Protein kinase activity associated with the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10).

The large subunit of the herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (RR1) is demonstrated to possess serine/threonine-specific kinase activity. Computer-assisted sequence analysis identified regions within the amino terminus of ICP10 that are homologous to the catalytic domain of known protein kinases (PKs). An in vitro kinase assay confirmed the ability of ICP10, immunoprecipitated from either HSV-2-infected cells or from cells transfected with an ICP10 expression vector, to autophosphorylate and transphosphorylate exogenous substrates in the presence of ATP and Mg2+. The HSV-1 RR1 was shown to be negative for PK activity under these conditions. PK activity was localized to a 57-kDa amino-terminal region within ICP10 that lacked RR activity.     

A novel human gene similar to the protein kinase (PK) coding domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) codes for a serine-threonine PK and is expressed in melanoma cells

The large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is a multifunctional protein that contains a serine-threonine protein kinase (PK) activity (Nelson, J. W., Zhu, J. , Smith, C. C., Kulka, M., and Aurelian, L. (1996) J. Biol. Chem. 271, 17021-17027). Phylogenetic analyses indicated that ICP10 PK belongs to a distinct subfamily of growth factor receptor serine-threonine PKs that are characterized by their ability to function with a limited number of conserved catalytic motifs (Hunter, J. C. R., Smith, C. C., and Aurelian, L. (1995) Int. J. Onc. 7, 515-522). Here, we report the isolation and characterization of a novel gene, designated H11, that contains an open reading frame of 588 nucleotides, which encodes a protein similar to ICP10 PK. The H11 protein has Mn(2+)-dependent serine-threonine-specific PK activity as determined with a GST-H11 fusion protein and by immununocomplex PK/immunoblotting assays of 293 cells transfected with a H11 eukaryotic expression vector. PK activity is ablated by mutation of Lys(113) within the presumtive catalytic motif II (invariant Lys). 293 cells stably transfected with H11 acquire anchorage-independent growth. Endogenous H11 RNA and the H11 phosphoprotein are expressed in melanoma cell lines and primary melanoma tissues at levels higher than in normal melanocytes and in benign nevi. Melanoma cell proliferation is inhibited by treatment with antisense oligonucleotides that inhibit H11 translation, suggesting that H11 expression is associated with cell growth.     

A physical domain of herpes simplex virus ICP8 is expressed and active in Escherichia coli.

In this report, we describe a series of procedures to assay the function of fusion genes in Escherichia coli and the specific application to the carboxy-terminal third of the herpes simplex virus type 1 (HSV-1) DNA-binding protein ICP8. E. coli cells containing the cloned HSV-1 BamHI G fragment with the HSV-1 BamHI-G-V site, map unit 0.388, nearest the tet promoter in pBR322 synthesized an active product containing a portion of ICP8. The new product induced phenotypic alterations in recipient hosts that were measurable and stable yet limited to the stability of the plasmid. The corresponding cloned DNA from the characterized HSV-1 DNA-binding protein mutant tsHA1 exhibited a predictable temperature-sensitive phenotype. Screening procedures based on the loss of induction of the parental plasmid-induced phenotype in E. coli cells allowed us to select additional mutations. One of these, which conferred a phenotype different from that of tsHA1, was transferred to the viral genome by marker transfer techniques. We suggest that any mutant could be isolated in any sequence, provided that the wild-type coding sequences induce alterations in E. coli cells. The observed alterations should have relevance in determining the mode of action of the protein in its normal environment.     

The transmembrane helical segment but not the invariant lysine is required for the kinase activity of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10).

The large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is a chimera consisting, at the amino terminus, of a Ser/Thr protein kinase (PK) with features of a signal peptide and a transmembrane (TM) helical segment, and at the carboxy-terminus, of the ribonucleotide reductase (Chung et al., 1989, 1990). Membrane immunofluorescence of ICP10 transformed cells with antibodies to synthetic peptides located upstream or downstream of the TM indicates that ICP10 is a membrane-spanning protein. Site-directed and deletion mutants were used to further characterize ICP10-PK. Mutation of Gly106 in catalytic motif I or of the invariant Lys in catalytic motif II, and deletion of both motifs (amino acids 106-178) did not eliminate kinase activity. PK activity was retained by the invariant Lys mutant expressed in bacteria and following protein separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to membrane filters. Both ICP10 and the invariant Lys mutant bound 14C-labeled rho-fluorosulfonylbenzoyl 5'-adenosine, an ATP affinity analog. The deletion mutant had 4-fold lower kinase activity than ICP10-PK, and it was insensitive to Mn2+, suggesting that these motifs are involved in Mn2+ activation of kinase activity. PK activity was lost by deletion of the TM segment (amino acid residues 85-106).     

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.     

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.     

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.     

Cloning of a ribonucleotide reductase gene of the herpes simplex virus type 2 strain G

The ribonucleotide reductase (RR) 2 gene of the HSV-2 strain G was cloned, sequenced, and expressed in an E. coli cell. The RR2 gene was located on the PstI 2.4 kb fragment, which was cloned and sequenced. The ORF of the gene was 1,011 bp and its termination codon was TAG; also, the CATATAA sequence was present in the promoter of the RR2 gene. A Poly A signal sequence (AATAAA) was found in the 3'-noncoding region. The RR2 proteins that were produced in the E. coli and Vero cells were confirmed using a Western blot analysis. SDS-PAGE revealed that the molecular weights of the fusion-RR2 that was produced in the E. coli cells were approximately 24 kDa and 38 kDa in the Vero cells. The RR2 proteins were soluble. The differences in the molecular weights might be due to modifications in the Vero cells.     

Cloning of the vaccinia virus ribonucleotide reductase small subunit gene. Characterization of the gene product expressed in Escherichia coli.

During its infectious cycle, vaccinia virus expresses a virus-encoded ribonucleotide reductase which is distinct from the host cellular enzyme (Slabaugh, M.B., and Mathews, C.K. (1984) J. Virol. 52, 501-506; Slabaugh, M.B., Johnson, T.L., and Mathews, C.K. (1984) J. Virol. 52, 507-514). We have cloned the gene for the small subunit of vaccinia virus ribonucleotide reductase (designated VVR2) into Escherichia coli and expressed the protein using a T7 RNA polymerase plasmid expression system. After isopropyl beta-D-thiogalactopyranoside induction, accumulation of a 37-kDa peptide was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and this peptide reacted with polyclonal antiserum raised against a TrpE-VVR2 fusion protein. The 37-kDa protein was purified to homogeneity, and gel filtration of the purified protein revealed that the recombinant protein existed as a dimer in solution. Purified recombinant VVR2 protein was shown to complement the activity of purified recombinant ribonucleotide reductase large subunit, with a specific activity that was similar to native VVR2 from a virus-infected cell extract. A CD spectrum of the recombinant viral protein showed that like the mouse protein, the vaccinia virus protein has 50% alpha-helical structure. Like other iron-containing ribonucleotide reductase small subunits, recombinant VVR2 protein contained a stable organic free radical that was detectable by EPR spectroscopy. The EPR spectrum of purified recombinant VVR2 was identical to that of vaccinia virus-infected mammalian cells. Both the hyperfine splitting character and microwave saturation behavior of VVR2 were similar to those of mouse R2 and distinct from E. coli R2. By using amino acid analysis to determine the concentration of VVR2, we determined that approximately 0.6 radicals were present per R2 dimer. Our results indicate that vaccinia virus small subunit is similar to mammalian ribonucleotide reductases.     

Purification and characterization of herpes simplex virus type 1 alkaline exonuclease expressed in Escherichia coli.

The alkaline exonuclease (AE) encoded by the herpes simplex virus type 1 (HSV-1) UL12 open reading frame was inducibly expressed in Escherichia coli and purified without the use of chromatographic separation. This recombinant AE was found to exhibit the same biochemical properties as the virus-encoded protein and was used to confirm the existence of a weak endonucleolytic activity in the enzyme. Antisera raised against the recombinant protein recognized several forms of the AE in HSV-1-infected cells. This expression and purification strategy will provide an economical and easily accessible alternative source of HSV-1 AE for future in vitro studies.     

Ribonucleotide reductase of herpes simplex virus type 2 resembles that of herpes simplex virus type 1.

The ribonucleotide reductase (ribonucleoside-diphosphate reductase; EC 1.17.4.1) induced by herpes simplex virus type 2 infection of serum-starved BHK-21 cells was purified to provide a preparation practically free of both eucaryotic ribonucleotide reductase and contaminating enzymes that could significantly deplete the substrates. Certain key properties of the herpes simplex virus type 2 ribonucleotide reductase were examined to define the extent to which it resembled the herpes simplex virus type 1 ribonucleotide reductase. The herpes simplex virus type 2 ribonucleotide reductase was inhibited by ATP and MgCl2 but only weakly inhibited by the ATP X Mg complex. Deoxynucleoside triphosphates were at best only weak inhibitors of this enzyme. ADP was a competitive inhibitor (K'i, 11 microM) of CDP reduction (K'm, 0.5 microM), and CDP was a competitive inhibitor (K'i, 0.4 microM) of ADP reduction (K'm, 8 microM). These key properties closely resemble those observed for similarly purified herpes simplex virus type 1 ribonucleotide reductase and serve to distinguish these virally induced enzymes from other ribonucleotide reductases.     

An autophosphorylating but not transphosphorylating activity is associated with the unique N terminus of the herpes simplex virus type 1 ribonucleotide reductase large subunit.

We report on a protein kinase function encoded by the unique N terminus of the herpes simplex virus type 1 (HSV-1) ribonucleotide reductase large subunit (R1). R1 expressed in Escherichia coli exhibited autophosphorylation activity in a reaction which depended on the presence of the unique N terminus. When the N terminus was separately expressed in E. coli and partially purified, a similar autophosphorylation reaction was observed. Importantly, transphosphorylation of histones and of proteins in HSV-1-infected cell extracts was also observed with purified R1 and with truncated R1 mutants in which most of the N terminus was deleted. Ion-exchange chromatography was used to separate the autophosphorylating activity of the N terminus from the transphosphorylating activity of an E. coli contaminant protein kinase. We propose a putative function for this activity of the HSV-1 R1 N terminus during the immediate-early phase of virus replication.     

Neutralization of herpes simplex virus ribonucleotide reductase activity by an oligopeptide-induced antiserum directed against subunit H2.

Herpes simplex virus type 1 ribonucleotide reductase is associated with two polypeptides of apparent molecular weights 136,000 and 38,000. The two polypeptides form a tight complex and, therefore, are often coprecipitated by monoclonal antibodies. We report here that immunoglobulins G purified from polyclonal rabbit antisera (P9) raised against a nonapeptide corresponding to the carboxy terminus of the 38,000-dalton polypeptide specifically neutralize the herpes simplex virus ribonucleotide reductase activity. We suggest that the P9 immunoglobulin G neutralizes the reductase activity by impairing the association of the two subunits (H1 and H2) of the enzyme.     

Immunological characterization of herpes simplex virus type 1 and 2 polypeptide(s) involved in viral ribonucleotide reductase activity

Mammalian cells infected with herpes simplex virus (HSV) express a novel ribonucleotide reductase which is biochemically and immunologically distinct from the uninfected-cell enzyme. Using polyvalent rabbit antiserum raised against partially purified HSV type 2 reductase as well as monoclonal antibodies to HSV type 1 and HSV type 2 early antigens, we have been able to show that in both serotypes reductase activity is associated with phosphoproteins of molecular weights 144,000 and 38,000 encoded between map units 0.566 and 0.602 in the viral genomes. The major antigenic species (144,000) have been tentatively identified as HSV type 1 ICP6 and HSV type 2 ICP10.     

Herpes simplex virus type 1 protease expressed in Escherichia coli exhibits autoprocessing and specific cleavage of the ICP35 assembly protein.

The UL26 gene of herpes simplex virus type 1 (HSV-1) encodes a protease which is responsible for the C-terminal cleavage of the nucleocapsid-associated proteins, ICP35 c and d, to their posttranslationally modified counterparts, ICP35 e and f. To further characterize the HSV-1 protease, the UL26 gene product was expressed in Escherichia coli. The expressed protease underwent autoproteolytic processing at two independent sites. The first site is shared with ICP35 and results in removal of 25 amino acids from the C terminus of the protease. The second unique site gives rise to protein species consistent with deletion of a 28-kDa fragment at the N terminus. A mutant protease, which showed no activity in a mammalian cell cotransfection assay (F. Liu and B. Roizman, Proc. Natl. Acad. Sci. USA 89:2076-2080, 1992), failed to exhibit autoproteolytic processing at either site when expressed in bacteria. The inactive mutant was able to serve as a substrate in a trans assay in which the substrate and protease were coexpressed in bacteria. This experiment demonstrated that the unique N-terminal processing was mediated exclusively by the HSV-1 protease. ICP35 c,d also served as a substrate in this assay and was correctly processed by HSV-1 protease in E. coli. This trans-cleavage assay will aid in the characterization of HSV-1 protease and assist in investigation of the role of proteolytic processing in the virus.     

Characterization of a major DNA-binding domain in the herpes simplex virus type 1 DNA-binding protein (ICP8).

We have studied the major DNA-binding protein (ICP8) from herpes simplex virus type 1 to identify its DNA-binding site. Since we obtained our protein from a cell line carrying multiple chromosomally located copies of the ICP8 gene, we first analyzed this protein to assess its similarity to the corresponding viral protein. Our protein resembled the viral protein by molecular weight, response to antibody, preference for binding single-stranded DNA, and ability to lower the melting temperature of poly(dA-dT). To define the DNA-binding domain, we subjected the protein to limited trypsin digestion and separated the peptide products on a sodium dodecyl sulfate-polyacrylamide gel. These fragments were then transferred to a nitrocellulose membrane, renatured in situ, and tested for their ability to bind DNA. From this assay, we identified four fragments which both bound DNA and exhibited the expected binding preference for single-stranded DNA. The sequence of the smallest of these fragments was determined and corresponds to a polypeptide spanning residues 300 to 849 in the intact protein. This peptide contains several regions which may be important for DNA binding based on sequence similarities in single-stranded DNA-binding proteins from other herpesviruses and, in one case, on a conserved sequence found in more distant procaryotic and eucaryotic proteins.     

A truncated v-abl-derived tyrosine-specific tyrosine kinase expressed in Escherichia coli.

Several biochemical properties of a 43 kDa v-abl-encoded tyrosine-specific protein kinase (p43v-abl) expressed in Escherichia coli were examined. p43v-abl is a fragment of a 60 kDa v-abl-encoded precursor, p60v-abl, and could be generated by limited proteolysis of a purified p60v-abl with trypsin. Tryptic cleavage of p60v-abl was prevented in the presence of ATP. These results suggest that the catalytic kinase domain of v-abl-derived protein can be separated from other (regulatory) domains by limited proteolysis. p43v-abl readily phosphorylated tyrosine residues on several different protein and peptide substrates, including peptides containing only two amino acid residues. However, the local sequence of the tyrosine-containing peptide substrate significantly affected its rate of phosphorylation. Thus the primary structure and local conformation at the tyrosine acceptor site can play an important role in determining the substrate specificity of v-abl-derived kinase. Phosphorylation by p43v-abl requires Mn2+, Co2+ or Mg2+ and exhibits a strong preference for ATP as phosphate donor. Analogues of ATP and the thiol-reactive reagent N-ethylmaleimide inhibited p43v-abl kinase activity. Purified p43v-abl is intrinsically thermolabile (t1/2 = 5 min at 40 degrees C) and phosphorylates glycerol inefficiently (Km = 1.4 M).