A recombinant bovine herpesvirus 5 defective in thymidine kinase and glycoprotein E is attenuated and immunogenic for calves

Bovine herpesvirus 5 (BoHV-5) is an important pathogen of cattle in South America and efforts have been made to produce safer and more effective vaccines. In addition to afford protection, herpesvirus vaccines should allow serological differentiation of vaccinated from naturally, latently infected animals. We previously reported the construction and characterization in vitro of a double mutant BoHV-5 (BoHV-5gE/TK Δ) lacking the genes encoding thymidine kinase (tk) for attenuation, and glycoprotein E (gE) as the antigenic marker, as a vaccine candidate strain (Brum et al. 2010a). The present article reports an investigation on the attenuation and immunogenicity of this recombinant in calves. In a first experiment, 80 to 90-day-old seronegative calves (n=6) inoculated intranasally with the recombinant (titer of 10(7.5)TCID50) shed virus in low to moderate titers in nasal secretions for up to 6 days, yet did not develop any respiratory, systemic or neurological signs of infection. At day 30 post-infection (pi) all calves had BoHV-5 specific neutralizing (VN) antibodies in titers of 4 to 8 and were negative for anti-gE antibodies in a commercial ELISA test. Administration of dexamethasone (0.1mg/kg/day during 5 days) to four of these calves at day 42 pi did not result in virus shedding or increase in VN titers, indicating lack of viral reactivation. Secondly, a group of 8-month-old calves (n=9) vaccinated intramuscularly (IM) with the recombinant virus (10(7.5)TCID50/animal) did not shed virus in nasal secretions, remained healthy and developed VN titers from 2 to 8 at day 42 post-vaccination (pv), remaining negative for gE antibodies. Lastly, 21 calves (around 10 months old) maintained under field conditions were vaccinated IM with the recombinant virus (titer of 10(7.3)TCID50). All vaccinated animals developed VN titers from 2 to 16 at day 30 pv. A boost vaccination performed at day 240 pv resulted in a rapid and strong anamnestic antibody response, with VN titers reaching from 16 to 256 at day 14 post-booster. Again, serum samples remained negative for gE antibodies. Selected serum samples from vaccinated animals showed a broad VN activity against nine BoHV-5 and eight BoHV-1 field isolates. These results show that the recombinant virus is attenuated, immunogenic for calves and induces an antibody response differentiable from that induced by natural infection. Thus, the recombinant BoHV-5gE/TKΔ is an adequate candidate strain for a modified live vaccine.

A thymidine kinase-negative bovine herpesvirus 5 is highly attenuated for rabbits, but is neuroinvasive and establishes latent infection

Mutant viral strains deleted in non-essential genes represent useful tools to study the function of specific gene products in the biology of the virus. We herein describe an investigation on the phenotype of a bovine herpesvirus 5 (BoHV-5) recombinant deleted in the gene encoding the enzyme thymidine kinase (TK) in rabbits, with special emphasis to neuroinvasiveness and the ability to establish and reactivate latent infection. Rabbits inoculated with the parental virus (SV-507/99) (n=18) at a low titer (10(5.5)TCID50) shed virus in nasal secretions in titers up to 10(4.5)TCID50 for up to 12 days (average: 9.8 days [5-12]) and 5/ 16 developed neurological disease and were euthanized in extremis. Rabbits inoculated with the recombinant BoHV-5TKΔ at a high dose (10(7.1)TCID50) also shed virus in nasal secretions, yet to lower titers (maximum: 10(2.3)TCID50) and for a shorter period (average: 6.6 days [2-11]) and remained healthy. PCR examination of brain sections of inoculated rabbits at day 6 post-infection (pi) revealed a widespread distribution of the parental virus, whereas DNA of the recombinant BoHV-5TKΔ-was detected only in the trigeminal ganglia [TG] and olfactory bulbs [OB]. Nevertheless, during latent infection (52pi), DNA of the recombinant virus was detected in the TGs, OBs and also in other areas of the brain, demonstrating the ability of the virus to invade the brain. Dexamethasone (Dx) administration at day 65 pi was followed by virus reactivation and shedding by 5/8 rabbits inoculated with the parental strain (mean duration of 4.2 days [1 - 9]) and by none of seven rabbits inoculated with the recombinant virus. Again, PCR examination at day 30 post-Dx treatment revealed the presence of latent DNA in the TGs, OBs and in other areas of the brain of both groups. Taken together, these results confirm that the recombinant BoHV-5TKΔ is highly attenuated for rabbits. It shows a reduced ability to replicate in the nose but retains the ability to invade the brain and to establish latent infection. Additional studies are underway to determine the biological and molecular mechanisms underlying the inability of BoHV-5TKΔ to reactivate from latency.

A thymidine kinase-deleted bovine herpesvirus 5 establishes latent infection but reactivates poorly in a sheep model

The ability of thymidine kinase (tk)-deleted recombinant bovine herpesvirus 5 (BoHV-5tkΔ) to establish and reactivate latent infection was investigated in lambs. During acute infection, the recombinant virus replicated moderately in the nasal mucosa, yet to lower titers than the parental strain. At day 40 post-infection (pi), latent viral DNA was detected in trigeminal ganglia (TG) of all lambs in both groups. However, the amount of recombinant viral DNA in TGs was lower (9.7-fold less) than that of the parental virus as determined by quantitative real time PCR. Thus, tk deletion had no apparent effect on the frequency of latent infection but reduced colonization of TG. Upon dexamethasone (Dx) administration at day 40 pi, lambs inoculated with parental virus shed infectious virus in nasal secretions, contrasting with lack of infectivity in secretions of lambs inoculated with the recombinant virus. Nevertheless, some nasal swabs from the recombinant virus group were positive for viral DNA by PCR, indicating low levels of reactivation. Thus, BoHV-5 TK activity is not required for establishment of latency, but seems critical for efficient virus reactivation upon Dx treatment.

Mapping the sites of latency and reactivation by bovine herpesvirus 5 (BoHV-5) and a thymidine kinase-deleted BoHV-5 in lambs

A thymidine kinase (tk)-deleted bovine herpesvirus 5 (BoHV-5tkΔ) was previously shown to establish latent infection and reactivate - even poorly - in a sheep model (Cadore et al. 2013). As TK-negative alphaherpesviruses are unlike to reactivate in neural tissue, this study investigated the sites of latency and reactivation by this recombinant in lambs. For this, groups of lambs were inoculated intranasally with the parental BoHV-5 strain (SV-507/99) or with the recombinant BoHV-5tkΔ. During latent infection (40 days post-inoculation, pi), the distribution of recombinant virus DNA in neural and non-neural tissues was similar to that of the parental virus. Parental and recombinant virus DNA was consistently detected by PCR in trigeminal ganglia (TGs); frequently in palatine and pharyngeal tonsils and, less frequently in the retropharyngeal lymph nodes. In addition, latent DNA of both viruses was detected in several areas of the brain. After dexamethasone (Dx) administration (day 40pi), the recombinant virus was barely detected in nasal secretions contrasting with marked shedding of the parental virus. In tissues of lambs euthanized at day 3 post-Dx treatment (pDx), reverse-transcription-PCR (RT-PCR) for a late viral mRNA (glycoprotein D gene) demonstrated reactivation of parental virus in neural (TGs) and lymphoid tissues (tonsils, lymph node). In contrast, recombinant virus mRNA was detected only in lymphoid tissues. These results demonstrate that BoHV-5 and the recombinant BoHV-5tkΔ do establish latent infection in neural and non-neural sites. Reactivation of the recombinant BoHV-5tkΔ, however, appeared to occur only in non-neural sites. In anyway, the ability of a tk-deleted strain to reactivate latent infection deserves attention in the context of vaccine safety.

Cellular responses to stress and kinase signaling activation : apoptosis and differentiation

Stressignaler avkänns många gånger av membranbundna proteiner som översätter signalerna till kemisk modifiering av molekyler, ofta proteinkinaser Dessa kinaser överför de avkodade budskapen till specifika transkriptionsfaktorer genom en kaskad av sekventiella fosforyleringshändelser, transkriptionsfaktorerna aktiverar i sin tur de gener som behövs för att reagera på stressen. En av de mest kända måltavlorna för stressignaler är transkriptionsfaktor AP-1 familjemedlemen c-Jun. I denna studie har jag identifierat den nukleolära proteinet AATF som en ny regulator av c-Jun-medierad transkriptionsaktivitet. Jag visar att stresstimuli inducerar omlokalisering av AATF vilket i sin tur leder till aktivering av c-Jun. Den AATF-medierad ökningen av c-Jun-aktiviteten leder till en betydande ökning av programmerad celldöd. Parallellt har jag vidarekarakteriserat Cdk5/p35 signaleringskomplexet som tidigare har identifierats i vårt laboratorium som en viktig faktor för myoblastdifferentiering. Jag identifierade den atypiska PKCξ som en uppströms regulator av Cdk5/p35-komplexet och visar att klyvning och aktivering av Cdk5 regulatorn p35 är av fysiologisk betydelse för differentieringsprocessen och beroende av PKCξ aktivitet. Jag visar att vid induktion av differentiering fosforylerar PKCξ p35 vilket leder till calpain-medierad klyvning av p35 och därmed ökning av Cdk5-aktiviteten. Denna avhandling ökar förståelsen för de regulatoriska mekanismer som styr c-Jun-transkriptionsaktiviteten och c-Jun beroende apoptos genom att identifiera AATF som en viktig faktor. Dessutom ger detta arbete nya insikter om funktionen av Cdk5/p35-komplexet under myoblastdifferentiering och identifierar PKCξ som en uppströms regulator av Cdk5 aktivitet och myoblast differentiering.

Modification of pyruvate kinase and lactate dehydrogenase in foot muscle of the sea mussel Mytilus galloprovincialis under anaerobiosis and recovery

The modification of pyruvate kinase (PK) and lactate dehydrogenase (LDH) activity in foot muscle of the mussel Mytilus galloprovincialis during exposure to air and recovery in water was investigated. In the course of exposure to air, the activity of these enzymes measured at high and low substrate concentrations showed successive increases and decreases. Returning the mussels to water after exposure to air affected enzyme activity in a manner similar to anaerobiosis. When measuring at saturated concentrations of substrates and substrate and coenzyme for PK and LDH, respectively, the maximum activation of PK (37%) was observed at 4 h of animal exposure to air, and for LDH (67%) at 6 h exposure to air. During 24 h of exposure of animals to air, PK activity practically reached the stock level, while LDH was still activated (148%). The change in lactate dehydrogenase activity in mussel muscle during anoxia and recovery is described here for the first time. Variation in pyruvate kinase activity during exposure to air and recovery is linked to the alteration of half-maximal saturation constants and maximal velocity for both substrates. The possible role of reversible phosphorylation in the regulation of pyruvate kinase and lactate dehydrogenase properties is discussed

Involvement of hippocampal AMPA glutamate receptor changes and the cAMP/protein kinase A/CREB-P signalling pathway in memory consolidation of an avoidance task in rats

Training in step-down inhibitory avoidance (0.3-mA footshock) is followed by biochemical changes in rat hippocampus that strongly suggest an involvement of quantitative changes in glutamate AMPA receptors, followed by changes in the dopamine D1 receptor/cAMP/protein kinase A (PKA)/CREB-P signalling pathway in memory consolidation. AMPA binding to its receptor and levels of the AMPA receptor-specific subunit GluR1 increase in the hippocampus within the first 3 h after training (20-70%). Binding of the specific D1 receptor ligand, SCH23390, and cAMP levels increase within 3 or 6 h after training (30-100%). PKA activity and CREB-P levels show two peaks: a 35-40% increase 0 h after training, and a second increase 3-6 h later (35-60%). The results correlate with pharmacological findings showing an early post-training involvement of AMPA receptors, and a late involvement of the D1/cAMP/PKA/CREB-P pathway in memory consolidation of this task

Agents that affect cAMP levels or protein kinase A activity modulate memory consolidation when injected into rat hippocampus but not amygdala

Male Wistar rats were trained in one-trial step-down inhibitory avoidance using a 0.4-mA footshock. At various times after training (0, 1.5, 3, 6 and 9 h for the animals implanted into the CA1 region of the hippocampus; 0 and 3 h for those implanted into the amygdala), these animals received microinfusions of SKF38393 (7.5 µg/side), SCH23390 (0.5 µg/side), norepinephrine (0.3 µg/side), timolol (0.3 µg/side), 8-OH-DPAT (2.5 µg/side), NAN-190 (2.5 µg/side), forskolin (0.5 µg/side), KT5720 (0.5 µg/side) or 8-Br-cAMP (1.25 µg/side). Rats were tested for retention 24 h after training. When given into the hippocampus 0 h post-training, norepinephrine enhanced memory whereas KT5720 was amnestic. When given 1.5 h after training, all treatments were ineffective. When given 3 or 6 h post-training, 8-Br-cAMP, forskolin, SKF38393, norepinephrine and NAN-190 caused memory facilitation, while KT5720, SCH23390, timolol and 8-OH-DPAT caused retrograde amnesia. Again, at 9 h after training, all treatments were ineffective. When given into the amygdala, norepinephrine caused retrograde facilitation at 0 h after training. The other drugs infused into the amygdala did not cause any significant effect. These data suggest that in the hippocampus, but not in the amygdala, a cAMP/protein kinase A pathway is involved in memory consolidation at 3 and 6 h after training, which is regulated by D1, ß, and 5HT1A receptors. This correlates with data on increased post-training cAMP levels and a dual peak of protein kinase A activity and CREB-P levels (at 0 and 3-6 h) in rat hippocampus after training in this task. These results suggest that the hippocampus, but not the amygdala, is involved in long-term storage of step-down inhibitory avoidance in the rat.

Insulin receptor has tyrosine kinase activity toward Shc in rat liver

Insulin induces tyrosine phosphorylation of Shc in cell cultures and in insulin-sensitive tissues of the intact rat. However, the ability of insulin receptor (IR) tyrosine kinase to phosphorylate Shc has not been previously demonstrated. In the present study, we investigated insulin-induced IR tyrosine kinase activity towards Shc. Insulin receptor was immunoprecipitated from liver extracts, before and after a very low dose of insulin into the portal vein, and incubated with immunopurified Shc from liver of untreated rats. The kinase assay was performed in vitro in the presence of exogenous ATP and the phosphorylation level was quantified by immunoblotting with antiphosphotyrosine antibody. The results demonstrate that Shc interacted with insulin receptor after infusion of insulin, and, more important, there was insulin receptor kinase activity towards immunopurified Shc. The description of this pathway in animal tissue may have an important role in insulin receptor tyrosine kinase activity toward mitogenic transduction pathways.

Perturbation of EGF-induced MAP kinase activation by TGF-ß1

TGF-ß1 regulates both cellular growth and phenotypic plasticity important for maintaining a growth advantage and increased invasiveness in progressively malignant cells. Recent studies indicate that TGF-ß-1 stimulates the conversion of epitheliod to fibroblastoid phenotype which presumably leads to the inactivation of growth-inhibitory effects by TGF-ß1 (Portella et al. (1998) Cell Growth and Differentiation, 9: 393-404). Therefore, the investigation of TGF-ß1 signaling that leads to altered growth and migration may provide novel targets for the prevention of increased cell growth and invasion. Although much attention has been paid to TGF-ß1 responses in epithelial cells, the above studies suggest that examination of signal transduction pathways in fibroblasts are important as well. Data from our laboratory are consistent with the concept that TGF-ß1 can act as a regulatory switch in density-dependent C3H 10T1/2 fibroblasts capable of either promoting or delaying G1 traverse. The regulation of this switch is proposed to occur prior to pRb phosphorylation, namely prior to activation of cyclin-dependent kinases. The current study is concerned with the evaluation of a key cyclin (cyclin D1) which activates cdk4 and p27KIP1 which in turn inhibit cdk2 in the proliferative responses of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) and their modulation by TGF-ß1. Although the molecular events that lead to elevation of cyclin D1 are not completely understood, it appears likely that activation of p42/p44MAPK kinases is involved in its transcriptional regulation. TGF-ß1 delayed EGF- or PDGF-induced cyclin D1 expression and blocked the induction of active p42/p44MAPK. The mechanism by which TGF-ß1 induces a block in p42/p44MAPK activation is being examined and the possibility that TGF-ß1 regulates phosphatase activity is being tested.

Identification of a protein kinase activity that phosphorylates connexin43 in a pH-dependent manner

The carboxyl-terminal (CT) domain of connexin43 (Cx43) has been implicated in both hormonal and pH-dependent gating of the gap junction channel. An in vitro assay was utilized to determine whether the acidification of cell extracts results in the activation of a protein kinase that can phosphorylate the CT domain. A glutathione S-transferase (GST)-fusion protein was bound to Sephadex beads and used as a target for protein kinase phosphorylation. A protein extract produced from sheep heart was allowed to bind to the fusion protein-coated beads. The bound proteins were washed and then incubated with 32P-ATP. Phosphorylation was assessed after the proteins were resolved by SDS-PAGE. Incubation at pH 7.5 resulted in a minimal amount of phosphorylation while incubation at pH 6.5 resulted in significant phosphorylation reaction. Maximal activity was achieved when both the binding and kinase reactions were performed at pH 6.5. The protein kinase activity was stronger when the incubations were performed with manganese rather than magnesium. Mutants of Cx43 which lack the serines between amino acids 364-374 could not be phosphorylated in the in vitro kinase reaction, indicating that this is a likely target of this reaction. These results indicate that there is a protein kinase activity in cells that becomes more active at lower pH and can phosphorylate Cx43.

Effects of lipopolysaccharide on low- and high-density cultured rabbit vascular smooth muscle cells: differential modulation of nitric oxide release, ERK1/ERK2 MAP kinase activity, protein tyrosine phosphatase activity, and DNA synthesis

Previous studies have shown that exogenously generated nitric oxide (NO) inhibits smooth muscle cell proliferation. In the present study, we stimulated rabbit vascular smooth muscle cells (RVSMC) with E. coli lipopolysaccharide (LPS), a known inducer of NO synthase transcription, and established a connection between endogenous NO, phosphorylation/dephosphorylation-mediated signaling pathways, and DNA synthesis. Non-confluent RVSMC were cultured with 0, 5, 10, or 100 ng/ml of the endotoxin. NO release was increased by 86.6% (maximum effect) in low-density cell cultures stimulated with 10 ng/ml LPS as compared to non-stimulated controls. Conversely, LPS (5 to 100 ng/ml) did not lead to enhanced NO production in multilayered (high density) RVSMC. DNA synthesis measured by thymidine incorporation showed that LPS was mitogenic only to non-confluent RVSMC; furthermore, the effect was prevented statistically by aminoguanidine (AG), a potent inhibitor of the inducible NO synthase, and oxyhemoglobin, an NO scavenger. Finally, there was a cell density-dependent LPS effect on protein tyrosine phosphatase (PTP) and ERK1/ERK2 mitogen-activated protein (MAP) kinase activities. Short-term transient stimulation of ERK1/ERK2 MAP kinases was maximal at 12 min in non-confluent RVSMC and was prevented by preincubation with AG, whereas PTP activities were inhibited in these cells after 24-h LPS stimulation. Conversely, no significant LPS-mediated changes in kinase or phosphatase activities were observed in high-density cells. LPS-induced NO generation by RVSMC may switch on a cell density-dependent proliferative signaling cascade, which involves the participation of PTP and the ERK1/ERK2 MAP kinases.

Interruption of the blood-stage cycle of the malaria parasite, Plasmodium chabaudi, by protein tyrosine kinase inhibitors

Malaria is a devastating disease caused by a unicellular protozoan, Plasmodium, which affects 3.7 million people every year. Resistance of the parasite to classical treatments such as chloroquine requires the development of new drugs. To gain insight into the mechanisms that control Plasmodium cell cycle, we have examined the effects of kinase inhibitors on the blood-stage cycle of the rodent malaria parasite, Plasmodium chabaudi. In vitro incubation of red blood cells for 17 h at 37ºC with the inhibitors led to a decrease in the percent of infected cells, compared to control treatment, as follows: genistein (200 µM - 75%), staurosporine (1 µM - 58%), R03 (1 µM - 75%), and tyrphostins B44 (100 µM - 66%) and B46 (100 µM - 68%). All these treatments were shown to retard or prevent maturation of the intraerythrocytic parasites. The diverse concentration ranges at which these inhibitors exert their effects give a clue as to the types of signals that initiate the transitions between the different developmental stages of the parasite. The present data support our hypothesis that the maturation of the intraerythrocytic cycle of malaria parasites requires phosphorylation. In this respect, we have recently reported a high Ca2+ microenvironment surrounding the parasite within red blood cells. Several kinase activities are modulated by Ca2+. The molecular identification of the targets of these kinases could provide new strategies against malaria.

The role of calcium, calcium-activated K+ channels, and tyrosine/kinase in psoralen-evoked responses in human melanoma cells

8-Methoxy psoralen (8-MOP) exerts a short-term (24 h) mitogenic action, and a long-term (48-72 h) anti-proliferative and melanogenic action on two human melanoma cell lines, SK-Mel 28 and C32TG. An increase of intracellular calcium concentration was observed by spectrofluorometry immediately after the addition of 0.1 mM 8-MOP to both cell lines, previously incubated with calcium probe fluo-3 AM (5 µM). The intracellular Ca2+ chelator BAPTA/AM (1 µM) blocked both early (mitogenic) and late (anti-proliferative and melanogenic) 8-MOP effects on both cell lines, thus revealing the importance of the calcium signal in both short- and long-term 8-MOP-evoked responses. Long-term biological assays with 5 and 10 mM tetraethylammonium chloride (TEA, an inhibitor of Ca2+-dependent K+ channels) did not affect the responses to psoralen; however, in 24-h assays 10 mM TEA blocked the proliferative peak, indicating a modulation of Ca2+-dependent K+ channels by 8-MOP. No alteration of cAMP basal levels or forskolin-stimulated cAMP levels was promoted by 8-MOP in SK-Mel 28 cells, as determined by radioimmunoassay. However, in C32TG cells forskolin-stimulated cAMP levels were further increased in the presence of 8-MOP. In addition, assays with 1 µM protein kinase C and calcium/calmodulin-dependent kinase inhibitors, Ro 31-8220 and KN-93, respectively, excluded the participation of these kinases in the responses evoked by 8-MOP. Western blot with antibodies anti-phosphotyrosine indicated a 92% increase of the phosphorylated state of a 43-kDa band, suggesting that the phosphorylation of this protein is a component of the cascade that leads to the increase of tyrosinase activity.

Pim-1 kinase inhibits the activation of reporter gene expression in Elk-1 and c-Fos reporting systems but not the endogenous gene expression: an artifact of the reporter gene assay by transient co-transfection

We have studied the molecular mechanism and signal transduction of pim-1, an oncogene encoding a serine-threonine kinase. This is a true oncogene which prolongs survival and inhibits apoptosis of hematopoietic cells. In order to determine whether the effects of Pim-1 occur by regulation of the mitogen-activated protein kinase pathway, we used a transcriptional reporter assay by transient co-transfection as a screening method. In this study, we found that Pim-1 inhibited the Elk-1 and NFkappaB transcriptional activities induced by activation of the mitogen-activated protein kinase cascade in reporter gene assays. However, Western blots showed that the induction of Elk-1-regulated expression of endogenous c-Fos was not affected by Pim-1. The phosphorylation and activation of neither Erk1/2 nor Elk-1 was influenced by Pim-1. Also, in the gel shift assay, the pattern of endogenous NFkappaB binding to its probe was not changed in any manner by Pim-1. These data indicate that Pim-1 does not regulate the activation of Erk1/2, Elk-1 or NFkappaB. These contrasting results suggest a pitfall of the transient co-transfection reporter assay in analyzing the regulation of transcription factors outside of the chromosome context. It ensures that results from reporter gene expression assay should be verified by study of endogenous gene expression.