Cysteinyl leukotrienes C4 and D4 downregulate human mast cell expression of toll-like receptors 1 through 7.

Cysteinyl leukotrienes (CysLT) are potent inflammatory lipid molecules that mediate some of the pathophysiological responses associated with asthma such as bronchoconstriction, vasodilation and increased microvascular permeability. As a result, CysLT receptor antagonists (LRA), such as montelukast, have been used to effectively treat patients with asthma. We have recently shown that mast cells are necessary modulators of innate immune responses to bacterial infection and an important component of this innate immune response may involve the production of CysLT. However, the effect of LRA on innate immune receptors, particularly on allergic effector cells, is unknown. This study determined the effect of CysLT on toll-like receptor (TLR) expression by the human mast cell line LAD2. Real-time PCR analysis determined that LTC4, LTD4 and LTE4 downregulated mRNA expression of several TLR. Specifically in human CD34+-derived human mast cells (HuMC), LTC4 inhibited expression of TLR1, 2, 4, 5, 6 and 7 while LTD4 inhibited expression of TLR1-7. Montelukast blocked LTC4-mediated downregulation of all TLR, suggesting that these effects were mediated by activation of the CysLT1 receptor (CysLT1R). Flow cytometry analysis confirmed that LTC4 downregulated surface expression of TLR2 which was blocked by montelukast. These data show that CysLT can modulate human mast cell expression of TLR and that montelukast may be beneficial for innate immune responses mediated by mast cells.

A novel eremophilane lactone inhibits FcεRI-dependent release of pro-inflammatory mediators: structure-dependent bioactivity.

BACKGROUND: Allergic inflammation is primarily mediated by immune effector cells such as mast cells and basophils that release proinflammatory cytokines. Both mast cells and basophils are activated via their high affinity IgE receptor (FcεRI) which initiates the release of proinflammatory mediators such as histamine and tumor necrosis factor (TNF). Considerable effort has been focused on finding an effective basophil stabilizer that inhibits the activation of FcεRI-activated mediator release. Recently, eremophilane lactones, a novel family of sesquiterpene compound originally isolated from Petasites japonicas (Sieb. et Zucc.), have been described, and it has been postulated that they may have anti-inflammatory activity, particularly in allergic disease. OBJECTIVE: Our objective was to determine the effect of two eremophilane lactones derived from 6ß-angeloyloxy-3ß,8-dihydroxyeremophil-7(11)-en-12,8ß-olide (F-1) on immunoglobulin E (IgE)-dependent release of pro-inflammatory mediators by a basophil cell line, RBL-2H3, a model system for FcεRI-mediated activation of pro-inflammatory mediator release. METHODS: The parent compound (F-1) was chemically modified to produce F-1a [6ß-angeloyloxy-3ß-benzoyloxy-8-hydroxyeremophil-7(11)-en-12,8ß-olide] and F-1b [6ß-angeloyloxy-3ß,8-diacetoxyeremophil-7(11)-en-12,8ß-olide]. RBL-2H3 cells were sensitized with DNP-specific IgE and then activated with DNP-BSA. The effect of these compounds on IgE-dependent basophil degranulation was assessed by measuring the release of ß-hexosaminidase (b-hex). In addition, TNF release was measured via ELISA. RESULTS: The phenylacetyl reaction modified C-8 and produced F-1a whereas acetylation of F-1 produced F-1b. F-1a was not cytotoxic to RBL-2H3 cells even at 50 µM, but F-1b was slightly cytotoxic at 50 µM, reducing viability of the cells by approximately 15 %. Neither F-1a nor F-1b inhibited FcεRI-dependent activation of RBL-2H3 cells when the cells were pretreated for only 30 min with the compounds. However, 24 h pretreatment with F-1a inhibited antigen-dependent degranulation by as much as 60 % and TNF production by as much as 90 %. F-1b had no effect on RBL-2H3 activation via FcεRI. CONCLUSIONS: These results indicate that F-1a inhibits degranulation of RBL-2H3 cells activated via the high affinity IgE receptor, FcεRI, and that this effect is dependent upon hydroxylation of the third carbon.

Pleurocidin, a novel antimicrobial peptide, induces human mast cell activation through the FPRL1 receptor.

Pleurocidins are a novel family of α-helical cationic antimicrobial peptides (CAPs) that are structurally and functionally similar to cathelicidins, one of the major CAP families. As cathelicidins stimulate mast cell chemotaxis and mediator release, we postulated that pleurocidins similarly activate mast cells. A screen of 20 pleurocidin peptides revealed that some were capable of degranulating the human mast cell line LAD2 (Laboratory of Allergic Diseases 2). Pleurocidin NRC-04 caused LAD2 to adhere, migrate, degranulate, and release cysteinyl leukotrienes and prostaglandin D2. Moreover, pleurocidin increased intracellular Ca(2+) mobilization in mast cells and induced the production of proinflammatory chemokines such as monocyte chemotactic protein-1/C-C motif chemokine ligand 2 (CCL2) and macrophage inflammatory protein-1ß/CCL4. Our evaluation of possible cellular mechanisms suggested that G proteins, phosphoinositol-3 kinase (PI3K), phospholipase C (PLC), and phosphokinase C (PKC) were involved in pleurocidin-induced mast cell activation as evidenced by the inhibitory effects of pertussis toxin (G protein inhibitor), wortmanin (PI3K inhibitor), U-73122 (PLC inhibitor), and Ro-31-8220 (PKC inhibitor), respectively. We also found that human mast cells expressed the N-formyl-peptide receptor 1 (FPRL1) receptor and FPRL1-specific inhibitor affected pleurocidin-mediated activation of mast cell. Our finding that the novel CAP pleurocidin activated human mast cell through G protein-coupled receptor signaling suggests that this peptide might have immunomodulatory functions.

Mineralization of the connective tissue: a complex molecular process leading to age-related loss of function.

Age-related metastatic mineralization of soft tissues has been considered a passive and spontaneous process. Recent data have demonstrated that calcium salt deposition in soft tissues could be a highly regulated process. Although calcification occurs in any tissue type, vascular calcification has been of particular interest due to association with atherosclerosis, chronic kidney disease (CKD), and osteoporosis. Different mechanisms underlying calcium apatite accumulation are explored with these age-related disorders. In the case of atherosclerotic plaques, oxy-lipids trigger release of the pro-inflammatory cytokines and inflammation that activate calcification processes in aorta intimae. In CKD patients, renal failure alters the balance between calcium and phosphate levels usually regulated by fibroblast growth factor-23 (FGF23), Klotho, and vitamin D, and vascular smooth muscle cells (VSMCs) begin to explore an osteoblastosteoblast-like phenotype. Calcification could affect extracellular matrix along with VSMCs. Collagen is a major component of extracellular matrix and its modifications accumulate with age. The formation of cross-links between collagen fibers is regulated by the action of lysine hydroxylases and lysyl oxidase and could occur spontaneously. Oxidation-induced advanced glycation end products (AGEs) are a major type of spontaneous cross-links that accelerate with age and may result in tissue stiffness, problems with recycling, and potential accumulation of calcium apatite. Applying strategies for clearing the AGEs proposed by de Grey may be more difficult in the highly mineralized extracellular matrix. We performed bioinformatic analysis of the molecular pathways underlying calcification in atherosclerotic and CKD patients, signaling pathways of collagen cross-links formation, and bone mineralization, and we propose new potential targets and review drugs for calcification treatment.

The cytopathic 18f strain of Hepatitis A virus induces RNA degradation in FrhK4 cells.

The mechanism responsible for the induction of apoptosis by the rapidly replicating HM175/18f strain of Hepatitis A virus (HAV) was investigated. Full length HAV RNA and viral capsid protein VP1 were detected in 18f infected cells at earlier times post-infection than in HM175/clone 1 infected cells. Analysis of total cellular RNA from HM175/18f infected FrhK4 cells by denaturing agarose gel electrophoresis and Northern blot hybridization revealed extensive degradation of both the 28S and 18S ribosomal RNA (rRNA) molecules. Similar degradation was observed when these cells were infected with Human coxsackievirus B1, a fast replicating enterovirus. In contrast, the parental strain of 18f, HM175/clone 1 did not induce RNA degradation. Inhibition of RNA degradation correlated with inhibition of virus replication. The pattern of rRNA degradation resembled degradation of rRNAs by RNase L, an enzyme activated in interferon-treated cells following infection with certain viruses. Ribosomal RNA degradation was accompanied by the reduction in the levels of several cellular RNAs including those for beta-actin and glyceraldehyde-3-phosphate dehydrogenase, while the levels of c-myc and c-jun were higher. Interferon mRNAs could not be detected in either infected or mock-infected control cells, and STAT1, a key regulator of interferon action was not phosphorylated following virus infection. These results reveal a heretofore-undescribed pathway that involves the regulation of RNA degradation and apoptosis following HAV/18f replication in FrhK4 cells.

Characteristics of nasal T/NK-cell lymphoma among Brazilians.

Nasal T/NK-cell lymphomas are highly associated with Epstein-Barr virus (EBV). They are more frequent in Asia than in Western countries. In Central and South America there are few studies about nasal T/NK-cell lymphoma and they have shown a strong predominance of this phenotype in Native American descents, supporting the hypothesis of a racial predisposition for the disease. We studied the lymphomas involving midline facial region at a Brazilian institution. T/NK cell lymphomas (16/25) were more frequently found compared to B lymphomas (9 cases, all B large cell). T/NK cell lymphomas involved predominantly the nasal region. Histologically they showed angioinvasion and necrosis. All of them were positive for CD3 and CD56 and showed numerous tumor cells labeled by EBER-1. Although disease was localized in 61% at diagnosis, there was no tendency to cure. The racial distribution of patients with T/NK-cell phenotype was similar to that found in B-cell lymphomas. EBV was more frequently found in adenoids than in palatine tonsils. In inflammatory lesions of the nasal and palatal regions EBV was not found. In the present study the relative frequency of T/NK versus B cell sinonasal lymphomas was high and similar to that observed in other Latin American countries. However, there was not any racial association with T/NK-cell phenotype and the tumor showed an agressive behavior similar to that reported in Asia. The high frequency of EBV-positive lymphocytes in nasopharyngeal lymphoid tissue (adenoids) suggests that they could serve as a reservoir for the virus.

Expression and functional characterization of CFTR in mast cells.

Mast cell activation requires Cl(-) flux, which maintains the driving force for entry of extracellular calcium and initiates release of mediators such as histamine. However, chloride channel expression in mast cells has been poorly understood. For the first time, reverse transcriptase-polymerase chain reaction shows that rat-cultured mast cells (RCMC) and peritoneal mast cells (PMC) contain mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR), an important chloride channel. Immunostaining with an anti-CFTR antibody indicates expression of CFTR in PMC and RCMC. Mast cell CFTR is a functional Cl(-) channel because it is capable of mediating Cl(-) flux in response to elevated cAMP. An inhibitor of CFTR-dependent Cl(-) flux, diphenylamine-2-carboxylate down-regulates mast cell mediator release. These results show that rat mast cells express a functional CFTR, which might be important in mediator release.

Modified adenovirus penton base protein (UTARVE) as a non-replicating vector for delivery of antisense oligonucleotides with antiviral and/or antineoplastic activity.

Antisense oligonucleotides that selectively inhibit gene expression are a genetic approach for disease treatment and prevention. However, their use as therapeutic agents is complicated by their low rate of transport across cellular membranes and their sequestration within endocytic-like vesicles. We report that the adenovirus type-2 penton base protein modified to include the fusogenic peptide of the influenza virus hemagglutinin protein is a non-replicating vector (designated UTARVE) that improves delivery of antisense oligonucleotides. Approximately 10-18% of the input vector was internalized by A549 and HeLa cells as determined by immunoblotting. It was cleared by proteolysis within 48 h. The vector had endosome disruptive potential as evidenced by erythrocyte lysis activity at low pH and a primarily diffuse cytoplasmic distribution in treated cells. Despite concentration and time-dependent cell detachment, UTARVE was not cytotoxic in the dye release assay. We used R1T1, an antisense oligonucleotide that inhibits expression of the multifunctional herpes simplex virus type-2 (HSV-2) R1 protein, HSV-2 growth and the proliferation of R1 PK transformed cells to examine vector-mediated delivery. Conjugated FITC-labeled R1T1 was rapidly (15-30 min) internalized by all cells treated at low (80 nM) concentration and the oligomer was intracellularly dissociated from the vector. This compares to 65-83% of cells internalizing the unconjugated R1T1 when treated for 24 h. In antiviral assays, the IC50 and time required to inhibit HSV-2 growth were significantly lower for the conjugated (2 nM; 30 min) as compared to unconjugated (100 nM; 24 h) R1T1. The data indicate that the bioavailability and biological activity of R1T1 were significantly increased by its delivery with UTARVE.

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.

ATP and SH3 binding sites in the protein kinase of the large subunit of herpes simplex virus type 2 of ribonucleotide reductase (ICP10).

The large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is a multifunctional protein. It consists of a ribonucleotide reductase and a serine/threonine protein kinase (PK) domain, which has three proline-rich motifs consistent with SH3-binding sites at positions 140, 149, and 396. We used site-directed mutagenesis to identify amino acids required for kinase activity and interaction with signaling proteins. Mutation of Lys176 or Lys259 reduced PK activity (5-8-fold) and binding of the 14C-labeled ATP analog rho-fluorosulfonylbenzoyl 5'-adenosine (FSBA) but did not abrogate them. Enzymatic activity and FSBA binding were abrogated by mutation of both Lys residues, suggesting that either one can bind ATP. Mutation of Glu209 (PK catalytic motif III) virtually abrogated kinase activity in the presence of Mg2+ or Mn2+ ions, suggesting that Glu209 functions in ion-dependent PK activity. ICP10 bound the adaptor protein Grb2 in vitro. Mutation of the ICP10 proline-rich motifs at positions 396 and 149 reduced Grb2 binding 20- and 2-fold, respectively. Binding was abrogated by mutation of both motifs. Grb2 binding to wild type ICP10 was competed by a peptide for the Grb2 C-terminal SH3 motif, indicating that it involves the Grb2 C-terminal SH3.

Herpes simplex virus-mediated activation of human immunodeficiency virus is inhibited by oligonucleoside methylphosphonates that target immediate-early mRNAs 1 and 3.

IE1 and IE3 mRNAs and their protein products (IE110 and IE175, respectively) were detected in HSV-1-infected U937 cells at 4-15 hours postinfection. In transient expression assays with infectious HIV or an HIV-LTR-directed chloramphenicol acetyltransferase construction (HIV-LTRcat), HSV-1 caused HIV activation (86.7% +/- 6.4% conversion). Electrophoretic mobility shift assays with DNA sequences that encompass the LBP-1 binding site revealed increased levels of DNA-protein complex formation with nuclear extracts from HSV-1 infected as compared with uninfected U937 cells. Novel bands were not seen. HSV-1 mutants respectively deleted in IE110 (dl1403) or IE175 (d120) activated HIV as well as wild-type virus. However, HSV-1-mediated activation was inhibited (26% conversion) by simultaneous treatment with oligonucleoside methylphosphonates (ONMP) that specifically inhibit expression of IE110 (IE1TI) or IE175 (IE3TI). ONMP did not inhibit activation when used individually (83.8% and 67.8% conversion with IETI1 and IE3TI, respectively). Combinations of mutant ONMP that do not inhibit IE110 or IE175 expression did not reduce the levels of HSV-1-mediated activation. These findings suggest that HSV genes IE1 and IE3 can independently activate HIV in monocytic cells and ONMP that target HSV IE genes can be used to inhibit HIV activation.

Intracellular internalization and signaling pathways triggered by the large subunit of HSV-2 ribonucleotide reductase (ICP10).

The large subunit of the HSV-2 ribonucleotide reductase (RR) (ICP10) is a chimera consisting of a serine threonine (Ser/Thr) protein kinase domain at the amino terminus and the RR domain at the carboxy terminus. Transformed human cells that constitutively express ICP10 (JHLa1) were stained with anti-LA-1 antibody (recognizes ICP10 amino acids 13-26) and immunogold-conjugated goat anti-rabbit IgG and were examined by electron microscopy. ICP10-associated gold particles were observed on the cell surface and in structures with ultrastructural characteristics of endocytic vesicles, multivesicular bodies, and lysosomes, consistent with endocytic internalization. ICP10 was also associated with the cytoskeleton fraction of JHLa1 cells and, at least in part, it colocalized with actin filaments. This was evidenced by immunoprecipitation of [35S]methionine-labeled cell fractions and immunofluorescent staining of Triton-treated cells with anti-LA-1 antibody and phalloidin. Endocytic localization of gold particles was not seen in cells that constitutively express the ICP10 transmembrane (TM)-deleted mutant p139TM (JHL15). p139TM did not associate with the cytoskeleton and was almost entirely localized within the cytoplasm. raf and Erk evidenced decreased mobility consistent with an activated state in JHLa1, but not JHL15, cells, and chloramphenicol acetyl transferase (CAT) expression from a c-fos/cat hybrid construct was significantly increased in JHLa1 but not JHL15 cells. The data indicate that effector molecules downstream of ras are activated in JHLa1 cells and the ICP10 TM segment plays a critical role in ICP10 intracellular localization and its ability to activate signaling pathways. This behavior is analogous to that of an activated growth factor receptor kinase.

Antiviral activity of an oligo(nucleoside methylphosphonate) that targets HSV-1 immediate-early pre-mRNA 4,5 is augmented by cotreatment with replication-defective adenovirus.

Replication-defective adenovirus p259A caused a 400-fold increase in the sequence-specific antiherpetic activity of oligo(nucleoside methylphosphonate) (ONMP) IE4,5SA. Herpes simplex virus type 1 (HSV-1) growth was not inhibited in cells exposed to p259A in the absence of IE4,5SA or in cells cotreated with IE4,5SA and heated (10 minutes, 90 degrees C) p259A virus. Fluorescent microscopy of Vero cells treated with BODIPY-conjugated IE4,5SA revealed intracellular localization within endocytic-like vesicles with minimal cytoplasmic and intranuclear distribution. Diffuse staining over the entire cell was observed in cell cotreated with the BODIPY-conjugated IE4,5SA and p259A virus. This effect was not observed in cells cotreated with the BODIPY-conjugated ONMP and heated p259A virus. We interpret these findings to indicate that p259A augments IE4,5SA antiherpetic activity presumably via its ability to increase ONMP uptake and release from endocytic-like vesicles.

Cutaneous ultraviolet radiation inhibits herpes simplex virus-induced lymphoproliferation in latently infected subjects.

Exposure of herpes simplex virus (HSV) latently infected subjects to ultraviolet irradiation (UVR) (1 minimum erythema dose, 90% body surface) caused a significant inhibition of HSV and phytohemagglutinin-induced lymphoproliferation. The inhibition was observed on Day 3 post-UVR and lasted at least 9 days. UVR-induced downregulation of HSV-specific lymphoproliferation was associated with increased levels of activated transforming growth factor beta. However, the relationship between UVR-induced immune downregulation and the development of recurrent HSV lesions was incomplete.

The GGTCA palindrome and cognate cellular factors in trans-regulation of human immunodeficiency virus long terminal repeat by herpes simplex virus.

Molecular interactions between herpes simplex virus type 1 (HSV-1) and human immunodeficiency virus (HIV) were investigated in the promonocytic cell line U937. HSV-1-mediated activation was observed in transient expression assays with hybrid constructions containing the HIV long terminal repeat (LTR)-directed chloramphenicol acetyltransferase gene. Comparison of constructions that differ in the GGTCA palindrome located within the negative regulatory region of the LTR revealed four- to eightfold lower activation levels for the wild-type as compared to the mutant sequence. Three protein species, 37K, 59K/64K and 75K, that bind to the wild-type GGTCA palindrome were resolved in nuclear extracts of uninfected U937 cells by gel retardation and u.v.-crosslinking experiments. The 37K protein did not bind to the mutant palindrome sequence. However, a distinct 120K protein was detected. The 37K and 59K/64K binding proteins were not resolved in similar experiments performed with nuclear extracts from HSV-1-infected U937 cells but there was a novel p50 species that binds only to the wild-type palindrome sequence. These findings raise the possibility that interaction of these proteins at the GGTCA palindrome is involved in HSV-1-mediated regulation of the HIV LTR in U937 cells.

NF-kappa B-binding proteins induced by HSV-1 infection of U937 cells are not involved in activation of human immunodeficiency virus.

Molecular interactions between the herpes simplex virus type 1 (HSV-1) and human immunodeficiency virus (HIV) were investigated in U937 cells. Nonpermissive HSV-1 infection of U937 cells activated HIV as determined in transient expression assays with hybrid constructions containing the HIV-LTR-directed chloramphenicol acetyltransferase gene. Activation was independent of kappa B-enhancer elements whereas these elements were required for HSV-1-mediated activation in another cell line (C6). kappa B-binding proteins were induced in U937 cells by HSV-1 infection. Four species (45, 55, 75, and 75/80 kDa) were identified by DNA-protein cross-linking. Methylation interference analysis defined close contact only with the third residue of the previously established critical contact triplet GGG. Transient expression assays using mutants in HIV-LTR revealed the presence a cis-response element (GGTCA palindrome) in the negative regulatory region.

Expression of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10) is required for virus growth and neoplastic transformation.

The amino-terminal domain of the large subunit of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (ICP10) has protein kinase (PK) activity and properties similar to those of growth factor receptor kinases which can be activated to transforming potential. DNA sequences that encode the PK domain cause neoplastic transformation of immortalized cells. The studies described in this report used a spontaneous mutant (ts5-152) temperature-sensitive for the synthesis of ICP10 and the previously described ICP10 expression vectors to study the role of ICP10 expression in HSV-2 growth and neoplastic potential. The titres of the ts5-152 mutant are 1000-fold lower at 39 degrees C compared to 34 degrees C after 12 h post-infection. The efficiency of plaquing is 0.003. The growth defect at 39 degrees C correlates with decreased ICP10 synthesis. Sequence analysis of the PK domain of the ts5-152 ICP10 gene identified a pair of frameshift mutations resulting in a 19 amino acid residue substitution at positions 275 to 293 and a downstream single base pair mutation causing a substitution at position 309. Cloning of the mutant ICP10 gene from ts5-152 into a wild-type HSV-2 isolate resulted in a recombinant (859/152) with growth properties and rates of ICP10 synthesis at 39 degrees C similar to those of ts5-152. Cells transformed with u.v.-inactivated ts5-152, or the recombinant 859/152, have significantly decreased cloning efficiency in agarose at 39 degrees C, but only during the first 250 post-transfer population doublings. Anchorage-independent growth was observed in cells transfected with expression vectors pJW17 or pJW32 that express ICP10 or its PK domain, respectively. Cells transfected with the frameshift mutant pJW21 or the ICP10 carboxy-terminal vector pJW31 did not form clones in agarose.

Immediate early and functional AP-1 cis-response elements are involved in the transcriptional regulation of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10).

Expression from the promoter of the herpes simplex virus type 2 (HSV-2) large subunit of ribonucleotide reductase (ICP10) is stimulated by co-transfection with DNA that encodes the virion protein Vmw65 previously shown to activate in trans the transcription of all IE genes (Wymer et al., 1989). Specific cis response elements involved in ICP10 transcriptional regulation were studied by chloramphenicol acetyltransferase analysis with hybrid ICP10 promoter/CAT structural gene constructions containing wild type or site-directed mutations of the promoter sequences. The data indicate that Vmw65 activation requires an intact TAAT-GARAT motif while complex formation requires an intact Oct-1 element, and the AP-1 consensus elements in the ICP10 promoter are functional in vitro. Thus, expression from the wild type and GA-rich mutant constructions was enhanced 10-20-fold by co-transfection with DNA encoding Vmw65. The GARAT and POU homeobox (PHB) binding motifs were required for Vmw65 mediated activation but the mutant in the POU specific box (PSB) binding motif was activated at higher concentrations of Vmw65 DNA (1.0-3.0 micrograms). The PHB and PSB binding motifs were necessary for complex formation as determined by gel retardation analysis with in vitro synthesized OTF-1 and Vmw65 proteins. The GARAT and GA-rich elements were not required. CAT expression from pICP10-cat was enhanced by co-transfection with jun and fos encoding DNA, and the ICP10 promoter complexed with in vitro synthesized jun protein.

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.

Myristylation and polylysine-mediated activation of the protein kinase domain of the large subunit of herpes simplex virus type 2 ribonucleotide reductase (ICP10).

The amino-terminal domain of the large subunit of herpes simplex virus type 2 (HSV-2) ribonucleotide reductase (ICP10) was previously shown to possess protein kinase (PK) activity that localizes to the cytosolic, cytoskeletal, and plasma membrane fractions. Further studies of the PK domain using computer-assisted sequence analysis have identified a single transmembrane segment and fatty acid incorporation findings indicate that ICP10 is myristylated. Myristylation does not depend on a viral enzyme, since myristic acid is incorporated into ICP10 precipitated from cells transfected with an ICP10 expression vector. It is also incorporated into the 57-kDa protein expressed by the amino-terminal PK expression vector. The myristyl moiety is linked through an amide bond. The basic protein polylysine stimulates the kinase activity and alters its divalent cation requirements resulting in 20- to 40-fold stimulation in the presence of 0.1 mM Mn2+. The PK activity is inhibited by antibody to synthetic peptides corresponding to residues 355-369 and 13-26, respectively, located within, and amino-terminal to, the predicted PK catalytic domain.