Biomedical subjects

J Conner

Publications and source records attributed to J Conner.

At least 19 recordsLinked to original sources

PubMed2001

Anti-GQ1b ganglioside antibodies mediate complement-dependent destruction of the motor nerve terminal.

Miller-Fisher syndrome is an autoimmune neuropathy characterized by ataxia, areflexia and ophthalmoplegia, and in the majority of cases the presence of high titres of anti-GQ1b ganglioside antibodies. In an ex vivo model, human and mouse anti-GQ1b antibodies have been shown previously to induce a complement-dependent alpha-latrotoxin-like effect on the murine motor endplate, i.e. they bring about massive quantal release of acetylcholine and eventually block neuromuscular transmission. Using immunofluorescence microscopy with image analysis, we show here that the late stages of this electrophysiological effect temporally coincide with the loss of heavy neurofilament (200 kDa) and type III beta-tubulin immunostaining and structural breakdown of the nerve terminal, as demonstrated by electron microscopy. Ultrastructurally, axon terminals were disorganized, depleted of vesicles, and subdivided by the infiltrating processes of capping Schwann cells. These findings provide clear pathological evidence to support a role for anti-ganglioside antibodies in mediating nerve terminal injury and further advance the view that this site may be of importance as a target in some human neuropathies.

Alkaline Phosphatase↗

PubMed2001

Herpes simplex virus type 1 blocks the apoptotic host cell defense mechanisms that target Bcl-2 and manipulates activation of p38 mitogen-activated protein kinase to improve viral replication.

Wild-type (wt) herpes simplex virus type 1 (HSV-1) suppresses cell death. We investigated the apoptotic pathways triggered during infection with mutant viruses tsk and 27lacZ (which lack functional ICP4 and ICP27 viral proteins, respectively) and examined the mechanisms used by wt HSV-1 to protect against programmed cell death induced by the DNA-damaging compound cisplatin. In our studies, we used BHK and HeLa cells, with similar results. We suggest that a decrease in the levels of Bcl-2 protein is a key event during apoptosis induced by the mutant viruses and that Bcl-2 levels are targeted by (i) a decrease of bcl-2 RNA, (ii) caspase-related proteolysis, and (iii) p38 mitogen-activated protein kinase (p38MAPK)-dependent destabilization of Bcl-2 protein. We show that wt HSV-1, but not the mutant viruses, maintains bcl-2 RNA and protein levels during infection and protects from the cisplatin-induced decrease in bcl-2 RNA; our data suggest that both ICP27 and ICP4 are required for this function. Additionally, wt HSV-1 evades but does not actively block activation of caspases. Although wt HSV-1 induces p38MAPK activation during infection, it prevents p38MAPK-dependent destabilization of Bcl-2 and exploits p38MAPK stimulation to enhance transcription of specific viral gene promoters to increase viral yields.

Animals↗

PubMed2000

Continuous intrathecal fluid infusions elevate nerve growth factor levels and prevent functional deficits after spinal cord ischemia.

Continuous intracerebroventricular or intrathecal infusions of neurotrophic factors have been reported to prevent neuronal degeneration, stimulate axonal sprouting and ameliorate behavioral deficits in various models of CNS injury and aging. In the present study, the ability of intrathecal infusions of recombinant human nerve growth factor (NGF) to reduce functional deficits following spinal cord ischemia was investigated. Adult rabbits underwent intrathecal cannulation and continuous infusions of either 300 microg/ml recombinant human NGF or artificial CSF (vehicle) at a rate of 143 microl/day for 7 days prior to induction of spinal cord ischemia. Continuous infusions were maintained after induction of ischemia. Four days later, both NGF-treated and vehicle-infused subjects showed a significant amelioration of functional motor deficits compared to lesioned, non-infused subjects (P<0.05). The average duration of tolerated ischemia increased from 23.4+/-1.8 min in lesioned, non-infused subjects to 35.5+/-3.1 min in lesioned, artificial CSF-infused subjects and 35.6+/-4.7 min in NGF-infused subjects (mean+/-S.E.M.). Significantly elevated NGF protein levels were attained within the spinal cords of both NGF-treated subjects and artificial CSF-infused subjects, although levels were substantially higher in NGF-treated subjects (9.8+/-3.8 ng/g in NGF-infused vs. 2.0+/-0.4 ng/g in vehicle-infused and only 0.4+/-0.2 ng/g in lesioned, non-infused animals). These findings indicate that the process of intrathecal cannulation and fluid infusion elicits alterations in the spinal cord environment that are neuroprotective, including spontaneous elevations in NGF levels.

Animals↗

PubMed2000

LEUNIG, a putative transcriptional corepressor that regulates AGAMOUS expression during flower development.

Regulation of homeotic gene expression is critical for proper developmental patterns in both animals and plants. LEUNIG is a key regulator of the Arabidopsis floral homeotic gene AGAMOUS. Mutations in LEUNIG cause ectopic AGAMOUS mRNA expression in the outer two whorls of a flower, leading to homeotic transformations of floral organ identity as well as loss of floral organs. We isolated the LEUNIG gene by using a map-based approach and showed that LEUNIG encodes a glutamine-rich protein with seven WD repeats and is similar in motif structure to a class of functionally related transcriptional corepressors including Tup1 from yeast and Groucho from Drosophila. The nuclear localization of LEUNIG-GFP is consistent with a role of LEUNIG as a transcriptional regulator. The detection of LEUNIG mRNA in all floral whorls at the time of their inception suggests that the restricted activity of LEUNIG in the outer two floral whorls must depend on interactions with other spatially restricted factors or on posttranslational regulation. Our finding suggests that both animals and plants use similar repressor proteins to regulate critical developmental processes.

AGAMOUS Protein, Arabidopsis↗

PubMed2000

Analysis of the HIV-1 LTR NF-kappaB-proximal Sp site III: evidence for cell type-specific gene regulation and viral replication.

It has been widely demonstrated that the human immunodeficiency virus type 1 (HIV-1) envelope, specifically the V3 loop of the gp120 spike, evolves to facilitate adaptation to different cellular populations within an infected host. Less energy has been directed at determining whether the viral promoter, designated the long terminal repeat (LTR), also exhibits this adaptive quality. Because of the unique nature of the cell populations infected during the course of HIV-1 infection, one might expect the opportunity for such adaptation to exist. This would permit select viral species to take advantage of the different array of conditions and factors influencing transcription within a given cell type. To investigate this hypothesis, the function of natural variants of the NF-kappaB-proximal Sp element (Sp site III) was examined in human cell line models of the two major cell types infected during the natural course of HIV-1 infection, T cells and monocytes. Utilizing the HIV-1 LAI molecular clone, which naturally contains a high-affinity Sp site III, substitution of low-affinity Sp sites in place of the natural site III element markedly decreased viral replication in Jurkat T cells. However, these substitutions had relatively small effects on viral replication in U-937 monocytic cells. Transient transfections of HIV-1 LAI-based LTR-luciferase constructs into these cell lines suggest that the large reduction in viral replication in Jurkat T cells, caused by low-affinity Sp site III variants, may result from reduced basal as well as Vpr- and Tat-activated LTR activities in Jurkat T cells compared to those in U-937 monocytic cells. When the function of Sp site III was examined in the context of HIV-1 YU-2-based LTR-luciferase constructs, substitution of a high-affinity element in place of the natural low-affinity element resulted in increased basal YU-2 LTR activity in Jurkat T cells and reduced activity in U-937 monocytic cells. These observations suggest that recruitment of Sp family members to Sp site III is of greater importance to the function of the viral promoter in the Jurkat T cell line as compared to the U-937 monocytic cell line. These observations also suggest that other regions of the LTR may compensate for Sp recruitment defects in specific cell populations.

Base Sequence↗

PubMed2000

Characterization of heterosubunit complexes formed by the R1 and R2 subunits of herpes simplex virus 1 and equine herpes virus 4 ribonucleotide reductase.

We report on the separate PCR cloning and subsequent expression and purification of the large (R1) and small (R2) subunits from equine herpes virus type 4 (EHV-4) ribonucleotide reductase. The EHV-4 R1 and R2 subunits reconstituted an active enzyme and their abilities to complement the R1 and R2 subunits from the closely related herpes simplex virus 1 (HSV-1) ribonucleotide reductase, with the use of subunit interaction and enzyme activity assays, were analysed. Both EHV-4 R1/HSV-1 R2 and HSV-1 R1/EHV-4 R2 were able to assemble heterosubunit complexes but, surprisingly, neither of these complexes was fully active in enzyme activity assays; the EHV-4 R1/HSV-1 R2 and HSV-1 R1/EHV-4 R2 enzymes had 50% and 5% of their respective wild-type activities. Site-directed mutagenesis was used to alter two non-conserved residues located within the highly conserved and functionally important C-termini of the EHV-4 and HSV-1 R1 proteins. Mutation of Pro-737 to Lys and Lys-1084 to Pro in EHV-4 and HSV-1 R1 respectively had no effects on subunit assembly. Mutation of Pro-737 to Lys in EHV-4 R1 decreased enzyme activity by 50%; replacement of Lys-1084 by Pro in HSV-1 R1 had no effect on enzyme activity. Both alterations failed to restore full enzyme activities to the heterosubunit enzymes. Therefore probably neither of these amino acids has a direct role in catalysis. However, mutation of the highly conserved Tyr-1111 to Phe in HSV-1 R1 inactivated enzyme activity without affecting subunit interaction.

Cloning, Molecular↗

PubMed2000

Anti-GQ1b antibodies and evoked acetylcholine release at mouse motor endplates.

Miller Fisher syndrome (MFS) is clinically characterized by ataxia, areflexia, and ophthalmoplegia, and is associated with serum anti-GQ1b-ganglioside antibodies. We have previously shown that anti-GQ1b antibodies induce complement-dependent, alpha-latrotoxin-like effects at mouse neuromuscular junctions (NMJs) in vitro. This effect comprises a massive increase in spontaneous quantal acetylcholine (ACh) release, accompanied by block of evoked release and muscle paralysis. This mechanism may contribute to the motor features of MFS. Whether the block of evoked ACh release is a primary effect of anti-GQ1b antibodies or occurs secondary to massive complement-dependent spontaneous release is unknown. Using conventional micro-electrode methods, we measured in detail ACh release evoked with low- and high-rate nerve stimulation, and studied the effect on it of a purified MFS IgG and a mouse monoclonal anti-GQ1b IgM (without added complement). We found that evoked transmitter release was unaffected. Control experiments proved binding of anti-GQ1b antibody at the NMJ. We conclude that the block of nerve-evoked ACh release at the NMJ is not a primary effect of anti-GQ1b antibodies, but is dependent on antibody-mediated complement activation. It remains to be determined whether the block of nerve-evoked ACh release is the consequence of massive spontaneous ACh release or occurs as a concomitant event.

Acetylcholine↗

PubMed2000

Nerve growth factor (NGF) influences differentiation and proliferation of myogenic cells in vitro via TrKA.

Classic studies have established that muscle cells exert trophic actions on neurons of the developing peripheral nervous system through the production of neurotrophins. For this reason neurotrophins are also known as 'target-derived factors'. During differentiation, muscle cells also express some neurotrophin receptors, such as the low-affinity p75 neurotrophin receptor, which binds all neurotrophins, and the high affinity tyrosine kinase receptor TrKA, nerve growth factor (NGF) transducing receptor. The functional roles of these receptors in muscle cells are still unclear and only fragmentary and controversial data are available regarding the responsiveness of muscle cells to NGF. The aim of the present study is to investigate the effects of NGF on cells of myogenic lineage. The rat myogenic cell line L6, primary cultures of adult human myoblasts, and the human rhabdomyosarcoma cell line TE-671 were used in this study. As expected, all the three cell types expressed NGF, p75 and TrKA. NGF was expressed by L6 and primary myoblasts following differentiation, but it was constitutively expressed at high levels in the TE-671 rhabdomyosarcoma cells. In L6 myoblasts, p75 receptor was expressed in myoblasts but not in myotubes early after plating; while some primary human myoblasts expressed it at all the time-points tested. Some fusiform cells of the TE-671 rhabdomyosarcoma cell line also expressed p75. TrKA was constitutively immunodetected in all the three cell lines, suggesting that these cells may respond to NGF. Addition of exogenous NGF increased the fusion rate of both primary and L6 myoblasts, as well as the proliferation of the slowly dividing primary myoblasts. Consistently, blocking the action of endogenously produced NGF with a specific neutralizing antibody decreased the percentage of fusion in both primary and L6 myoblasts. On the contrary, blocking the binding of NGF to p75 did not affect the percentage of fusion. Furthermore, neither exogenous NGF nor NGF- or p75-neutralizing antibodies appeared to affect the rhabdomyosarcoma cells, which have a high proliferation rate and do not fuse. Pharmacological inhibition of TrKA signal transduction with K252a (in the nM range) and tyrphostin AG879 (in the low microM range) resulted in a dramatic dose-dependent decrease in proliferation of all of the myogenic cell lines tested. Interestingly, this was especially evident in the rapidly dividing rhabdomyosarcoma cell line. The TrKA inhibitors also blocked fusion of L6 and primary myoblasts and induced morphological changes characterized by the flattening of the cells and a 'spider-like' rearrangement of the intermediate filaments in all three cell lines with some minor differences. A transfection study showed that p75-overexpressing L6 cells do not fuse and present changes in their morphology similar to the TrKA-inhibitors treated L6 cells. These data support the notion that NGF expression in skeletal muscle is not only associated with a classical target-derived neurotrophic function for peripheral nervous system neurons, but also with an autocrine action which affects the proliferation, fusion into myotubes, and cell morphology of developing myoblasts. The present data also suggest that these effects of NGF are mediated by TrKA receptors and that a sustained presence of NGF is needed for increase fusion into myotubes. Lastly, the dramatic anti-proliferative effect of TrKA inhibitors on myogenic cells, and especially on the TE-671 rhabdomyosarcoma cell line, suggests that pharmacological interference with NGF signal transduction could be effective in the control of these malignancies.

Animals↗

PubMed1999

Herpes simplex virus type 1 infection stimulates p38/c-Jun N-terminal mitogen-activated protein kinase pathways and activates transcription factor AP-1.

Cells respond to environmental stress and proinflammatory cytokines by stimulating the Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) and the p38 mitogen-activated protein kinase cascades. Infection of eukaryotic cells with herpes simplex virus type 1 (HSV-1) resulted in stimulation of both JNK/SAPK and p38 mitogen-activated protein kinase after 3 h of infection, and activation reached a maximum of 4-fold by 9 h post-infection. By using a series of mutant viruses, we showed that the virion transactivator protein VP16 stimulates p38/JNK, whereas no immediate-early, early, or late viral expressed gene is involved. We identified the stress-activated protein kinase kinase 1 as an upstream activator of p38/JNK, and we demonstrated that activation of AP-1 binding proceeded p38/JNK stimulation. During infection, the activated AP-1 consisted mainly of JunB and JunD with a simultaneous decrease in the cellular levels of Jun protein. We suggest that activation of the stress pathways by HSV-1 infection either represents a cascade triggered by the virus to facilitate the lytic cycle or a defense mechanism of the host cell against virus invasion.

Animals↗

PubMed1999

The unique N terminus of herpes simplex virus type 1 ribonucleotide reductase large subunit is phosphorylated by casein kinase 2, which may have a homologue in Escherichia coli.

Studies were performed to determine if the unique N-terminal domain of the R1 subunit from herpes simplex virus (HSV) type 1 ribonucleotide reductase is a substrate for casein kinase 2 (CK2). Transphosphorylation assays demonstrated that R1 was highly phosphorylated by this enzyme with multiple phosphorylation sites mapped to the N terminus between residues 1 and 245. Immunoprecipitation pull-down assays using R1-specific antisera failed to demonstrate a stable interaction between R1 and CK2 but residual amounts of CK2 present after immunoprecipitation efficiently transphosphorylated R1. Activity assays with a peptide substrate identified CK2 in R1 immunoprecipitated from infected-cell extracts but did not detect activity in R1 proteins immunoprecipitated from bacterial extracts. However, Western blotting identified potential E. coli homologues of the CK2 alpha and beta subunits. These results support conclusions that the N-terminal domain of HSV R1 is not a protein kinase and that all previous results can be explained by contaminating kinases, principally CK2.

Autoradiography↗

PubMed1999

Monoclonal antibodies raised against Guillain-Barré syndrome-associated Campylobacter jejuni lipopolysaccharides react with neuronal gangliosides and paralyze muscle-nerve preparations.

Guillain-Barré syndrome and its variant, Miller-Fisher syndrome, are acute, postinfectious, autoimmune neuropathies that frequently follow Campylobacter jejuni enteritis. The pathogenesis is believed to involve molecular mimicry between sialylated epitopes on C. jejuni LPSs and neural gangliosides. More than 90% of Miller-Fisher syndrome cases have serum anti-GQ1b and anti-GT1a ganglioside antibodies that may also react with other disialylated gangliosides including GD3 and GD1b. Structural studies on LPS from neuropathy-associated C. jejuni strains have revealed GT1a-like and GD3-like core oligosaccharides. To determine whether this structural mimicry results in pathogenic autoantibodies, we immunized mice with GT1a/GD3-like C. jejuni LPS and then cloned mAb's that reacted with both the immunizing LPS and GQ1b/GT1a/GD3 gangliosides. Immunohistology demonstrated antibody binding to ganglioside-rich sites including motor nerve terminals. In ex vivo electrophysiological studies of nerve terminal function, application of antibodies either ex vivo or in vivo via passive immunization induced massive quantal release of acetylcholine, followed by neurotransmission block. This effect was complement-dependent and associated with extensive deposits of IgM and C3c at nerve terminals. These data provide strong support for the molecular mimicry hypothesis as a mechanism for the induction of cross-reactive pathogenic anti-ganglioside/LPS antibodies in postinfectious neuropathies.

Animals↗

PubMed1998

A novel nested reverse transcription PCR detects bovine viral diarrhoea virus in fluids from aborted bovine fetuses.

A nested reverse transcription-PCR (RT-PCR) was developed to detect pestivirus nucleic acid in fetal fluids and to study the number of bovine abortions associated with BVDV infection. Three techniques for the extraction of viral RNA from fetal fluids were compared; phenol:chloroform method, treatment with Catrimox-14 followed by guanidium isothiocyanate buffer and the Qiagen total RNA kit. The Qiagen kit was the most sensitive and reproducible and therefore adopted. After cDNA synthesis, initial amplification of a 288-base pair product using existing primers derived from the highly conserved 5'-untranslated region of the BVDV genome was achieved. Newly designed internal primers yielded a 171-base pair fragment which was visualised after electrophoresis on an ethidium bromide-stained gel. This assay detected 6.0 TCID50 of BVDV per 300 microl of artificially contaminated fetal fluid. One hundred fetal fluids were screened for the presence of BVDV RNA and the results compared with existing virus isolation methods. The BVDV antibody status of each fetus was determined. The nested RT-PCR detected BVDV RNA in eight of the hundred fetal fluids screened, whereas BVD virus was isolated from only one sample. The use of the nested RT-PCR will provide us with a more accurate picture of bovine embryonic infection due to BVDV.

Abortion, Veterinary↗

PubMed1998

Sp1 and related factors fail to interact with the NF-kappaB-proximal G/C box in the LTR of a replication competent, brain-derived strain of HIV-1 (YU-2).

The HIV-1 LTR promoter proximal G/C box array has been demonstrated to function by interacting with the Sp1 transcription factor family whose members can act as either activators or repressors of transcription. In this regard, we have examined the interaction of the HIV-1 Sp binding sites with nuclear factors that are present in cell types that support HIV-1 replication, including those of lymphocytic, monocytic, and astrocytic origin. As determined by electrophoretic mobility shift (EMS) competition analyses using oligonucleotides containing the sequences of each of the Sp1 sites of HIV-1 strain LAI, the NF-kappaB-proximal Sp site (site III) displayed the highest binding activity compared to sites I and II with regard to Sp1 and related factors present in lymphocytic (Jurkat) and astrocytic (U-373 MG) nuclear extracts. Sp1 and two Sp3 isoforms were detected as the primary cellular constituents of DNA-protein complexes formed with the NF-kappaB-proximal site. Only modest differences in Sp1:Sp3 binding ratios were observed when this site was reacted with either astrocytic or lymphocytic nuclear extract. However, when nuclear extracts derived from two monocytic cell types that differ in the degree of differentiation were reacted with the HIV-1 LAI Sp site III, a large difference in Sp1 and Sp3 binding was observed. To determine if naturally occurring and replication-competent strains of HIV-1 contain base pair alterations within the Sp elements that affect the ability of the site to interact with Sp1 and related factors, a series of Sp site III variants were constructed and examined by EMS analyses. One of these sites, obtained from the published sequence of the YU-2 strain (a brain-derived macrophage tropic strain of HIV-1), displayed almost no Sp1 or Sp3 binding activity as a result of a single base pair alteration in Sp site III. This base-pair alteration, when placed in the context of an HIV-1 LAI LTR-luciferase construct, resulted in a 40-50% reduction in LTR activity in transiently transfected Jurkat and U-373 MG cells. Overall, these results suggest that specific G/C box sequence alterations present in the brain-derived HIV-1 variant YU-2, or possibly other brain-derived variants, may exhibit altered replication properties as a result of the low affinity of the NF-kappaB-proximal G/C box for members of the Sp transcription factor family.

Astrocytes↗

PubMed1995

Intracellular localisation of herpes simplex virus type 1 ribonucleotide reductase subunits during infection of cultured cells.

We have analysed the intracellular localisation of herpes simplex virus type 1 ribonucleotide reductase during infection of cultured cells by indirect immunofluorescence using polyclonal and monoclonal antibodies specific for the R1 and R2 subunits. Three different viruses were used to infect cells, wild-type strain 17+ and two temperature-sensitive mutants, ts 1222, which produces R1 only, and ts 1207, which expresses a normal R2 and an altered R1 that fails to interact with R2 at the nonpermissive temperature because of an amino acid substitution in R1. R1 was detected 2 hr postinfection with all three viruses and remained evenly distributed throughout the cytoplasm. R2 was not observed until 4 hr postinfection and, in contrast to the even distribution of R1, was localised in discrete cytoplasmic foci close to the nucleus. In double-labelling experiments both R1 and R2 were found in these foci where they presumably associate to form the active enzyme. As expected R2 was not detectable in cells infected with ts 1222. In ts 1207-infected cells it formed wild-type-like foci, indicating that interaction with R1 is not required for R2 focus formation. R1 was present in a twofold excess over R2 in wild-type-infected cells. We suggest that the uncomplexed R1 could perform a role associated with the protein kinase present in the N-terminal domain.

Amino Acid Sequence↗

PubMed1995

Evolution in heterogeneous environments: genetic variability within and across different grains in Tribolium castaneum.

The course of adaptation to heterogeneous environments is influenced by the magnitude of genetic variation for ecologically important characters within each environment and the extent of genotype x environment interaction. Using the genetic correlation between the expression of characters in different environments as a measure of genotype x environment interaction is particularly useful for evolutionary interpretation. In this study, we estimated patterns of genetic variability and cross-environment genetic correlations for pupal weight and development time in two strains of the flour beetle Tribolium castaneum in five flours (wheat with brewer's yeast, wheat, rice, corn and oat). Wheat plus yeast is the standard medium in which the strains have been reared for hundreds of generations; other flours are novel environments. The results indicated moderate levels of genetic variation within the various flours for pupal weight but not for development time. Performance varied considerably across flours, with the highest performance for both strains found in the standard medium and the poorest in oat flour. The genetic correlations of pupal weight across flours in both strains were generally not significantly different from + 1. This suggests that evolution of body size in different flours cannot proceed independently, and that improved performance in the novel flours may produce declines in fitness in the standard environment.

Animals↗

PubMed1995

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.

Adenosine↗

PubMed1994

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

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

Amino Acid Sequence↗

PubMed1993

Herpes simplex virus type 1 ribonucleotide reductase large subunit: regions of the protein essential for subunit interaction and dimerization.

We have constructed a series of random N-terminal deletions of the large subunit (R1) of the herpes simplex virus type 1 ribonucleotide reductase. Deletions extended throughout the R1 gene open reading frame and, in total, 31 different truncated polypeptides were expressed in Escherichia coli using the T7 expression system. N-Terminal truncations were analyzed for their interaction with the small subunit (R2) of ribonucleotide reductase using a sensitive enzyme-linked immunosorbent assay (ELISA) method and for their ability to complement R2 in ribonucleotide reductase assays. Truncated proteins were also tested for homodimerization using gel-filtration chromatography. The results identified a region of R1 between amino acids 349 and 373 which was essential for subunit interaction. Proteins lacking up to 348 amino-terminal residues associated with R2 and complemented R2 in ribonucleotide reductase assays. Proteins commencing at amino acid 373 and beyond did not interact with R2 and were inactive in enzyme assays. Using a plasmid which expressed an N-terminal deleted protein commencing at amino acid 247, we constructed two defined C-terminal deletions to give proteins comprising amino acids 247-434 and 247-996 of R1. Neither of these truncated proteins bound R2 and we concluded that a second region between amino acids 996 and 1137 (the C-terminus) is required for interaction with R2. Gel-filtration studies indicated that deletion of the first 420 amino acids from R1 did not affect dimerization. However, deletions of 457 amino acids and larger gave proteins which existed as monomers.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗