63,123 research outputs found
Molecular and immunogenic analysis of Jembrana disease virus Tat
No full textJembrana disease is an acute and severe disease of Bali cattle (Bos javanicus) endemic in Indonesia that is caused by a bovine lentivirus designated Jembrana disease virus (JDV). Previous studies have demonstrated that it is possible to induce a protective immunity against the disease by immunisation with a crude whole virus vaccine prepared from the tissues of infected cattle. This vaccine has been demonstrated to ameliorate the clinical signs of disease resulting from exposure to virus infection but a safer vaccine amenable to commercial production techniques is required.
JDV, like all lentiviruses, encodes a transcriptional trans-activator Tat protein that is encoded from one or both of two exons of the tat gene. Tat is particularly essential for virus replication and it was hypothesised that the induction of an immune response in cattle against JDV Tat may effect protection against virus infection. Investigations were therefore conducted on JDV Tat to provide basic information on the protein that would enable it to be further investigated as a potential immunogen for incorporation into vaccines for the control of Jembrana disease.
Analysis of tat transcripts obtained from tissues of cattle infected with three strains of JDV suggested that, during the acute clinical disease, Tat produced at this stage of the disease process was translated from the first coding exon only. Nucleotide variation in this exon, which would have translated into amino acid variations in the Tat protein, was evident especially between strains from geographically different regions of Indonesia. There was; however, conservation of the essential functional domains of cysteine-rich, core and basic regions, which suggested immunity to a single Tat protein might protect against infection by heterologous strains. Subsequent studies on Tat reported in the thesis therefore concentrated on the protein encoded by tat exon 1 of a single strain of JDV. The exon 1 of tat was cloned into the pGEX vector and recombinant Tat expressed in Escherichia coli. Methods for the purification of the expressed protein were developed. Immunogenicity of the recombinant protein was initially demonstrated by inoculation of the protein into a sheep which developed a high titred specific antibody response. Antibodies induced by this recombinant protein recognised native Tat proteins produced by three JDV strains in Bali cattle and provided a valuable reagent for the subsequent detection of Tat in vitro and in vivo.
Aspects of the antibody response to Tat were determined in cattle that had been infected naturally or experimentally with JDV, and compared with the levels of antibody to the immunodominant capsid protein. Tat antibodies were detected in 23 % of 128 Bali cattle from Jembrana disease-endemic areas of Indonesia; in all these cattle, evidence of previous virus infection had been demonstrated by detection of antibody to the JDV capsid protein by Western blot analysis. In cattle experimentally infected with JDV, low levels of serum antibody to Tat were detected by Western blot in the first month post-infection but the levels of antibody then decreased; levels of antibody to the JDV capsid protein increased over the 6-month observation period following infection. The detection of Tatantibody soon after the acute clinical disease suggested that this protein is secreted extracellularly during JDV infection in cattle. In contrast to the antibody response to Tat in JDV-infected cattle, an apparently greater antibody response to Tat was induced by injection of recombinant Tat in Bali cattle. The strong antibody response resulting from inoculation of the recombinant Tat and low levels of Tat antibody in animals that had been naturally or experimentally infected with virus suggested there might be a conformational difference in the recombinant and native Tat protein and that the native protein was a poor immunogen, or that the levels of Tat in infected cattle were too low to induce a strong antibody response. As an alternative means of inducing an immune response to JDV Tat, perhaps one associated with a greater cell-mediated rather than an antibody response, a candidate tat DNA vaccine was produced by insertion of tat exon 1 into a DNA vaccine vector. Transfection of this naked DNA plasmid into mammalian cells induced the expression of a functional Tat protein which maintained antigenicity. The results suggested this construct merits further animal studies attempting to induce a protective immune response against Jembrana disease in cattle. A method of assaying the trans-acting function of Tat was also developed which will have application for quality control procedures for large-scale production of tat DNA vaccine
Transient-mediated fate determination in a transcriptional circuit of HIV
No full textSteady-state behavior and bistability have been proposed as mechanisms for decision-making in gene circuits. However, transient gene expression has also been proposed to control cell fate with the decision arbitrated by the lifetime of the expression transient. Here, we report that transcriptional positive-feedback plays a critical role in determining HIV infected cell-fate by extending the duration of Tat expression transients far beyond what protein half-life modulation can achieve. To directly quantify feedback strength and its effects on the duration of Tat transcriptional pulses, we exploit the noise inherent to gene-expression and measure shifts in the autocorrelation of expression noise. The results indicate that transcriptional positive-feedback extends the single-cell Tat expression lifetime by ~6-fold for both minimal Tat circuits and full-length, actively-replicating HIV-1. Importantly, artificial weakening of Tat positive-feedback shortened the duration of Tat expression transients and biased the probability in favor of latency. Thus, transcriptional positive-feedback appears to modulate transient expression lifetime and thereby control cell-fate in HIV
Interaction of HIV-1 Tat protein with heparin. Role of the backbone structure, sulfation, and size.
No full textHuman immunodeficiency virus type 1 (HIV-1) Tat protein is released from infected cells. Extracellular Tat enters the cell where it stimulates the transcriptional activity of HIV-long terminal repeat (LTR) and of endogenous genes. Heparin modulates the angiogenic (Albini, A., Benelli, R., Presta, M., Rusnati, M., Ziche, M., Rubartelli, A., Paglialunga, G., Bussolino, F., and Noonan, D. (1996) Oncogene 12, 289-297) and transcriptional (Mann, D. A., and Frankel, A. D. (1991) EMBO J. 10, 1733-1739) activity of extracellular Tat. Here we demonstrate that heparin binds specifically to recombinant HIV-1 Tat produced as glutathione S-transferase (GST) fusion protein and immobilized on glutathione-agarose beads. Heparin and heparan sulfate (HS), but not dermatan sulfate, chondroitin sulfates A and C, hyaluronic acid, and K5 polysaccharide, competed with 3H-labeled heparin for binding to immobilized GST-Tat and inhibited HIV-LTR transactivation induced by extracellular GST-Tat. Selective 2-O-, 6-O-, total-O-desulfation, or N-desulfation/N-acetylation dramatically reduced the capacity of heparin to bind GST-Tat. Totally-O-desulfated and 2-O-desulfated heparins also showed a reduced capacity to inhibit the transactivating activity of GST-Tat. Very low molecular weight heparins showed a significant decrease in their capacity to bind GST-Tat and to inhibit its LTR transactivating activity when compared with conventional 13.6-kDa heparin. However, when 3.0-kDa heparin was affinity chromatographed on immobilized GST-Tat to isolate binding and non-binding subfractions, the Tat-bound fraction was >/=1,000 times more potent than the unbound fraction in inhibiting the transactivating activity of GST-Tat. The results demonstrate that Tat interacts in a size-dependent manner with heparin/HS and that high affinity Tat-heparin interaction requires at least some 2-O-, 6-O-, and N-positions to be sulfated. The Tat binding activity of the glycosaminoglycans tested correlates with their capacity to affect the transactivating activity of extracellular Tat, indicating the possibility to design specific heparin/HS-like structures with Tat-antagonist activity
TAT
No full textHistograms representing the proportion of time a tag spent in 12 temperature bins define in 2C increments between 4C and 24C TAT histograms were returned by Argos satellites from SPOT5 tags produced by Wildlife Computers. TAT were collected in 6-hour sampling periods, and sampling periods were programmed to begin at 01:00, 07:00, 13:00, or 21:00 local time, so that the majority (>80%) of sampling of each TAT histogram fell within either daytime or nighttime. TAT histograms that were translated into units of depth using the hydrographic data and interpolation methods detailed in Joyce et al. (2016)
Environmental salinity determines the specificity and need for tat-dependent secretion of the YwbN protein in bacillus subtilis
No full textTwin-arginine protein translocation (Tat) pathways are required for transport of folded proteins across bacterial, archaeal and chloroplast membranes. Recent studies indicate that Tat has evolved into a mainstream pathway for protein secretion in certain halophilic archaea, which thrive in highly saline environments. Here, we investigated the effects of environmental salinity on Tat-dependent protein secretion by the Gram-positive soil bacterium Bacillus subtilis, which encounters widely differing salt concentrations in its natural habitats. The results show that environmental salinity determines the specificity and need for Tat-dependent secretion of the Dyp-type peroxidase YwbN in B. subtilis. Under high salinity growth conditions, at least three Tat translocase subunits, namely TatAd, TatAy and TatCy, are involved in the secretion of YwbN. Yet, a significant level of Tat-independent YwbN secretion is also observed under these conditions. When B. subtilis is grown in medium with 1% NaCl or without NaCl, the secretion of YwbN depends strictly on the previously described “minimal Tat translocase” consisting of the TatAy and TatCy subunits. Notably, in medium without NaCl, both tatAyCy and ywbN mutants display significantly reduced exponential growth rates and severe cell lysis. This is due to a critical role of secreted YwbN in the acquisition of iron under these conditions. Taken together, our findings show that environmental conditions, such as salinity, can determine the specificity and need for the secretion of a bacterial Tat substrate
Twin arginine translocase (Tat) : structural and functional insight
No full textThe twin arginine tranlocation (Tat) pathway is responsible for the transport of folded protein across the membrane. In bacteria, this occurs at the cytoplasmic membrane. In Gram-negative bacteria, Tat forms a 3-component machinery named TatABC. The current hypothesised mechanism is compiled from the model organism Escherichia coli.
In Gram-positive bacteria the Tat machinery lack the TatB component and so raises the question on the validity of the mechanism assumed from the TatABC system. To date there is limited research available on the TatAC system with in Gram-postive bacteria. Recent characterisation has been focused on the Bacillus subtilis system, which contain two TatAC systems; the TatAdCd and TatAyCy.
This thesis focuses on the structural characterisation of the TatAC system of B. subtilis and their similarity to the TatABC system in E. coli. The TatAdCd complex was studies by electron microscopy (EM) to show structure similarity to the TatBC complex. Mutation within the N-terminus region of the TatAy protein showed functional involved in complex assembly. The TatAyCy was also analysed by EM show conserved round complexes similar to the TatAdCd and TatBC. The similarity in structure may suggest the Gram-negatives and the Gram-Positives share a similar mechanism of transport despite difference in the components
A structural investigation of bacterial twin-arginine translocation (tat) complexes by single-particle electron microscopy
No full textThe Twin arginine translocase (Tat) pathway was first characterised in chloroplast
thylakoid membranes in the late 1990s. It has since been identified in the plasma
membranes of both Gram-positive and Gram-negative bacteria. Substrates of this
transport system contain a critical twin-arginine motif within their cleavable Nterminal
signal sequence and the majority are large co-factor containing proteins.
There is now considerable evidence that Tat systems can transport such globular
proteins in a fully folded state. The minimal components required for transport in
E.coli are TatA, TatB and TatC; these three integral membrane proteins are thought
to form an active translocon. In Bacillus subtilis only TatA and TatC subunits are
present, with TatA acting in a bifunctional manner to replace TatB. Little structural
information is known about these multimeric integral membrane protein complexes
due to the inherent difficulty in purifying them and their compositional variability.
Complexes formed by B. subtilis TatAd and TatAyCy and E. coli TatE were
investigated by single-particle EM analysis. An image processing protocol was
developed to analyse and separate out individual Tat complexes based on their size.
Using this method 3D electron density maps were generated of TatAd and TatE,
which appear as small, ring-shaped complexes. Unlike E. coli TatA complexes, that
have been shown to vary widely in size, those observed here appear small and
homogeneous. These data conflict with the widely accepted ‘size-fitting pore’ model
of Tat mediated translocation and rather support the alternative transient coalescent
model.
Additionally the first structural characterisation of a TatA-type mutant protein was
performed revealing a dramatic polymerisation phenotype and indicating a primary
role for the N-terminus in forming protein-protein interactions
Multiple interactions of HIV-1 Tat protein with size-defined heparin oligosaccharides
No full textTat protein, a transactivating factor of the human immunodeficiency virus type I, acts also as an extracellular molecule. Heparin affects the bioavailability and biological activity of extracellular Tat (Rusnati, M., Coltrini, D., Oreste, P., Zoppetti, G., Albini, A., Noonan, D., D'Adda di Fagagna, F., Giacca, M., and Presta, M. (1997) J. Biol. Chem. 272, 11313-11320). Here, a series of homogeneously sized, (3)H-labeled heparin fragments were evaluated for their capacity to bind to free glutathione S-transferase (GST)-Tat protein and to immobilized GST-Tat. Hexasaccharides represent the minimum sized heparin fragments able to interact with GST-Tat at physiological ionic strength. Also, the affinity of binding increases with increasing the molecular size of the oligosaccharides, with large fragments (>/=18 saccharides) approaching the affinity of full-size heparin. 6-Mer heparin binds GST-Tat with a dissociation constant (K(d)) equal to 0.7 +/- 0.4 microM and a molar oligosaccharide:GST-Tat ratio of about 1:1. Interaction of GST-Tat with 22-mer or full-size heparin is consistent instead with two-component binding. At subsaturating concentrations, a single molecule of heparin interacts with 4-6 molecules of GST-Tat with high affinity (K(d) values in the nanomolar range of concentration); at saturating concentrations, heparin binds GST-Tat with lower affinity (K(d) values in the micromolar range of concentration) and a molar oligosaccharide:GST-Tat ratio of about 1:1. In agreement with the binding data, a positive correlation exists between the size of heparin oligosaccharides and their capacity to inhibit cell internalization, long terminal repeat-transactivating activity of extracellular Tat in HL3T1 cells, and its mitogenic activity in murine adenocarcinoma T53 Tat-less cells. The data demonstrate that the modality of heparin-Tat interaction is strongly affected by the size of the saccharide chain. The possibility of establishing multiple interactions increases the affinity of large heparin fragments for Tat protein and the capacity of the glycosaminoglycan to modulate the biological activity of extracellular Tat
The basic domain in HIV-1 Tat protein as a target for polysulfonated heparin-mimicking extracellular Tat antagonists
No full textHeparin binds extracellular HIV-1 Tat protein and modulates its HI long terminal repeat (LTR)-transactivating activity (M. Rusnati, D. Coltrini, P. Oreste, G. Zoppetti, A. Albini, D. Noonan, F. d'Adda di Fagagna, M. Giacca, and M. Presta (1997) J. Biol. Chem. 272, 11313-11320). On this basis, the glutathione S-transferase (GST)-Tat(R49/52/53/55/56/57A) mutant, in which six arginine residues within the basic domain of Tat were mutagenized to alanine residues, was compared with GSTTat for its capacity to bind immobilized heparin. Dissociation of the GST-Tat(R49/52/53/55/56/57A) · heparin complex occurred at ionic strength significantly lower than that required to dissociate the GST-Tat-heparin complex. Accordingly, heparin binds immobilized GST-Tat and GSTTat(R49/52/53/55/56/57A) with a dissociation constant equal to 0.3 and 1.0 μM, respectively. Also, the synthetic basic domain Tat-(41-60) competes with GST-Tat for heparin binding. Suramin inhibits [3H]heparin/Tat interaction, 125I-GST-Tat internalization, and the LTR-transactivating activity of extracellular Tat in HL3T1 cells and prevents 125I-GST-Tat binding and cell proliferation in Tat-overexpressing T53 cells. The suramin derivative 14CPNU 145156E binds immobilized GST-Tat with a dissociation constant 5 times higher than heparin and is unable to bind GST-Tat(R49-52/53/55/56/57A). Although heparin was an antagonist more potent than suramin, modifications of the backbone structure in selected suramin derivatives originated Tat antagonists whose potency was close to that shown by heparin. In conclusion, suramin derivatives bind the basic domain of Tat, prevent Tat/heparin and Tat/cell surface interactions, and inhibit the biological activity of extracellular Tat. Our data demonstrate that tailored polysulfonated compounds represent potent extracellular Tat inhibitors of possible therapeutic value
Flow cytometric quantification of HIV-1 Tat protein in tat-transfected Jurkat T cell lines
No full textThe transactivator Tat protein represents a pivotal factor for the replication of human immunodeficiency virus type 1 (HIV-1). In this report, we describe a flow cytometry procedure designed to quantify the intracellular content of Tat protein in Jurkat CD4+ T lymphoblastoid cell lines, stably transfected with plasmids expressing full-length Tat protein. Various expression vectors were compared for their effectiveness to yield Tat protein in Jurkat cells, and several technical parameters were analyzed to optimize the assay. This method offers a quick and efficient approach to select stably transfected cell lines expressing different levels of specific protein
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