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Atze T Das

Publications and source records attributed to Atze T Das.

At least 19 recordsLinked to original sources
PubMed2006

Hepatocyte-specific interplay of transcription factors at the far-upstream enhancer of the carbamoylphosphate synthetase gene upon glucocorticoid induction.

Carbamoylphosphate synthetase-I is the flux-determining enzyme of the ornithine cycle, and neutralizes toxic ammonia by converting it to urea. An 80 bp glucocorticoid response unit located 6.3 kb upstream of the transcription start site mediates hormone responsiveness and liver-specific expression of carbamoylphosphate synthetase-I. The glucocorticoid response unit consists of response elements for the glucocorticoid receptor, forkhead box A, CCAAT/enhancer-binding protein, and an unidentified protein. With only four transcription factor response elements, the carbamoylphosphate synthetase-I glucocorticoid response unit is a relatively simple unit. The relationship between carbamoylphosphate synthetase-I expression and in vivo occupancy of the response elements was examined by comparing a carbamoylphosphate synthetase-I-expressing hepatoma cell line with a carbamoylphosphate synthetase-I-negative fibroblast cell line. DNaseI hypersensitivity assays revealed an open chromatin configuration of the carbamoylphosphate synthetase-I enhancer in hepatoma cells only. In vivo footprinting assays showed that the accessory transcription factors of the glucocorticoid response unit bound to their response elements in carbamoylphosphate synthetase-I-positive cells, irrespective of whether carbamoylphosphate synthetase-I expression was induced with hormones. In contrast, the binding of glucocorticoid receptor to the carbamoylphosphate synthetase-I glucocorticoid response unit was dependent on treatment of the cells with glucocorticoids. Only forkhead box A was exclusively present in hepatoma cells, and therefore appears to be an important determinant of the observed tissue specificity of carbamoylphosphate synthetase-I expression. As the glucocorticoid receptor is the only DNA-binding protein specifically recruited to the glucocorticoid response unit upon stimulation by glucocorticoids, it is likely to be directly responsible for the transcriptional activation mediated by the glucocorticoid response unit.

Animals↗
PubMed2006

Modification of the Tet-On regulatory system prevents the conditional-live HIV-1 variant from losing doxycycline-control.

BACKGROUND: We have previously constructed a doxycycline (dox)-dependent HIV-1 variant by incorporating the Tet-On gene regulatory system into the viral genome. Replication of this HIV-rtTA virus is driven by the dox-inducible transactivator protein rtTA, and can be switched on and off at will. We proposed this conditional-live virus as a novel vaccine approach against HIV-1. Upon vaccination, replication of HIV-rtTA can be temporarily activated by transient dox administration and controlled to the extent needed for optimal induction of the immune system. However, subsequent dox-withdrawal may impose a selection for virus variants with reduced dox-dependence. RESULTS: We simulated this on/off switching of virus replication in multiple, independent cultures and could indeed select for HIV-rtTA variants that replicated without dox. Nearly all evolved variants had acquired a typical amino acid substitution at position 56 in the rtTA protein. We developed a novel rtTA variant that blocks this undesired evolutionary route and thus prevents HIV-rtTA from losing dox-control. CONCLUSION: The loss of dox-control observed upon evolution of the dox-dependent HIV-1 variant was effectively blocked by modification of the Tet-On regulatory system.

Binding Sites↗
PubMed2006

A conditionally replicating HIV-based vector that stably expresses an antiviral shRNA against HIV-1 replication.

Human pathogenic viruses can be targeted by therapeutic strategies based on RNA interference. Whereas the administration of synthetic short interfering RNAs (siRNAs) may transiently inhibit viral replication, long-term inhibition may be achieved through stable intracellular expression of siRNAs or short hairpin RNAs (shRNAs). Both approaches face serious problems with delivery to the right cells in an infected individual. We explored the potential of a replicating HIV-based vector to deliver an antiviral shRNA cassette into HIV-1-susceptible target cells to block chronic HIV-1 infection. The vector is based on a doxycycline (dox)-dependent HIV-1 variant that we previously proposed as a conditional-live HIV-1 vaccine. With dox, this virus spreads efficiently to all HIV-susceptible cells. Subsequent dox withdrawal generates cells with a transcriptionally silent integrated provirus, but with an active shRNA expression cassette. Because the shRNA targets viral sequences that are removed from the vector construct, there is no self-targeting, yet there is specific shutdown of HIV-1 replication.

Cell Line, Tumor↗
PubMed2006

A novel splice donor site in the gag-pol gene is required for HIV-1 RNA stability.

Productive infection and successful replication of human immunodeficiency virus 1 (HIV-1) requires the balanced expression of all viral genes. This is achieved by a combination of alternative splicing events and regulated nuclear export of viral RNA. Because viral splicing is incomplete and intron-containing RNAs must be exported from the nucleus where they are normally retained, it must be ensured that the unspliced HIV-1 RNA is actively exported from the nucleus and protected from degradation by processes such as nonsense-mediated decay. Here we report the identification of a novel 178-nt-long exon located in the gag-pol gene of HIV-1 and its inclusion in at least two different mRNA species. Although efficiently spliced in vitro, this exon appears to be tightly repressed and infrequently used in vivo. The splicing is activated or repressed in vitro by the splicing factors ASF/SF2 and heterogeneous nuclear ribonucleoprotein A1, respectively, suggesting that splicing is controlled by these factors. Interestingly, mutations in the 5'-splice site resulted in a dramatic reduction in the steady-state level of HIV-1 RNA, and this effect was partially reversed by expression of U1 small nuclear RNA harboring the compensatory mutation. This implies that U1 small nuclear RNA binding to optimal but non-functional splice sites might have a role in protecting unspliced HIV-1 mRNA from degradation.

Amino Acid Sequence↗
PubMed2006

Hairpin-induced tRNA-mediated (HITME) recombination in HIV-1.

Recombination due to template switching during reverse transcription is a major source of genetic variability in retroviruses. In the present study we forced a recombination event in human immunodeficiency virus type 1 (HIV-1) by electroporation of T cells with DNA from a molecular HIV-1 clone that has a 300 bp long hairpin structure in the Nef gene (HIV-lhNef). HIV-lhNef does not replicate, but replication-competent escape variants emerged in four independent cultures. The major part of the hairpin was deleted in all escape viruses. In three cases, the hairpin deletion was linked to patch insertion of tRNA(asp), tRNA(glu) or tRNA(trp) sequences. The tRNAs were inserted in the viral genome in the antisense orientation, indicating that tRNA-mediated recombination occurred during minus-strand DNA synthesis. We here propose a mechanistic model for this hairpin-induced tRNA-mediated (HITME) recombination. The transient role of the cellular tRNA molecule as enhancer of retroviral recombination is illustrated by the eventual removal of inserted tRNA sequences by a subsequent recombination/deletion event.

Base Sequence↗
PubMed2006

The genetic stability of a conditional live HIV-1 variant can be improved by mutations in the Tet-On regulatory system that restrain evolution.

Live attenuated human immunodeficiency virus type 1 (HIV-1) vaccines are considered unsafe because more quickly replicating pathogenic virus variants may evolve after vaccination. As an alternative vaccine approach, we have previously presented a doxycycline (dox)-dependent HIV-1 variant that was constructed by incorporating the tetracycline-inducible gene expression system (Tet-On system) into the viral genome. Replication of this HIV-rtTA variant is driven by the dox-inducible transcriptional activator rtTA and can be switched on and off at will. A large scale evolution study was performed to test the genetic stability of this conditional live vaccine candidate. In several long term cultures, we selected for HIV-rtTA variants that no longer required dox for replication. These evolved variants acquired a typical amino acid substitution either at position 19 or 37 in the rtTA protein. Both mutations caused rtTA activity and viral replication in the absence of dox. We designed a novel rtTA variant with a higher genetic barrier toward these undesired evolutionary routes. The corresponding HIV-rtTA variant did not lose dox control in long term cultures, demonstrating its improved genetic stability.

AIDS Vaccines↗
PubMed2006

Factor correction as a tool to eliminate between-session variation in replicate experiments: application to molecular biology and retrovirology.

BACKGROUND: In experimental biology, including retrovirology and molecular biology, replicate measurement sessions very often show similar proportional differences between experimental conditions, but different absolute values, even though the measurements were presumably carried out under identical circumstances. Although statistical programs enable the analysis of condition effects despite this replication error, this approach is hardly ever used for this purpose. On the contrary, most researchers deal with such between-session variation by normalisation or standardisation of the data. In normalisation all values in a session are divided by the observed value of the 'control' condition, whereas in standardisation, the sessions' means and standard deviations are used to correct the data. Normalisation, however, adds variation because the control value is not without error, while standardisation is biased if the data set is incomplete. RESULTS: In most cases, between-session variation is multiplicative and can, therefore, be removed by division of the data in each session with a session-specific correction factor. Assuming one level of multiplicative between-session error, unbiased session factors can be calculated from all available data through the generation of a between-session ratio matrix. Alternatively, these factors can be estimated with a maximum likelihood approach. The effectiveness of this correction method, dubbed "factor correction", is demonstrated with examples from the field of molecular biology and retrovirology. Especially when not all conditions are included in every measurement session, factor correction results in smaller residual error than normalisation and standardisation and therefore allows the detection of smaller treatment differences. Factor correction was implemented into an easy-to-use computer program that is available on request at: biolab-services@amc.uva.nl?subject=factor. CONCLUSION: Factor correction is an effective and efficient way to deal with between-session variation in multi-session experiments.

Female↗
PubMed2005

Comparison of single regulated lentiviral vectors with rtTA expression driven by an autoregulatory loop or a constitutive promoter.

Regulated expression of a therapeutic gene is crucial for safe and efficacious gene therapy. Many inducible regulatory systems use a constitutive promoter to express a regulatory protein, such as rtTA in the Tet-On system, which may restrict their use because of cytotoxicity and immunogenicity. Autoregulatory expression of rtTA provides extremely low levels of rtTA when transgene expression is off, with rapid transgene induction upon addition of doxycycline. Lentiviral vectors efficiently transfer genes to dividing and non-dividing cells with long-term gene expression both in vitro and in vivo. We compared regulatory function in a single lentiviral vector where rtTA was either expressed from a constitutive promoter or placed in an autoregulatory loop. Autoregulatory expression of rtTA was superior to constitutive promoter expression, resulting in higher viral titers, undetectable levels of both rtTA and transgene expression in the absence of doxycycline, improved induction kinetics and increased induction levels in all cells tested. We further expanded the utility of the autoregulatory vector by using an improved rtTA variant with an increased sensitivity to doxycycline. This lentiviral vector with doxycycline-regulated transgene expression may be useful for gene therapy applications and in experimental settings where strict temporal expression of a transgene is required.

Cell Line↗
PubMed2005

HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome.

HIV-1 replication can be efficiently inhibited by intracellular expression of an siRNA targeting the viral RNA. However, HIV-1 escape variants emerged after prolonged culturing. These RNAi-resistant viruses contain nucleotide substitutions or deletions in or near the targeted sequence. We observed an inverse correlation between the level of resistance and the stability of the siRNA/target-RNA duplex. However, two escape variants showed a higher level of resistance than expected based on the duplex stability. We demonstrate that these mutations induce alternative folding of the RNA such that the target sequence is occluded from binding to the siRNA, resulting in reduced RNAi efficiency. HIV-1 can thus escape from RNAi-mediated inhibition not only through nucleotide substitutions or deletions in the siRNA target sequence, but also through mutations that alter the local RNA secondary structure. The results highlight the enormous genetic flexibility of HIV-1 and provide detailed molecular insight into the sequence specificity of RNAi and the impact of target RNA secondary structure.

Anti-HIV Agents↗
PubMed2005

Alternative tRNA priming of human immunodeficiency virus type 1 reverse transcription explains sequence variation in the primer-binding site that has been attributed to APOBEC3G activity.

It is generally assumed that human immunodeficiency virus type 1 (HIV-1) uses exclusively the cellular tRNA(3)(Lys) molecule as a primer for reverse transcription. We demonstrate that HIV-1 uses not only tRNA(3)(Lys) but also an alternative tRNA primer. This tRNA was termed tRNA(5)(Lys), and the near completion of the human genome project has allowed the identification of four tRNA(5)(Lys)encoding genes. Priming with tRNA(5)(Lys) results in a single nucleotide polymorphism in the viral primer-binding site that is present in multiple natural and laboratory HIV isolates. This sequence variation was recently attributed to APOBEC3G activity. However, our results show that alternative tRNA priming can cause this mutation in the absence of APOBEC3G.

APOBEC-3G Deaminase↗
PubMed2005

Improving the safety of a conditional-live human immunodeficiency virus type 1 vaccine by controlling both gene expression and cell entry.

Live attenuated human immunodeficiency virus type 1 (HIV-1) vaccines are considered unsafe because faster-replicating pathogenic virus variants may evolve after vaccination. We previously presented a conditional-live HIV-1 variant of which replication can be switched off as an alternative vaccination strategy. To improve the safety of such a vaccine, we constructed a new HIV-1 variant that depends not only on doxycycline for gene expression but also on the T20 peptide for cell entry. Replication of this virus can be limited to the level required to induce the immune system by transient administration of doxycycline and T20. Subsequent withdrawal of these inducers efficiently blocks viral replication and evolution.

AIDS Vaccines↗
PubMed2004

Depletion of CD4 T lymphocytes in human lymphoid tissue infected ex vivo with doxycycline-dependent HIV-1.

We investigated whether CD4+ T cells that do not produce HIV-1 are killed in HIV-infected human lymphoid tissue. Tissue blocks were inoculated with high amount of doxycycline-dependent HIV-rtTA. Doxycycline triggered productive infection and loss of CD4+ T cells in these tissues, whereas without doxycycline, neither productive infection nor CD4+ T cell depletion was detected in spite of the massive presence of virions in the tissue and of viral DNA in the cells. Thus, HIV-1 alone is sufficient to deplete productively infected CD4+ T cells but is not sufficient to cause the death of uninfected or latently infected CD4+ T cells.

Anti-Infective Agents↗
PubMed2004

Structural requirements of the glucocorticoid-response unit of the carbamoyl-phosphate synthase gene.

The GRU (glucocorticoid-response unit) within the distal enhancer of the gene encoding carbamoyl-phosphate synthase, which comprises REs (response elements) for the GR (glucocorticoid receptor) and the liver-enriched transcription factors FoxA (forkhead box A) and C/EBP (CCAAT/enhancer-binding protein), and a binding site for an unknown protein denoted P3, is one of the simplest GRUs described. In this study, we have established that the activity of this GRU depends strongly on the positioning and spacing of its REs. Mutation of the P3 site within the 25 bp FoxA-GR spacer eliminated GRU activity, but the requirement for P3 could be overcome by decreasing the length of this spacer to < or =12 bp, by optimizing the sequence of the REs in the GRU, and by replacing the P3 sequence with a C/EBPbeta sequence. With spacers of < or =12 bp, the activity of the GRU depended on the helical orientation of the FoxA and GR REs, with highest activities observed at 2 and 12 bp respectively. Elimination of the 6 bp C/EBP-FoxA spacer also increased GRU activity 2-fold. Together, these results indicate that the spatial positioning of the transcription factors that bind to the GRU determines its activity and that the P3 complex, which binds to the DNA via a 75 kDa protein, functions to facilitate interaction between the FoxA and glucocorticoid response elements when the distance between these transcription factors means that they have difficulties contacting each other.

Animals↗
PubMed2004

Viral evolution as a tool to improve the tetracycline-regulated gene expression system.

We present viral evolution as a novel and powerful method to optimize non-viral proteins. We used this approach to optimize the tetracycline (Tc)-regulated gene expression system (Tet system) for its function in mammalian cells. The components of the Tet system were incorporated in the human immunodeficiency virus (HIV)-1 virus such that viral replication is controlled by this regulatory system. Upon long term replication of this HIV-rtTA virus in human T cells, we obtained a virus variant with an enhanced replication potential resulting from an improved rtTA component of the introduced Tet system. We identified a single amino acid exchange, F86Y, which enhances the transcriptional activity and doxycycline (dox) sensitivity of rtTA. We generated a new rtTA variant that is 5-fold more active at high dox levels than the initial rtTA, and 25-fold more sensitive to dox, whereas the background activity in the absence of dox is not increased. This new rtTA variant will be very useful in biological applications that require a more sensitive or active Tet system. Our results demonstrate that the viral evolution strategy can be used to improve the activity of genes by making them an integral and essential part of the virus.

Biotechnology↗
PubMed2004

Human immunodeficiency virus type 1 escapes from RNA interference-mediated inhibition.

Short-term assays have suggested that RNA interference (RNAi) may be a powerful new method for intracellular immunization against human immunodeficiency virus type 1 (HIV-1) infection. However, RNAi has not yet been shown to protect cells against HIV-1 in long-term virus replication assays. We stably introduced vectors expressing small interfering RNAs (siRNAs) directed against the HIV-1 genome into human T cells by retroviral transduction. We report here that an siRNA directed against the viral Nef gene (siRNA-Nef) confers resistance to HIV-1 replication. This block in replication is not absolute, and HIV-1 escape variants that were no longer inhibited by siRNA-Nef appeared after several weeks of culture. These RNAi-resistant viruses contained nucleotide substitutions or deletions in the Nef gene that modified or deleted the siRNA-Nef target sequence. These results demonstrate that efficient inhibition of HIV-1 replication through RNAi is possible in stably transduced cells. Therefore, RNAi could become a realistic gene therapy approach with which to overcome the devastating effect of HIV-1 on the immune system. However, as is known for antiviral drug therapy against HIV-1, antiviral approaches involving RNAi should be used in a combined fashion to prevent the emergence of resistant viruses.

Base Sequence↗
PubMed2004

A human immunodeficiency virus type 1-infected individual with low viral load harbors a virus variant that exhibits an in vitro RNA dimerization defect.

We investigated the in vitro RNA dimerization properties of the untranslated leader RNA derived from human immunodeficiency virus type 1 variants circulating in an individual with a low viral load and slow disease progression. The leader sequences of these viruses contain highly unusual polymorphisms within the dimerization initiation site (DIS): an insert that abolishes dimerization and a compensatory substitution. The dimerization of leader RNA from late stages of infection is further improved by additional mutations outside the DIS motif that facilitate a secondary structure switch from a dimerization-incompetent to a dimerization-competent RNA conformation.

Base Sequence↗
PubMed2003

The apical loop of the HIV-1 TAR RNA hairpin is stabilized by a cross-loop base pair.

The TAR hairpin of the HIV-1 RNA genome is indispensable for trans-activation of the viral promoter and virus replication. The TAR structure has been studied extensively, but most attention has been directed at the three-nucleotide bulge that constitutes the binding site of the viral Tat protein. In contrast, the conformational properties of the apical loop have remained elusive. We performed biochemical studies and molecular dynamics simulations, which indicate that the TAR loop is structured and stabilized by a cross-loop base pair between residues C30 and G34. Mutational disruption of the cross-loop base pair results in reduced Tat response of the LTR promoter, which can be rescued by compensatory mutations that restore the base pair. Thus, Tat-mediated transcriptional activation depends on the structure of the TAR apical loop. The C30-G34 cross-loop base pair classes TAR in a growing family of hairpins with a structured loop that was recently identified in ribosomal RNA, tRNA, and several viral and cellular mRNAs.

2-Aminopurine↗