Herpes simplex virus ribonucleotide reductase: expression in Escherichia coli and purification to homogeneity of a tyrosyl free radical-containing, enzymatically active form of the 38-kilodalton subunit.

Infection of mammalian cells with herpes simplex virus (HSV) induces a virus-encoded ribonucleotide reductase which is different from the cellular enzyme. This essential viral enzyme consists of two nonidentical subunits of 140 and 38 kilodaltons (kDa) which have not previously been purified to homogeneity. The small subunit of ribonucleotide reductases from other species contains a tyrosyl free radical essential for activity. We have cloned the gene for the small subunit of HSV-1 ribonucleotide reductase into a tac expression plasmid vector. After transfection of Escherichia coli, expression of the 38-kDa protein was detected in immunoblots with a specific monoclonal antibody. About 30 micrograms of protein was produced per liter of bacterial culture. The 38-kDa protein was purified to homogeneity in an almost quantitative yield by immunoaffinity chromatography. It contained a tyrosyl free radical which gave a specific electron paramagnetic resonance spectrum identical to that we have observed in HSV-infected mammalian cells and clearly different from that produced by the E. coli and mammalian ribonucleotide reductases. The recombinant 38-kDa subunit had full activity when assayed in the presence of HSV-infected cell extracts deficient in the native 38-kDa subunit.     

Ribonucleotide reductase from the higher plant Arabidopsis thaliana : expression of the R2 component and characterization of its iron-radical center

 Deoxyribonucleotides synthesis has not been biochemically characterized in higher plants. From a cDNA of the small component (protein R2) of ribonucleotide reductase from Arabidopsis thaliana, an inducible overexpression plasmid has been constructed. A recombinant 78-kDa homodimeric protein containing very little iron was purified to homogeneity. Addition of ferrous iron and oxygen resulted in a protein containing 1.2 tyrosyl radicals and 4 iron atoms per dimer. Light absorption and low-temperature EPR spectra indicated close similarity of the iron-radical centers in plant and mouse R2 proteins. It is then suggested that, as in all class I eukaryotic ribonucleotide reductase, the active site of R2 component contains a μ-oxo bridged di-iron center in strong interaction with a tyrosyl radical. The stability of the radical seems, however, to be larger in the plant R2 protein, as shown by its resistance to hydroxyurea. Received: 20 March 1997 / Accepted: 5 June 1997     

Mouse ribonucleotide reductase control: influence of substrate binding upon interactions with allosteric effectors

Using ribonucleotide reductase encoded by vaccinia virus as a model for the mammalian enzyme, our laboratory developed an assay that allows simultaneous monitoring of the reduction of ADP, CDP, GDP, and UDP. That study found ADP reduction to be specifically inhibited by ADP itself. To learn whether this effect is significant for cellular regulation, we have analyzed recombinant mouse ribonucleotide reductase. We report that allosteric control properties originally described in single-substrate assays operate also under our four-substrate assay conditions. Three distinctions from the vaccinia enzyme were seen: 1) higher sensitivity to allosteric modifiers; 2) higher activity with UDP as substrate; and 3) significant inhibition by ADP of GDP reduction as well as that of ADP itself. Studies of the effects of ADP and other substrates upon binding of effectors indicate that binding of ribonucleoside diphosphates at the catalytic site influences dNTP binding at the specificity site. We also examined the activities of hybrid ribonucleotide reductases, composed of a mouse subunit combined with a vaccinia subunit. As previously reported, a vaccinia R1/mouse R2 hybrid has low but significant activity. Surprisingly, a mouse R1/vaccinia R2 hybrid was more active than either mouse R1/R2 or vaccinia R1/R2, possibly explaining why mutations affecting vaccinia ribonucleotide reductase have only small effects upon viral DNA replication.     

Identification of ribonucleotide reductase protein R1 as an activator of microtubule nucleation in Xenopus egg mitotic extracts

Microtubule nucleation on the centrosome and the fungal equivalent, the spindle pole body (SPB), is activated at the onset of mitosis. We previously reported that mitotic extracts prepared from Xenopus unfertilized eggs convert the interphase SPB of fission yeast into a competent state for microtubule nucleation. In this study, we have purified an 85-kDa SPB activator from the extracts and identified it as the ribonucleotide reductase large subunit R1. We further confirmed that recombinant mouse R1 protein was also effective for SPB activation. On the other hand, another essential subunit of ribonucleotide reductase, R2 protein, was not required for SPB activation. SPB activation by R1 protein was suppressed in the presence of anti-R1 antibodies or a partial oligopeptide of R1; the oligopeptide also inhibited aster formation on Xenopus sperm centrosomes. In accordance, R1 was detected in animal centrosomes by immunofluorescence and immunoblotting with anti-R1 antibodies. In addition, recombinant mouse R1 protein bound to gamma- and alpha/beta-tubulin in vitro. These results suggest that R1 is a bifunctional protein that acts on both ribonucleotide reduction and centrosome/SPB activation.     

Extracellular vaccinia virus envelope glycoprotein encoded by the A33R gene.

With the aid of three monoclonal antibodies (MAbs), a glycoprotein specifically localized to the outer envelope of vaccinia virus was shown to be encoded by the A33R gene. These MAbs reacted with a glycosylated protein that migrated as 23- to 28-kDa and 55-kDa species under reducing and nonreducing conditions, respectively. The protein recognized by the three MAbs was synthesized by all 11 orthopoxviruses tested: eight strains of vaccinia virus (including modified vaccinia virus Ankara) and one strain each of cowpox, rabbitpox, and ectromelia viruses. The observation that the protein synthesized by ectromelia virus-infected cells reacted with only one of the three MAbs provided a means of mapping the gene encoding the glycoprotein. By transfecting vaccinia virus DNA into cells infected with ectromelia virus and assaying for MAb reactivity, we mapped the glycoprotein to the A33R open reading frame. The amino acid sequence and hydrophilicity plot predicted that the A33R gene product is a type II membrane protein with two asparagine-linked glycosylation sites. Triton X-114 partitioning experiments indicated that the A33R gene product is an integral membrane protein. The ectromelia virus homolog of the vaccinia virus A33R gene was sequenced, revealing 90% predicted amino acid identity. The vaccinia and variola virus homolog sequences predict 94% identical amino acids, the latter having one fewer internal amino acid. Electron microscopy revealed that the A33R gene product is expressed on the surface of extracellular enveloped virions but not on the intracellular mature form of virus. The conservation of this protein and its specific incorporation into viral envelopes suggest that it is important for virus dissemination.     

The role of herpes simplex virus ribonucleotide reductase small subunit carboxyl terminus in subunit interaction and formation of iron-tyrosyl center structure.

Herpes simplex virus ribonucleotide reductase consists of two nonidentical subunits, proteins R1 and R2, which are required together for activity. Active R2 protein contains a tyrosyl free radical and a binuclear iron center. A truncated form of the R2 subunit, lacking 7 amino acid residues in the carboxyl terminus, was constructed, overexpressed in Escherichia coli and purified to homogeneity. In the presence of ferrous iron and oxygen, the truncated protein readily generated similar amounts of tyrosyl free radical as the intact protein. However, the radical showed differences in EPR characteristics in the truncated protein compared with the normal one, indicating an altered structural arrangement of the radical relative to the iron center. The truncated R2* protein was completely devoid of binding affinity to the R1 protein, demonstrating that the subunit interaction is totally dependent on the 7 outermost carboxyl-terminal amino acids of protein R2.     

Characterization of a vaccinia virus-encoded 42-kilodalton class I membrane glycoprotein component of the extracellular virus envelope.

Using a reverse genetic approach, we have demonstrated that the product of the B5R open reading frame (ORF), which has homology with members of the family of complement control proteins, is a membrane glycoprotein present in the extracellular enveloped (EEV) form of vaccinia virus but absent from the intracellular naked (INV) form. An antibody (C'-B5R) raised to a 15-amino-acid peptide from the translated B5R ORF reacted with a 42-kDa protein (gp42) found in vaccinia virus-infected cells and cesium chloride-banded EEV but not INV. Under nonreducing conditions, an 85-kDa component, possibly representing a hetero- or homodimeric form of gp42, was detected by both immunoprecipitation and Western immunoblot analysis. Metabolic labeling with [3H]glucosamine and [3H]palmitate revealed that the B5R product is glycosylated and acylated. The C-terminal transmembrane domain of the protein was identified by constructing a recombinant vaccinia virus that overexpressed a truncated, secreted form of the B5R ORF product. By N-terminal sequence analysis of this secreted protein, the site of signal peptide cleavage of gp42 was determined. A previously described monoclonal antibody (MAb 20) raised to EEV, which immunoprecipitated a protein with biochemical characteristics similar to those of wild-type gp42, reacted with the recombinant, secreted product of the B5R ORF. Immunofluorescence of wild-type vaccinia virus-infected cells by using either MAb 20 or C'-B5R revealed that the protein is expressed on the cell surface and within the cytoplasm. Immunogold labeling of EEV and INV with MAb 20 demonstrated that the protein was found exclusively on the EEV membrane.     

Purification and characterization of a protein synthesis inhibitor associated with vaccinia virus

A protein synthesis inhibitor, solubilized from vaccinia virus (Ben-Hamida, F., Person, A., and Beaud, G. (1983) J. Virol. 45, 452-455), has been purified to homogeneity, yielding a basic protein with molecular mass of 11 kDa. This purified protein migrates as a single spot in two-dimensional gel analysis (isoelectric point above 8.6). It is phosphorylated by the vaccinia-associated protein kinase, and it aggregates in the absence of reducing agents. This 11-kDa protein inhibits protein synthesis when added to a reticulocyte lysate at a stoichiometric ratio of approximately one protein molecule/ribosome, and it associates with the ribosome fraction after incubation in reticulocyte lysates or in Ehrlich ascites tumor cell lysates. As previously described for the inhibitor associated with vaccinia cores, the purified inhibitor inhibits the formation of the 40 S ribosomal subunit X Met-tRNAi ribosomal initiation complex. It has no detectable effect on the formation of the ternary complex (Met-tRNAi X GTP X eucaryotic initiation factor 2). This inhibitor associated with vaccinia virus particles may be involved in the shutoff of host protein synthesis and may also be responsible for the absence of virus replication in some cell-virus systems.     

Early loss of the tyrosyl radical in ribonucleotide reductase of adenocarcinoma cells producing nitric oxide.

Nitric oxide (NO) has been previously shown to inhibit crude preparations of ribonucleotide reductase, a key enzyme in DNA synthesis, and to destroy the essential tyrosyl free radical in pure recombinant R2 subunit of the enzyme. In R2-overexpressing TA3 cells, a decrease in the tyrosyl radical was observed by whole-cell EPR spectroscopy, as soon as 4 h after NO synthase induction by immunological stimuli. Complete loss of the tyrosyl EPR signal occurred after 7 h in cells cultured at a high density. Disappearance of the tyrosyl radical was prevented by N omega-nitro-L-arginine, a specific inhibitor of NO synthesis, and by oxyhemoglobin, which reacts rapidly with NO. It was reproduced by S-nitrosoglutathione, a NO-releasing molecule. Stable end products of NO synthase metabolism did not affect the radical. Immunoblot analysis of the R2 subunit indicated that expression of the protein was not influenced by NO synthase activity. These results establish that NO, or a labile product of NO synthase, induces the disappearance of the R2-centered tyrosyl radical. Since the radical is necessary for ribonucleotide reductase activity, its destruction by NO would contribute markedly to the antiproliferative action exerted by macrophage-type NO synthase.     

Structural and functional properties of the 14-kDa envelope protein of vaccinia virus synthesized in Escherichia coli

Vaccinia virus is a highly cytocidal virus, but the steps that lead to virus penetration into cells, the first event in virus pathogenesis, have not been elucidated. We have shown that a 14-kDa envelope protein of vaccinia virus might play a major role in virus-penetration acting at the level of cell fusion (Rodriguez, J. F., Paez, E., and Esteban, M. (1987) J. Virol. 61, 395-404; Gong, S., Lai, C., and Esteban, M. (1990) Virology 178, 81-91). To carry out structural and functional studies on the vaccinia 14-kDa protein, it would be desirable to have a high level expression system, since the amount of protein that can be obtained from purified virus or from infected cells is very limited. In this investigation we demonstrate that the 14-kDa envelope protein of vaccinia virus is expressed in Escherichia coli in soluble form and at high levels. We establish, by several criteria, that the 14-kDa vaccinia virus protein expressed in E. coli is similar to the protein found in the virus particle based on apparent molecular mass, occurrence of disulfide-linked oligomers, reactivity against specific monoclonal antibody, and identity in amino-terminal sequence with the predicted DNA sequence of the gene. We define several structural and functional properties concerning the 14-kDa envelope protein of vaccinia virus. 1) 14 kDa is a trimer of identical subunits. 2) A monomer binds to itself more strongly than to a dimer or a trimer. 3) Oligomerization does not require cellular factors. 4) Trimers induce high titer neutralizing antibodies in animals which correlate with overall immunogenicity. 5) 14-kDa binds with specificity to the cell surface of cultured cells.     

Purification and characterization of recombinant mouse and herpes simplex virus ribonucleotide reductase R2 subunit.

Overexpression of recombinant mouse and herpes simplex virus ribonucleotide reductase small subunit (protein R2) has been obtained by using the T7 RNA polymerase expression system. Both proteins, which constitute about 30% of the soluble Escherichia coli proteins, have been purified to homogeneity by a rapid and simple procedure. At this stage, few of the molecules contain the iron-tyrosyl free-radical center necessary for activity; however, addition of ferrous iron and oxygen under controlled conditions resulted in a mouse R2 protein containing 0.8 radical and 2 irons per polypeptide chain. In this reaction, one oxygen molecule was needed to generate each tyrosyl radical. Both proteins had full enzymatic activity. EPR spectroscopy showed that iron-center/radical interactions are considerably stronger in both mouse and viral proteins than in E. coli protein R2. CD spectra showed that the bacterial protein contains 70% alpha-helical structure compared to only about 50% in the mouse and viral proteins. Light absorption spectra between 310 and 600 nm indicate close similarity of the mu-oxo-bridged binuclear iron centers in all three R2 proteins. Furthermore, the paramagnetically shifted iron ligand proton NMR resonances show that the antiferromagnetic coupling and ligand arrangement in the iron center are nearly identical in all three species.     

Vaccinia virus gene H5R encodes a protein that is phosphorylated by the multisubstrate vaccinia virus B1R protein kinase.

Vaccinia virus gene B1R encodes a protein kinase, the previously identified substrates of which include the proteins S2 and Sa of 40S ribosomal subunits. This work characterizes another substrate of the B1R kinase: a 36-kDa protein induced at the early stage of infection. Partially purified 36-kDa protein, eluted from a single-stranded DNA-cellulose column by 0.5 M NaCl, was separated by two-dimensional gel electrophoresis. Phosphorylation in vitro yielded multiple forms of the 36-kDa protein with approximate isoelectric points (pI) of 5.5, 5.7, 5.9, and 6.3, in addition to the apparently unphosphorylated form with a pI of approximately 6.8. The tryptic peptides derived from 36-kDa proteins with pI values of 5.7, 5.9, and 6.3 yielded almost identical high-pressure liquid chromatography profiles, strongly suggesting that the 36-kDa protein was modified by the phosphorylation of at least four sites, which were characterized as threonine residues. The amino acid sequence of several tryptic peptides derived from the 36-kDa protein showed that the 36-kDa protein was encoded by gene H5R of vaccinia virus. Consistent with this, the B1R kinase--either expressed in Escherichia coli or highly purified from HeLa cells--phosphorylated a recombinant trpE-H5R fusion protein in vitro. Fingerprints of the trpE-H5R and 36-kDa proteins phosphorylated by recombinant B1R kinase revealed common sites of phosphorylation, although some tryptic peptides were specific to either protein. Comparison was made of fingerprints of tryptic phosphopeptides derived from 36-kDa single-stranded DNA-binding protein labelled in vivo or in vitro. A common subset of peptides was observed, suggesting that some sites on H5R protein are phosphorylated by the B1R kinase in infected cells. These results suggest that some of the multiple threonine sites in the H5R protein are phosphorylated in vivo by the B1R protein kinase.     

Subunit M2 of mammalian ribonucleotide reductase. Characterization of a homogeneous protein isolated from M2-overproducing mouse cells

The M2 subunit of mammalian ribonucleotide reductase was purified to homogeneity from hydroxyurea-resistant, M2-overproducing mouse cells. The purification procedure involved affinity chromatography on an anti-tubulin antibody-Sepharose column and high performance gel permeation chromatography. The pure protein is a dimer of Mr = 88,000, containing stoichiometric amounts of a non-heme iron center and a tyrosyl free radical. The radical is destroyed by hydroxyurea but can readily be regenerated on incubation of the radical-free protein alone with iron-dithiothreitol in the presence of air. The ability to spontaneously regenerate the tyrosyl radical distinguishes protein M2 from the corresponding subunit of Escherichia coli ribonucleotide reductase, protein B2, but apart from that the two proteins are very similar.     

A truncated protein kinase domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) expressed in Escherichia coli.

The amino-terminal domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) contains a serine/threonine-specific protein kinase that has characteristics of a growth factor receptor (Chung, T. D., Wymer, J. P., Smith, C. C., Kulka, M., and Aurelian, L. (1989) J. Virol. 63, 3389-3398; Chung, T. D., Wymer, J. P., Kulka, M. Smith, C. C., and Aurelian, L. (1990) Virology 179, 168-178). To characterize this protein kinase (PK) domain further we constructed a bacterial expression vector (pJL11) containing DNA sequences encoding ICP10 amino acid residues 1-445. Bacteria containing pJL11 were induced to express a 29-kDa protein (designated pp29la1) that represents a truncated portion of the ICP10-PK domain (includes PK catalytic motifs I-V) as demonstrated by immunoprecipitation with antibodies that recognize different antigenic domains, competition studies with extracts of ICP10-positive eukaryotic cells, and peptide mapping.pp29la1 has autophosphorylating and transphosphorylating activity for calmodulin. The enzyme is activated by Mn2+ but not by Mg2+ ions, and autophosphorylation is inhibited by histone. It differs from the authentic ICP10-PK in that phosphorylation is specific only for threonine.     

Affinity purification of active subunit 1 of herpes simplex virus type 1 ribonucleotide reductase exhibiting a protein kinase activity

Herpes simplex virus (HSV) ribonucleotide reductase is formed by the association of two distinct dimeric subunits, R1 and R2. Attempts to purify either the HSV holoenzyme or its R1 subunit in their active form have been unsuccessful until now. The C terminus of the R2 protein being involved in the association with R1, the synthetic nonapeptide corresponding to this terminus, impedes the formation of the holoenzyme by competing with R2 for a critical site on R1. Based upon these observations, we developed an affinity chromatographic procedure to purify the R1 protein from HSV-1-infected baby hamster kidney cells. Specific binding of R1 to an affinity column made by linking the peptide HSV R2-(326-337) to Affi-Gel 10, followed by specific elution with an excess of an analogous peptide exhibiting a higher affinity for R1 yielded, in a single step, highly purified R1 protein. The purified R1 preparations contained approximately 95% of intact R1, the remaining 5% consisting of two R1 copurifying proteolytic breakdown products. The purified R1 protein exhibited a high reductase specific activity when mixed with an excess of the R2 subunit. Moreover, in vitro kinase assays revealed that the purified R1 protein of HSV-1 possesses an autophosphorylating activity also able to phosphorylate alpha-casein and histone II-S. The intrinsic protein kinase activity of HSV R1 is associated with its unique N-terminal domain which is absent from all other reductase subunits 1 and contains consensus motifs found in Ser/Thr protein kinases. A preliminary characterization of the kinase activity of the R1 protein of HSV-1 ribonucleotide reductase is presented.     

Neutralizing antibodies induced by recombinant vaccinia virus expressing varicella-zoster virus gpIV.

Monoclonal antibodies generated against varicella-zoster virus (VZV) glycoprotein I (gpI) also recognize VZV gpIV (A. Vafai, Z. Wroblewska, R. Mahalingam, G. Cabirac, M. Wellish, M. Cisco, and D. Gilden, J. Virol. 62:2544-2551, 1988). To determine whether the virus-neutralizing activity of these antibodies belongs to gpI, gpIV, or both, the open reading frame encoding gpIV was inserted into the vaccinia virus genome. Immunoprecipitation of recombinant vaccinia virus-infected cells with anti-gpIV monoclonal antibody yielded synthesis and processing of gpIV similar to those expressed in VZV-infected cells. Antibodies raised against VVgpIV in a rabbit recognized both native gpI and gpIV and neutralized VZV infectivity. In addition, antibodies raised against recombinant vaccinia virus carrying VZV gpI neutralized VZV infection. These results indicate a structural relationship between VZV gpI and gpIV and show that gpI and gpIV each induce virus-neutralizing antibody.     

Deoxyadenosine reverses hydroxyurea inhibition of vaccinia virus growth.

Hydroxyurea, an inhibitor of ribonucleotide reductase, blocks replication of vaccinia virus. However, when medium containing hydroxyurea and dialyzed serum was supplemented with deoxyadenosine, the block to viral reproduction was circumvented, provided that an inhibitor of adenosine deaminase was also present. Deoxyguanosine, deoxycytidine, and deoxythymidine were ineffective alone and did not augment the deoxyadenosine effect. In fact, increasing concentrations of deoxyguanosine and deoxythymidine, but not deoxycytidine, eliminated the deoxyadenosine rescue, an effect that was reversed by the addition of low concentrations of deoxycytidine. These results suggested that the inhibition of viral replication by hydroxyurea was primarily due to a deficiency of dATP. Deoxyribonucleoside triphosphate pools in vaccinia virus-infected cells were measured at the height of viral DNA synthesis after a synchronous infection. With 0.5 mM hydroxyurea, the dATP pool was greater than 90% depleted, the dCTP and dGTP pools were 40 to 50% reduced, and the dTTP pool was increased. Assay of ribonucleotide reductase activity in intact virus-infected cells suggested that hydroxyurea may differentially affect reduction of the various substrates of the enzyme.