Early Protein Synthesis Research Articles

Protein Synthesis Is Required for Synaptic Immunity to Depotentiation

De novo protein synthesis and transcription are necessary for the expression of long-lasting synaptic potentiation [long-term potentiation (LTP)] in hippocampal area CA1 and for the consolidation of long-term memory. The stability of LTP and its longevity require macromolecular synthesis at later stages, but a specific role for early protein synthesis has not been identified. Using electrophysiological recording methods in mouse hippocampal slices, we show that multiple trains of high-frequency stimulation provide immediate synaptic immunity to depotentiation. This immunity to depotentiation is dependent on the amount of synaptic stimulation used to induce LTP, it is input specific, and it is prevented by inhibitors of protein synthesis. We propose that local translation mediates input-specific synaptic immunity against depotentiation. We also present evidence suggesting that, in addition to translation, products of transcription can provide cell-wide immunity to depotentiation via heterosynaptic transfer of synaptic immunity between distinct pathways in area CA1. Protein synthesis and transcription may importantly regulate long-term storage of information by conferring synaptic immunity to depotentiation at previously potentiated synapses.

Regulation of proteolysis during reloading of the unweighted soleus muscle

There is little information on the mechanisms responsible for muscle recovery following a catabolic condition. To address this point, we reloaded unweighted animals and investigated protein turnover during recovery from this highly catabolic state and the role of proteolysis in the reorganization of the soleus muscle. During early recovery (18 h of reloading) both muscle protein synthesis and breakdown were elevated (+65%, P<0.001 and +22%, P<0.05, respectively). However, only the activation of non-lysosomal and Ca 2+-independent proteolysis was responsible for increased protein breakdown. Accordingly, mRNA levels for ubiquitin and 20S proteasome subunits C8 and C9 were markedly elevated (from +89 to +325%, P<0.03) and actively transcribed as shown by the analysis of polyribosomal profiles. In contrast, both cathepsin D and 14-kDa-ubiquitin conjugating enzyme E2 mRNA levels decreased, suggesting that the expression of such genes is an early marker of reversed muscle wasting. Following 7 days of reloading, protein synthesis was still elevated and there was no detectable change in protein breakdown rates. Accordingly, mRNA levels for all the proteolytic components tested were back to control values even though an accumulation of high molecular weight ubiquitin conjugates was still detectable. This suggests that soleus muscle remodeling was still going on. Taken together, our observations suggest that enhanced protein synthesis and breakdown are both necessary to recover from muscle atrophy and result in catch-up growth. The observed non-coordinate regulation of proteolytic systems is presumably required to target specific classes of substrates (atrophy-specific protein isoforms, damaged proteins) for replacement and/or elimination.

Azathioprine Inhibits Vaccinia Virus Replication in Both BSC-40 and Rag Cell Lines Acting on Different Stages of Virus Cycle

In the present study we demonstrate that azathioprine (AZA) inhibits vaccinia virus (VV) replication in both BSC-40 and RAG cell lines, acting on different stages of virus cycle. In BSC-40 cells, early protein synthesis was not significantly affected, but late gene expression was severely impaired. In RAG cells all stages of gene expression were completed during synchronous infection in the presence of the drug. The onset of DNA replication was not affected in RAG cells, but a severe inhibition was observed in BSC-40 cells. Electron microscopic analysis of VV-infected RAG cells treated with AZA revealed brick-shaped particles presenting abnormal definition of the internal structure. Purified virions from AZA-treated RAG cells presented several modifications of the protein content, a lesser amount of DNA, and a lower PFU:particle ratio. Our results suggest that in VV-infected RAG cells AZA interfered with virus morphogenesis, whereas in BSC-40 cells the replicative cycle was inhibited at the DNA replication stage.

The use of linezolid in the treatment of vancomycin-resistant enterococcal septicaemia in two patients with burn injuries

Vancomycin-resistant enterococci (VRE) are multi-resistant micro-organisms that have emerged as important nosocomial pathogens during the last decade. Emergence of this organism has been blamed mainly on the increased and inappropriate use of antibiotics, in particular, the cephalosporins and the glycopeptide, vancomycin. Burns patients are highly vulnerable to acquiring VRE infections, being both debilitated and immunocompromised, and often receiving antibiotics that further diminish their intrinsic microbial flora. We report on two patients with large burn injuries who acquired vancomycin-resistant enterococcal septicaemia during their in-patient stay. Both patients were successfully treated using the antibiotic, linezolid. Linezolid is the first in a new class of antibiotics known as the oxazolidinones whose mode of action inhibits early bacterial protein synthesis. Linezolid has a spectrum of activity against Gram-positive micro-organisms including methicillin-resistant Staphylococcus aureus (MRSA) and VRE, and can provide a useful treatment alternative to the glycopeptides.

Effects of PKR/RNase L-dependent and alternative antiviral pathways on alphavirus replication and pathogenesis.

Type I interferons (IFN-alpha/beta) rapidly confer resistance to alphavirus infection in macrophages and dendritic cells (DC) as evidenced by the dramatically increased susceptibility of these cells in mice with the IFNAR1 subunit of the IFN-alpha/beta receptor ablated (IFNAR1-/-). Normal adult mice develop only a subclinical Sindbis virus infection, whereas infected IFNAR1-/- mice rapidly succumb to a fatal disease. Here, we investigated the individual and combined contributions of the two best characterized INF-alpha/beta-mediated antiviral pathways to the control of Sindbis virus replication: (1) the coupled 2-5A synthetase/RNase L pathway and (2) the double-stranded RNA-dependent protein kinase (PKR) pathway. Surprisingly, mice deficient in PKR, RNase L, and Mx-1 (triply-deficient [TD]) developed only subclinical infection. Although the permissivity of cells in lymph nodes draining the inoculation site was increased in the absence of PKR/RNase L, systemic dissemination of the virus infection was restricted by an alternative IFN-alpha/beta receptor-dependent mechanism. In vitro, suppression of early virus protein synthesis and virion production in primary bone marrow-derived dendritic cells (BMDC) was largely dependent on the PKR pathway. However, later in infection virion production was reduced even in the absence of PKR/RNase L by an IFN-alpha/beta receptor-dependent mechanism. Priming of BMDC with IFN-alpha/beta or IFN-gamma resulted in dose-dependent restriction of virus replication, largely independent of PKR and/or RNase L expression.

Abundant early expression of gpUL4 from a human cytomegalovirus mutant lacking a repressive upstream open reading frame.

The human cytomegalovirus UL4 gene encodes a 48-kDa glycoprotein, expression of which is repressed at the translational level by a short upstream open reading frame (uORF2) within the UL4 transcript leader. Mutation of the uORF2 initiation codon in the viral genome eliminates ribosomal stalling at the uORF2 termination site, resulting in early and abundant gpUL4 protein synthesis. This mutation does not appear to affect viral replication kinetics in human fibroblasts. These results reveal that the unusual uORF2 inhibitory mechanism is a principal determinant of the abundance and timing of gpUL4 expression but is nonessential for replication in cell culture.

Activation of IκB Kinase by Herpes Simplex Virus Type 1: A NOVEL TARGET FOR ANTI-HERPETIC THERAPY

Herpes simplex viruses (HSV) are ubiquitous pathogens causing a variety of diseases ranging from mild illness to severe life-threatening infections. HSV utilize cellular signaling pathways and transcription factors to promote their replication. Here we report that HSV type 1 (HSV-1) induces persistent activation of transcription factor NF-kappa B, a critical regulator of genes involved in inflammation, by activating the I kappa B kinase (IKK) in the early phase of infection. Activated NF-kappa B enhances HSV-1 gene expression. HSV-1-induced NF-kappa B activation is dependent on viral early protein synthesis and is not blocked by the anti-herpetic drug acyclovir. IKK inhibition by the anti-inflammatory cyclopentenone prostaglandin A(1) blocks HSV-1 gene expression and reduces virus yield by more than 3000-fold. The results identify IKK as a potential target for anti-herpetic drugs and suggest that cyclopentenone prostaglandins or their derivatives could be used in the treatment of HSV infection.

Vaccinia virus A30L protein is required for association of viral membranes with dense viroplasm to form immature virions.

The previously uncharacterized A30L gene of vaccinia virus has orthologs in all vertebrate poxviruses but no recognizable nonpoxvirus homologs or functional motifs. We determined that the A30L gene was regulated by a late promoter and encoded a protein of approximately 9 kDa. Immunoelectron microscopy of infected cells indicated that the A30L protein was associated with viroplasm enclosed by crescent and immature virion membranes. The A30L protein was also present in mature virions and was partially released by treatment with a nonionic detergent and reducing agent, consistent with a location in the matrix between the core and envelope. To determine the role of the A30L protein, we constructed a stringent conditional lethal mutant with an inducible A30L gene. In the absence of inducer, synthesis of viral early and late proteins occurred but the proteolytic processing of certain core proteins was inhibited, suggesting an assembly block. Inhibition of virus maturation was confirmed by electron microscopy. Under nonpermissive conditions, we observed aberrant large, dense, granular masses of viroplasm with clearly defined margins; viral crescent membranes that appeared normal except for their location at a distance from viroplasm; empty immature virions; and an absence of mature virions. The data indicated that the A30L protein is needed for vaccinia virus morphogenesis, specifically the association of the dense viroplasm with viral membranes.

Phosphorylation of the herpes simplex virus type 1 origin binding protein.

The herpes simplex virus type 1 (HSV-1) origin binding protein (OBP), the product of the UL9 gene, is one of seven HSV-encoded proteins required for viral DNA replication. OBP performs multiple functions characteristic of a DNA replication initiator protein, including origin-specific DNA binding and ATPase and helicase activities, as well as the ability to interact with viral and cellular proteins involved in DNA replication. Replication initiator proteins in other systems, including those of other DNA viruses, are known to be regulated by phosphorylation; however, the role of phosphorylation in OBP function has been difficult to assess due to the low level of OBP expression in HSV-infected cells. Using a metabolic labeling and immunoprecipitation approach, we obtained evidence that OBP is phosphorylated during HSV-1 infection. Kinetic analysis of metabolically labeled cells indicated that the levels of OBP expression and phosphorylation increased at approximately 4 h postinfection. Notably, when expressed from a transfected plasmid, a recombinant baculovirus, or a recombinant adenovirus (AdOBP), OBP was phosphorylated minimally, if at all. In contrast, superinfection of AdOBP-infected cells with an OBP-null mutant virus increased the level of OBP phosphorylation approximately threefold, suggesting that HSV-encoded viral or HSV-induced cellular factors enhance the level of OBP phosphorylation. Using HSV mutants inhibited at sequential stages of the viral life cycle, we demonstrated that this increase in OBP phosphorylation is dependent on early protein synthesis and is independent of viral DNA replication. Based on gel mobility shift assays, phosphorylation does not appear to affect the ability of OBP to bind to the HSV origins.

Role of protein phosphorylation in the inhibition of protein synthesis caused by hypoxia in rat hepatocytes.

Hypoxia causes a rapid and reversible inhibition of translation in freshly isolated rat hepatocytes. This inhibition is neither due to an ATP loss nor to an increase in cell death. Because protein synthesis is mainly regulated by reversible phosphorylation of initiation and/or elongation factors, we investigated whether translation inhibition by hypoxia may be related to changes in the phosphorylation status of proteins. Whatever the incubation conditions, three phosphoreactive bands (molecular weights 220, 129, and 83 kDa) were detected by antiphosphotyrosine antibodies. The phosphorylation in the 129- and 83-kDa bands, however, was significantly and progressively decreased under hypoxia. Although this time-dependent decrease was sensitive to changes in oxygen tension, it occurred after the early protein synthesis inhibition caused by hypoxia. Moreover, sodium orthovanadate prevented tyrosine dephosphorylation in hypoxic cells, but did not restore the depressed protein synthesis caused by hypoxia. Under aerobic conditions, orthovanadate inhibited the synthesis of proteins, confirming that protein phosphorylation is a major mechanism involved in translational regulation. Once again, this inhibitory effect occurred only after 90 minutes of incubation whereas hypoxia inhibits the protein synthesis at the beginning of the incubation. Labeling cells with [33-32P]-ortho-phosphoric acid allowed detection of several phosphorylated proteins that appeared under hypoxia. Because they were not recognized by the phosphotyrosine antibodies, we suggest that serine/threonine residues of key proteins may be the putative hypoxic targets.

Signal transduction pathways that contribute to increased protein synthesis during T-cell activation

Protein synthesis rates were maximally stimulated in human lymphocytes by ionomycin and the phorbol ester PMA (I+P), which promotes proliferation, whereas PMA alone, which does not promote proliferation, stimulated protein synthesis to a lesser degree. Three translation-associated activities, eIF4E phosphorylation, eIF2B activity and 4E-BP1 phosphorylation also increased with stimulation by I+P and PMA, but only 4E-BP1 phosphorylation was differentially stimulated by these conditions. Correspondingly, signaling pathways activated in T cells were probed for their connection to these activities. Immunosuppressants FK506 and rapamycin partially blocked the protein synthesis rate increases by I+P stimulation. FK506 had less of an inhibitory effect with PMA stimulation suggesting that its mechanism mostly affected ionomycin-activated signals. I+P and PMA equally stimulated phosphorylation of ERK1/2, but I+P more strongly stimulated Akt, and p70 S6K phosphorylation. An inhibitor that blocks ERK1/2 phosphorylation only slightly reduced protein synthesis rates stimulated by I+P or PMA, but greatly reduced eIF4E phosphorylation and eIF2B activity. In contrast, inhibitors of the PI-3 kinase and mTOR pathways strongly blocked early protein synthesis rate stimulated by I+P and PMA and also blocked 4E-BP1 phosphorylation and release of eIF4E suggesting that these pathways regulate protein synthesis activities, which are important for proliferation in T cells.

Free DNA induces modification on the protein synthesis profile of human peripheral blood mononuclear cells of healthy donors.

Understanding how free DNA might act as a signal between cells is important for knowing how DNA orchestrates immune responses and for optimizing the therapeutic of cancer, infection and immunologic diseases. This communication demonstrates that DNAs from different origins (bacteria, T. cruzi, HeLa cells) and synthetic oligonucleotide containing an unmethylated CpG motif are capable of inducing alterations in the protein profile of normal human leukocytes. As far as we know there have been no similar studies regarding the comparative effects of different free DNAs on early protein synthesis of human peripheral blood mononuclear cells.

Characterization of vaccinia virus intracellular cores: implications for viral uncoating and core structure.

The entry of vaccinia virus (VV) into the host cell results in the delivery of the double-stranded DNA genome-containing core into the cytoplasm. The core is disassembled, releasing the viral DNA in order to initiate VV cytoplasmic transcription and DNA replication. Core disassembly can be prevented using the VV early transcription inhibitor actinomycin D (actD), since early VV protein synthesis is required for core uncoating. In this study, VV intracellular cores were accumulated in the presence of actD and isolated from infected cells. The content of these cores was analyzed by negative staining EM and by Western blotting using a collection of antibodies to VV core and membrane proteins. By Western blot analyses, intracellular actD cores, as well as cores prepared by NP-40-dithiothreitol treatment of purified virions (NP-40/DTT cores), contained the core proteins p25 (encoded by L4R), 4a (A10L), 4b (A3L), and p39 (A4L) as well as small amounts of the VV membrane proteins p32 (D8L) and p35 (H3L). While NP-40/DTT cores contained the major putative DNA-binding protein p11 (F17R), actD cores entirely lacked this protein. Labeled cryosections of cells infected for different periods of time in the presence or absence of actD were subsequently used to follow the fate of VV core proteins by EM. These EM images confirmed that p11 was lost at the plasma membrane upon core penetration. The cores that accumulated in the presence of actD were labeled with antibodies to 4a, p39, p25, and DNA at all times examined. In the absence of the drug the cores gradually lost their electron-dense inner part, concomitant with the loss of p25 and DNA labeling. The remaining core shell still labeled with antibodies to p39 and 4a/4b, implying that these proteins are part of this structure. These combined data are discussed with respect to the structure of VV as well as core disassembly.

Endoribonuclease RegB from bacteriophage T4 is necessary for the degradation of early but not middle or late mRNAs

The RegB endoribonuclease from bacteriophage T4 cleaves early mRNAs specifically in the middle of the sequence GGAG. We show here that RegB is required for the degradation of bulk T4 early mRNA. In the absence of RegB, the chemical half-life of early transcripts is increased nearly fourfold, whereas their functional half-life is increased twofold. RegB also regulates the translation of several prereplicative genes. The synthesis of several early proteins is down-regulated, probably as a consequence of RegB cleavages in the Shine-Dalgarno sequence of these genes. The synthesis of several other proteins is up-regulated, suggesting that processing by RegB might improve translation by changing the conformation of a transcript. In contrast, RegB does not affect the average half-life of middle and late mRNA. An analysis of the susceptibility to RegB of many GGAG motifs carried by these mRNA species showed that most middle and all late GGAG-carrying mRNAs escape RegB processing in spite of the fact that the enzyme is acting at least until ten minutes post-infection. The sensitivity or resistance to RegB observed during phage infection could be reproduced in uninfected Escherichia coli cells and in vitro. This shows that the GGAG-carrying RNAs that are uncut during T4 infection are not substrates, whatever the period of the T4 cycle when the transcripts are made.

Role of interferon alpha (IFN-alpha) and interferon gamma (IFN-gamma) in the control of the infection of monocyte-like cells with human cytomegalovirus (HCMV).

The role of cytokines in the control of HCMV infection has been studied in THP-1 cells, a macrophage-like cell model and in MRC-5 cells. HCMV replication was studied by immune detection of viral immediate-early antigens (IEA) and virus yield was evaluated in MRC-5 cells by immunoperoxidase staining. Pretreatment of MRC-5 and phorbol 12-myristate 13-acetate (PMA)-treated THP-1 cells with IFN-alpha or IFN-gamma for 24 hr prior to the infection reduced the number of infected cells and virus yield. A synergistic anti-CMV activity in synthesis of early proteins was obtained with these cytokines in combination with TNF-alpha in differentiated THP-1 cells only. Treatment of HCMV-infected differentiated THP-1 cells or MRC-5 cells with IFN-alpha or IFN-gamma alone had no inhibitory effect on virus replication, however the virus yield was reduced with ganciclovir. A synergistic anti-CMV activity in virus yield was obtained only when infected differentiated THP-1 cells were treated with ganciclovir in combination with IFN-gamma. The current study shows that IFN-alpha and IFN-gamma can play a role in the reduction of HCMV replication in macrophage-like cells and in the efficiency of therapies with ganciclovir in this cell type and that the anti-CMV effect of cytokines may be different in fibroblasts and in macrophage-like cells.

Transcriptional control of development, protein synthesis, and heat-induced heat shock protein 70 synthesis in 2-cell bovine embryos.

Experiments were performed to evaluate the role of transcription in early development of bovine embryos. Two transcription inhibitors-5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) and actinomycin D-were used to test whether 1) the inhibitors alter the rate of early embryonic development and protein synthesis, 2) heat shock increases the steady-state amounts of mRNA for the inducible form of heat shock protein 70 (HSP70) in embryos, and 3) this latter effect is blocked by transcription inhibitors. Addition of either DRB or actinomycin D to culture medium beginning 8 h postinsemination (hpi) reduced the proportion of oocytes that had undergone cleavage by 32-34 hpi. Both transcription inhibitors also reduced the proportion of cleaved embryos that reached the 4-cell stage by 32-34 hpi. Incorporation of (35)S-labeled amino acids into de novo synthesized protein by bovine 2-cell embryos was lower for embryos cultured with DRB. Using reverse transcription-polymerase chain reaction, HSP70 mRNA in 2- and 4-cell embryos was increased by exposure to 42 degrees C. Both inhibitors reduced amounts of HSP70 mRNA at 42 degrees C. Results indicate that bovine embryos can undergo transcription in response to heat shock as early as the 2-cell stage. Moreover, the observations that transcription inhibitors reduce rates of cleavage and early development point out the importance of transcription for development from the earliest period of embryonic life.

A non-cytotoxic herpes simplex virus vector which expresses Cre recombinase directs efficient site specific recombination

The coding sequences for the bacteriophage P1 recombinase Cre were cloned into the genome of a herpes simplex virus type 1 (HSV-1) mutant which is severely impaired for the synthesis of immediate early (IE) proteins. The resulting recombinant, virus in1372, expressed functional Cre which mediated the excision in trans of loxP-flanked sequences located in the HSV-1 genome, both in tissue culture cells and in vivo in mouse sensory neurons. Infection with in1372 also resulted in recombination, at high efficiency, between loxP sequences in the cellular genome without causing detectable cytotoxicity. Mutant in1372 is a versatile vector for the delivery of Cre in tissue culture and in vivo.

Cocaine Potentiates the Switch between Latency and Replication of Epstein–Barr Virus in Raji Cells

This paper shows that cocaine amplifies Epstein–Barr virus (EBV) reactivation in Raji cells. Its effect on early viral protein synthesis was maximal when it was added with 12-O-tetradecanoyl phorbol-13-acetate (TPA) plus n-butyrate, but nil when added alone. The enhancing effect of cocaine on early replicative stages of latent EBV was associated with an increase of Ca2+ mobilization induced by the drug and with an induction of cellular protein phosphorylation in chemicals and cocaine-treated Raji cells. Cocaine also acted synergistically with TPA and n-butyrate to induce Z Epstein–Barr replication activator (ZEBRA), a nuclear phosphoprotein responsible for the activation of early viral gene expression. These findings provide the first evidence that cocaine may represent an important co-factor in the reactivation of early stages of latent EBV infection.

Antiherpetic activity and mode of action of natural carrageenans of diverse structural types

The λ-carrageenan 1T1, the κ/ ι-carrageenan 1C1 and the μ/ν-type 1C3, isolated from the red seaweed Gigartina skottsbergii, proved to be potent and selective inhibitors of herpes simplex virus (HSV) types 1 and 2. The antiviral IC 50 values determined by virus yield inhibition assay in different cell lines ranged from 0.4 to 3.3 μg/ml, and no cytotoxic effects, measured by trypan blue exclusion on stationary or proliferating cells, tetrazolium salt method or cell protein synthesis, were observed. Time of addition and attachment studies suggested that the main target for antiviral action of the three carrageenans was virus adsorption, whereas no effect on virus internalization, or early or late protein synthesis was detected. However, the λ-carrageenan 1T1 was still significantly inhibitory when added any time after adsorption. The pretreatment of virions with the carrageenans showed that 1C1 and 1C3 lacked direct inactivating effect at concentrations near the antiviral IC 50 but 1T1 exerted virucidal action. The cyclization of 1T1 to afford the derivative 1T1T1 maintained the antiviral activity but eliminated the virucidal properties. Thus, the structure of 1T1 seems to be responsible for its differential behavior from 1C1 and 1C3, probably allowing a more stable binding to HSV, leading to virion inactivation. In contrast, 1C1 and 1C3 fail to bind with high affinity to virus alone, but are able to interfere with the interaction between HSV particles and the cell.

Differential control of xanthophylls and light-induced stress proteins, as opposed to light-harvesting chlorophyll a/b proteins, during photosynthetic acclimation of barley leaves to light irradiance

Barley (Hordeum vulgare L.) plants were grown at different photon flux densities ranging from 100 to 1800 &mgr;mol m-2 s-1 in air and/or in atmospheres with reduced levels of O2 and CO2. Low O2 and CO2 partial pressures allowed plants to grow under high photosystem II (PSII) excitation pressure, estimated in vivo by chlorophyll fluorescence measurements, at moderate photon flux densities. The xanthophyll-cycle pigments, the early light-inducible proteins, and their mRNA accumulated with increasing PSII excitation pressure irrespective of the way high excitation pressure was obtained (high-light irradiance or decreased CO2 and O2 availability). These findings indicate that the reduction state of electron transport chain components could be involved in light sensing for the regulation of nuclear-encoded chloroplast gene expression. In contrast, no correlation was found between the reduction state of PSII and various indicators of the PSII light-harvesting system, such as the chlorophyll a-to-b ratio, the abundance of the major pigment-protein complex of PSII (LHCII), the mRNA level of LHCII, the light-saturation curve of O2 evolution, and the induced chlorophyll-fluorescence rise. We conclude that the chlorophyll antenna size of PSII is not governed by the redox state of PSII in higher plants and, consequently, regulation of early light-inducible protein synthesis is different from that of LHCII.