-IP) and colocalization analyses. Furthermore, the interaction amino acid (aa) domains in pUL39 and pUL40 were mapped using a series of truncated proteins. Consequently, the 90―210 aa in pUL39 was identified to be responsible for the interaction with pUL40. In turn, the 66―152, 218―258 and 280―303 aa in pUL40 could interact with pUL39, respectively. Deletion of 90―210 aa in pUL39 completely abrogated the interaction with pUL40. Deletion of 66―152, 218―258 and 280―303 aa in pUL40 remarkably weakened the interaction with pUL39, whereas a weak interaction could still be observed. Amino acid sequence alignments showed that the interaction domains identified in PRV pUL39/pUL40 were relatively non-conserved among the selected RNR subunits in alphaherpesviruses HSV1, HSV2, HHV3(VZV), BHV1, EHV1 and DEV. However, they were relatively conserved among PRV, HSV1 and HSV2. Collectively, our findings provided some molecular targets for inhibition of pUL39―pUL40 interaction to antagonize viral replication in PRV infected hosts.

Feedback inhibition of bovine herpesvirus 5 replication by dual-copy bhv5-miR-B10-3p.

Bovine herpesvirus 5 (BoHV-5) is a pathogen of cattle responsible for fatal meningoencephalitis. Like alpha herpesvirus subfamily members, BoHV-5 also encodes microRNA in lytic infections of epithelial cells. BoHV-5-miR-B10 was the most abundant miRNA detected in a high-throughput sequencing study. Here, we evaluated the kinetics of miR-B10 expression after BoHV-5 productive infection by stem-loop real-time quantitative PCR. miR-B10 candidate target sites in the virus were predicted, and BoHV-5 UL39 was confirmed as a target gene by dual-luciferase assay with the design of an miR-B10 tough decoy (TuD). The UL39 gene encoding ribonucleotide reductase (RR) large subunit plays an important role in the early stage of BoHV-5 lytic infection. As BoHV-5-miR-B10 is located in internal and terminal repeat regions, we generated a TuD gene-integrated BoHV-5 strain, which effectively down-regulated miR-B10-3p. Strikingly, the suppression of miR-B10-3p significantly improved BoHV-5 replication. Taking these findings together, our study established an efficient method to deliver and express TuD RNA for viral miRNA suppression, and demonstrated that virus-encoded miRNA suppresses viral-genome biogenesis with a feedback mode, which might serve as a brake for viral replication. Herpesviruses infect humans and a variety of animals. Almost all herpesviruses can encode miRNAs, but the functions of these miRNAs remain to be elucidated. Most herpesvirus-encoded miRNA harbours dual copies, which is difficult to be deleted by current genetic modulation. Here, we developed an efficient method to deliver and express TuD RNA to efficiently suppress viral miRNA with multiple copies. Using this method, we demonstrated for the first time that viral miRNA feedback regulates viral replication by suppressing the expression of RR.

A Conserved Mechanism of APOBEC3 Relocalization by Herpesviral Ribonucleotide Reductase Large Subunits.

An integral part of the antiviral innate immune response is the APOBEC3 family of single-stranded DNA cytosine deaminases, which inhibits virus replication through deamination-dependent and -independent activities. Viruses have evolved mechanisms to counteract these enzymes, such as HIV-1 Vif-mediated formation of a ubiquitin ligase to degrade virus-restrictive APOBEC3 enzymes. A new example is Epstein-Barr virus (EBV) ribonucleotide reductase (RNR)-mediated inhibition of cellular APOBEC3B (A3B). The large subunit of the viral RNR, BORF2, causes A3B relocalization from the nucleus to cytoplasmic bodies and thereby protects viral DNA during lytic replication. Here, we use coimmunoprecipitation and immunofluorescence microscopy approaches to ask whether this mechanism is shared with the closely related gammaherpesvirus Kaposi's sarcoma-associated herpesvirus (KSHV) and the more distantly related alphaherpesvirus herpes simplex virus 1 (HSV-1). The large RNR subunit of KSHV, open reading frame 61 (ORF61), coprecipitated multiple APOBEC3s, including A3B and APOBEC3A (A3A). KSHV ORF61 also caused relocalization of these two enzymes to perinuclear bodies (A3B) and to oblong cytoplasmic structures (A3A). The large RNR subunit of HSV-1, ICP6, also coprecipitated A3B and A3A and was sufficient to promote the relocalization of these enzymes from nuclear to cytoplasmic compartments. HSV-1 infection caused similar relocalization phenotypes that required ICP6. However, unlike the infectivity defects previously reported for BORF2-null EBV, ICP6 mutant HSV-1 showed normal growth rates and plaque phenotypes. Combined, these results indicate that both gamma- and alphaherpesviruses use a conserved RNR-dependent mechanism to relocalize A3B and A3A and furthermore suggest that HSV-1 possesses at least one additional mechanism to neutralize these antiviral enzymes.IMPORTANCE The APOBEC3 family of DNA cytosine deaminases constitutes a vital innate immune defense against a range of different viruses. A novel counterrestriction mechanism has recently been uncovered for the gammaherpesvirus EBV, in which a subunit of the viral protein known to produce DNA building blocks (ribonucleotide reductase) causes A3B to relocalize from the nucleus to the cytosol. Here, we extend these observations with A3B to include a closely related gammaherpesvirus, KSHV, and a more distantly related alphaherpesvirus, HSV-1. These different viral ribonucleotide reductases also caused relocalization of A3A, which is 92% identical to A3B. These studies are important because they suggest a conserved mechanism of APOBEC3 evasion by large double-stranded DNA herpesviruses. Strategies to block this host-pathogen interaction may be effective for treating infections caused by these herpesviruses.

Deoxyribonucleotide salvage falls short in whole animals

Ribonucleotide reductase (RNR) catalyzes the first committed reaction in DNA synthesis. Most of what we know about RNR regulation comes from studies with cultured cells and with purified proteins. In this study, Tran et al. use Cre-Lox technology to inactivate RNR large subunit expression in heart and skeletal muscle of mouse embryos. Analysis of these mutants paints a picture of dNTP regulation in whole animals quite different from that seen in studies of purified proteins and cultured cells.

SiSTL1, encoding a large subunit of ribonucleotide reductase, is crucial for plant growth, chloroplast biogenesis, and cell cycle progression in Setaria italica.

The activity of ribonucleotide reductase (RNR), which catalyses the transformation of four ribonucleoside diphosphates (NDPs) to their corresponding deoxyribonucleoside diphosphates (dNDPs), is the main determiner of the cellular concentration of dNTP pools and should be tightly coordinated with DNA synthesis and cell-cycle progression. Constitutively increased or decreased RNR activity interferes with DNA replication and leads to arrested cell cycle progression; however, the mechanisms underlying these disruptive effects in higher plants remain to be uncovered. In this study, we identified a RNR large subunit mutant, sistl1, in Setaria italica (foxtail millet), which exhibited growth retardation as well as striped leaf phenotype, i.e. irregularly reduced leaf vein distances and decreased chloroplast biogenesis. We determined that a Gly737 to Glu substitution occurring in the C-terminus of the SiSTL1 protein slightly affected its optimal function, leading in turn to the reduced expression of genes variously involved in the assembly and activation of the DNA pre-replicative complex, elongation of replication forks and S phase entry. Our study provides new insights into how SiSTL1 regulates plant growth, chloroplast biogenesis, and cell cycle progression in Poaceae crops.

Species-independent contribution of ZBP1/DAI/DLM-1-triggered necroptosis in host defense against HSV1

Necroptosis complements apoptosis as a host defense pathway to stop virus infection. Herpes simplex virus shows a propensity to trigger necroptosis of mouse cells and mice even though cell death is blocked in human cells through UL39-encoded ICP6. This ribonucleotide reductase large subunit (R1) nucleates RHIM-dependent oligomerization of RIP3 kinase (RIPK3, also known as RIP3) in mouse cells but inhibits activation in cells from the natural human host. By interrogating the comparative behavior of ICP6-deficient viruses in mouse and human cells, here we unveil virus-induced necroptosis mediated by Z-DNA-binding protein 1 (ZBP1, also known as DAI). ZBP1 acts as a pathogen sensor to detect nascent RNA transcripts rather than input viral DNA or viral DNA generated through replication. Consistent with the implicated role of virus-induced necroptosis in restricting infection, viral pathogenesis is restored in Zbp1−/−, Ripk3−/− and Mlkl−/− mice. Thus, in addition to direct activation of RIPK3 via ICP6, HSV1 infection in mice and mouse cells triggers virus-induced necroptosis through ZBP1. Importantly, virus-induced necroptosis is also induced in human HT-29 cells by ICP6 mutant viruses; however, ZBP1 levels must be elevated for this pathway to be active. Thus, our studies reveal a common, species-independent role of this nucleic acid sensor to detect the presence of this virus. HSV1 ICP6 functions as a bona fide RHIM signaling inhibitor to block virus-induced necroptosis in its natural host. Altogether, ZBP1-dependent restriction of herpesvirus infection emerges as a potent antiviral armament of the innate immune system.

Adenoviral shRNA vector targeting ribonucleotide reductase M1 (RRM1) has a strong antitumor activity and decrease the drug resistance to GEM in RRM1-overexpressing bladder cells.

e24237Background: Ribonucleotide reductase large subunit (RRM1) is the main enzyme responsible for synthesis of the deoxyribonucleotides and involved in cell proliferation. It is also the primary c...

Herpes Simplex Virus 1 Mutant with Point Mutations in UL39 Is Impaired for Acute Viral Replication in Mice, Establishment of Latency, and Explant-Induced Reactivation.

In the process of generating herpes simplex virus 1 (HSV-1) mutations in the viral regulatory gene encoding infected cell protein 0 (ICP0), we isolated a viral mutant, termed KOS-NA, that was severely impaired for acute replication in the eyes and trigeminal ganglia (TG) of mice, defective in establishing a latent infection, and reactivated poorly from explanted TG. To identify the secondary mutation(s) responsible for the impaired phenotypes of this mutant, we sequenced the KOS-NA genome and noted that it contained two nonsynonymous mutations in UL39, which encodes the large subunit of ribonucleotide reductase, ICP6. These mutations resulted in lysine-to-proline (residue 393) and arginine-to-histidine (residue 950) substitutions in ICP6. To determine whether alteration of these amino acids was responsible for the KOS-NA phenotypes in vivo, we recombined the wild-type UL39 gene into the KOS-NA genome and rescued its acute replication phenotypes in mice. To further establish the role of UL39 in KOS-NA's decreased pathogenicity, the UL39 mutations were recombined into HSV-1 (generating UL39mut), and this mutant virus showed reduced ocular and TG replication in mice comparable to that of KOS-NA. Interestingly, ICP6 protein levels were reduced in KOS-NA-infected cells relative to the wild-type protein. Moreover, we observed that KOS-NA does not counteract caspase 8-induced apoptosis, unlike wild-type strain KOS. Based on alignment studies with other HSV-1 ICP6 homologs, our data suggest that amino acid 950 of ICP6 likely plays an important role in ICP6 accumulation and inhibition of apoptosis, consequently impairing HSV-1 pathogenesis in a mouse model of HSV-1 infection.IMPORTANCE HSV-1 is a major human pathogen that infects ∼80% of the human population and can be life threatening to infected neonates or immunocompromised individuals. Effective therapies for treatment of recurrent HSV-1 infections are limited, which emphasizes a critical need to understand in greater detail the events that modulate HSV-1 replication and pathogenesis. In the current study, we identified a neuroattenuated HSV-1 mutant (i.e., KOS-NA) that contains novel mutations in the UL39 gene, which codes for the large subunit of ribonucleotide reductase (also known as ICP6). This mutant form of ICP6 was responsible for the attenuation of KOS-NA in vivo and resulted in diminished ICP6 protein levels and antiapoptotic effect. Thus, we have determined that subtle alteration of the UL39 gene regulates expression and functions of ICP6 and severely impacts HSV-1 pathogenesis, potentially making KOS-NA a promising vaccine candidate against HSV-1.

Rnr1's role in telomere elongation cannot be replaced by Rnr3: a role beyond dNTPs?

Telomeres, the nucleoprotein complexes at the end of eukaryotic chromosomes, protect them from degradation and ensure the replicative capacity of cells. In most human tumors and in budding yeast, telomere length is maintained by the activity of telomerase, an enzyme that adds dNTPs according to an internal RNA template. The dNTPs are generated with the help of the ribonucleotide reductase (RNR) complex. We have recently generated strains lacking the large subunit of RNR, Rnr1, which were kept viable by the expression of RNR complexes containing the Rnr1 homolog, Rnr3. Interestingly, we found that these Rnr1-deficient strains have short telomeres that are stably maintained, but cannot become efficiently elongated by telomerase. Thus, a basic maintenance of short telomeres is possible under conditions, where Rnr1 activity is absent, but a sustained elongation of short telomeres fully depends on Rnr1 activity. We show that Rnr3 cannot compensate for this telomeric function of Rnr1 even when overall cellular dNTP values are restored. This suggests that Rnr1 plays a role in telomere elongation beyond increasing cellular dNTP levels. Furthermore, our data indicate that telomerase may act in two different modes, one that is capable of coping with the "end-replication problem" and is functional even in the absence of Rnr1 and another required for the sustained elongation of short telomeres, which fully depends on the presence of Rnr1. Supply of dNTPs for telomere elongation is provided by the Mec1ATR checkpoint, both during regular DNA replication and upon replication fork stalling. We discuss the implications of these results on telomere maintenance in yeast and cancer cells.

Cooperative inhibition of RIP1-mediated NF-κB signaling by cytomegalovirus-encoded deubiquitinase and inactive homolog of cellular ribonucleotide reductase large subunit.

Several viruses have been found to encode a deubiquitinating protease (DUB). These viral DUBs are proposed to play a role in regulating innate immune or inflammatory signaling. In human cytomegalovirus (HCMV), the largest tegument protein encoded by UL48 contains DUB activity, but its cellular targets are not known. Here, we show that UL48 and UL45, an HCMV-encoded inactive homolog of cellular ribonucleotide reductase (RNR) large subunit (R1), target receptor-interacting protein kinase 1 (RIP1) to inhibit NF-κB signaling. Transfection assays showed that UL48 and UL45, which binds to UL48, interact with RIP1 and that UL48 DUB activity and UL45 cooperatively suppress RIP1-mediated NF-κB activation. The growth of UL45-null mutant virus was slightly impaired with showing reduced accumulation of viral late proteins. Analysis of a recombinant virus expressing HA-UL45 showed that UL45 interacts with both UL48 and RIP1 during virus infection. Infection with the mutant viruses also revealed that UL48 DUB activity and UL45 inhibit TNFα-induced NF-κB activation at late times of infection. UL48 cleaved both K48- and K63-linked polyubiquitin chains of RIP1. Although UL45 alone did not affect RIP1 ubiquitination, it could enhance the UL48 activity to cleave RIP1 polyubiquitin chains. Consistently, UL45-null virus infection showed higher ubiquitination level of endogenous RIP1 than HA-UL45 virus infection at late times. Moreover, UL45 promoted the UL48-RIP1 interaction and re-localization of RIP1 to the UL48-containing virion assembly complex. The mouse cytomegalovirus (MCMV)-encoded DUB, M48, interacted with mouse RIP1 and M45, an MCMV homolog of UL45. Collectively, our data demonstrate that cytomegalovirus-encoded DUB and inactive R1 homolog target RIP1 and cooperatively inhibit RIP1-mediated NF-κB signaling at the late stages of HCMV infection.

Physical interaction between human ribonucleotide reductase large subunit and thioredoxin increases colorectal cancer malignancy

Ribonucleotide reductase (RR) is the rate-limiting enzyme in DNA synthesis, catalyzing the reduction of ribonucleotides to deoxyribonucleotides. During each enzymatic turnover, reduction of the active site disulfide in the catalytic large subunit is performed by a pair of shuttle cysteine residues in its C-terminal tail. Thioredoxin (Trx) and glutaredoxin (Grx) are ubiquitous redox proteins, catalyzing thiol-disulfide exchange reactions. Here, immunohistochemical examination of clinical colorectal cancer (CRC) specimens revealed that human thioredoxin1 (hTrx1), but not human glutaredoxin1 (hGrx1), was up-regulated along with human RR large subunit (RRM1) in cancer tissues, and the expression levels of both proteins were correlated with cancer malignancy stage. Ectopically expressed hTrx1 significantly increased RR activity, DNA synthesis, and cell proliferation and migration. Importantly, inhibition of both hTrx1 and RRM1 produced a synergistic anticancer effect in CRC cells and xenograft mice. Furthermore, hTrx1 rather than hGrx1 was the efficient reductase for RRM1 regeneration. We also observed a direct protein-protein interaction between RRM1 and hTrx1 in CRC cells. Interestingly, besides the known two conserved cysteines, a third cysteine (Cys779) in the RRM1 C terminus was essential for RRM1 regeneration and binding to hTrx1, whereas both Cys32 and Cys35 in hTrx1 played a counterpart role. Our findings suggest that the up-regulated RRM1 and hTrx1 in CRC directly interact with each other and promote RR activity, resulting in enhanced DNA synthesis and cancer malignancy. We propose that the RRM1-hTrx1 interaction might be a novel potential therapeutic target for cancer treatment.