Inhibition of Tat activity by the HEXIM1 protein

BACKGROUND: The positive transcription elongation factor b (P-TEFb) composed by CDK9/CyclinT1 subunits is a dedicated co-factor of HIV transcriptional transactivator Tat protein. Transcription driven by the long terminal repeat (LTR) of HIV involves formation of a quaternary complex between P-TEFb, Tat and the TAR element. This recruitment is necessary to enhance the processivity of RNA Pol II from the HIV-1 5' LTR promoter. The activity of P-TEFb is regulated in vivo and in vitro by the HEXIM1/7SK snRNA ribonucleic-protein complex. RESULTS: Here we report that Tat transactivation is effectively inhibited by co-expression of HEXIM1 or its paralog HEXIM2. HEXIM1 expression specifically represses transcription mediated by the direct activation of P-TEFb through artificial recruitment of GAL4-CycT1. Using appropriate HEXIM1 mutants we determined that effective Tat-inhibition entails the 7SK snRNA basic recognition motif as well as the C-terminus region required for interaction with cyclin T1. Enhanced expression of HEXIM1 protein modestly affects P-TEFb activity, suggesting that HEXIM1-mediated repression of Tat activity is not due to a global inhibition of cellular transcription. CONCLUSION: These results point to a pivotal role of P-TEFb for Tat's optimal transcription activity and suggest that cellular proteins that regulate P-TEFb activity might exert profound effects on Tat function in vivo

Inhibition of HIV-1 replication by P-TEFb inhibitors DRB, seliciclib and flavopiridol correlates with release of free P-TEFb from the large, inactive form of the complex

<p>Abstract</p> <p>Background</p> <p>The positive transcription elongation factor, P-TEFb, comprised of cyclin dependent kinase 9 (Cdk9) and cyclin T1, T2 or K regulates the productive elongation phase of RNA polymerase II (Pol II) dependent transcription of cellular and integrated viral genes. P-TEFb containing cyclin T1 is recruited to the HIV long terminal repeat (LTR) by binding to HIV Tat which in turn binds to the nascent HIV transcript. Within the cell, P-TEFb exists as a kinase-active, free form and a larger, kinase-inactive form that is believed to serve as a reservoir for the smaller form.</p> <p>Results</p> <p>We developed a method to rapidly quantitate the relative amounts of the two forms based on differential nuclear extraction. Using this technique, we found that titration of the P-TEFb inhibitors flavopiridol, DRB and seliciclib onto HeLa cells that support HIV replication led to a dose dependent loss of the large form of P-TEFb. Importantly, the reduction in the large form correlated with a reduction in HIV-1 replication such that when 50% of the large form was gone, HIV-1 replication was reduced by 50%. Some of the compounds were able to effectively block HIV replication without having a significant impact on cell viability. The most effective P-TEFb inhibitor flavopiridol was evaluated against HIV-1 in the physiologically relevant cell types, peripheral blood lymphocytes (PBLs) and monocyte derived macrophages (MDMs). Flavopiridol was found to have a smaller therapeutic index (LD₅₀/IC₅₀) in long term HIV-1 infectivity studies in primary cells due to greater cytotoxicity and reduced efficacy at blocking HIV-1 replication.</p> <p>Conclusion</p> <p>Initial short term studies with P-TEFb inhibitors demonstrated a dose dependent loss of the large form of P-TEFb within the cell and a concomitant reduction in HIV-1 infectivity without significant cytotoxicity. These findings suggested that inhibitors of P-TEFb may serve as effective anti-HIV-1 therapies. However, longer term HIV-1 replication studies indicated that these inhibitors were more cytotoxic and less efficacious against HIV-1 in the primary cell cultures.</p

LARP7 is a stable component of the 7SK snRNP while P-TEFb, HEXIM1 and hnRNP A1 are reversibly associated

Regulation of the elongation phase of RNA polymerase II transcription by P-TEFb is a critical control point for gene expression. The activity of P-TEFb is regulated, in part, by reversible association with one of two HEXIMs and the 7SK snRNP. A recent proteomics survey revealed that P-TEFb and the HEXIMs are tightly connected to two previously-uncharacterized proteins, the methyphosphate capping enzyme, MEPCE, and a La-related protein, LARP7. Glycerol gradient sedimentation analysis of lysates from cells treated with P-TEFb inhibitors, suggested that the 7SK snRNP reorganized such that LARP7 and 7SK remained associated after P-TEFb and HEXIM1 were released. Immunodepletion of LARP7 also depleted most of the 7SK regardless of the presence of P-TEFb, HEXIM or hnRNP A1 in the complex. Small interfering RNA knockdown of LARP7 in human cells decreased the steady-state level of 7SK, led to an initial increase in free P-TEFb and increased Tat transactivation of the HIV-1 LTR. Knockdown of LARP7 or 7SK ultimately caused a decrease in total P-TEFb protein levels. Our studies have identified LARP7 as a 7SK-binding protein and suggest that free P-TEFb levels are determined by a balance between release from the large form and reduction of total P-TEFb

DNA intercalator stimulates influenza transcription and virus replication

Influenza A virus uses its host transcription machinery to facilitate viral RNA synthesis, an event that is associated with cellular RNA polymerase II (RNAPII). In this study, various RNAPII transcription inhibitors were used to investigate the effect of RNAPII phosphorylation status on viral RNA transcription. A low concentration of DNA intercalators, such as actinomycin D (ActD), was found to stimulate viral polymerase activity and virus replication. This effect was not observed in cells treated with RNAPII kinase inhibitors. In addition, the loss of RNAPIIa in infected cells was due to the shift of nonphosphorylated RNAPII (RNAPIIa) to hyperphosphorylated RNAPII (RNAPIIo)

Metabolic stress-induced microRNA and mRNA expression profiles of human fibroblasts

Metabolic and oxidative stresses induce physiological adaptation processes, disrupting a finely tuned, coordinated network of gene expression. To better understand the interplay between the mRNA and miRNA transcriptomes, we examined how two distinct metabolic stressors alter the expression profile of human dermal fibroblasts.Primary fibroblast cultures were obtained from skin biopsies of 17 healthy subjects. Metabolic stress was evoked by growing subcultured cells in glucose deprived, galactose enriched (GAL) or lipid reduced, cholesterol deficient (RL) media, and compared to parallel-cultured fibroblasts grown in standard (STD) medium. This was followed by mRNA expression profiling and assessment of >1000 miRNAs levels across all three conditions. The miRNA expression levels were subsequently correlated to the mRNA expression profile.Metabolic stress by RL and GAL both produced significant, strongly correlated mRNA/miRNA changes. At the single gene level four miRNAs (miR-129-3p, miR-146b-5p, miR-543 and miR-550a) showed significant and comparable expression changes in both experimental conditions. These miRNAs appeared to have a significant physiological effect on the transcriptome, as nearly 10% of the predicted targets reported changes at mRNA level. The two distinct metabolic stressors induced comparable changes in the miRNome profile, suggesting a common defensive response of the fibroblasts to altered homeostasis. The differentially expressed miR-129-3p, miR-146b-5p, miR-543 and miR-550a regulated multiple genes (e.g. NGEF, NOVA1, PDE5A) with region- and age-specific transcription in the human brain, suggesting that deregulation of these miRNAs might have significant consequences on CNS function. The overall findings suggest that analysis of stress-induced responses of peripheral fibroblasts, obtained from patients with psychiatric disorders is a promising avenue for future research endeavors. © 2013 Elsevier Inc

Beyond Implications and Applications: the Story of ‘Safety by Design’

Using long-term anthropological observations at the Center for Biological and Environmental Nanotechnology in Houston, Texas, the article demonstrates in detail the creation of new objects, new venues and new modes of veridiction which have reoriented the disciplines of materials chemistry and nanotoxicology. Beginning with the confusion surrounding the meaning of ‘implications’ and ‘applications’ the article explores the creation of new venues (CBEN and its offshoot the International Council on Nanotechnology); it then demonstrates how the demands for a responsible, safe or ethical science were translated into new research and experiment in and through these venues. Finally it shows how ‘safety by design’ emerged as a way to go beyond implications and applications, even as it introduced a whole new array of controversies concerning its viability, validity and legitimacy

On decoding and rewriting genomes: a psychoanalytical reading of a scientific revolution

In various documents the view emerges that contemporary biotechnosciences are currently experiencing a scientific revolution: a massive increase of pace, scale and scope. A significant part of the research endeavours involved in this scientific upheaval is devoted to understanding and, if possible, ameliorating humankind: from our genomes up to our bodies and brains. New developments in contemporary technosciences, such as synthetic biology and other genomics and “post-genomics” fields, tend to blur the distinctions between prevention, therapy and enhancement. An important dimension of this development is “biomimesis”: i.e. the tendency of novel technologies and materials to mimic or plagiarize nature on a molecular and microscopic level in order to optimise prospects for the embedding of technological artefacts in natural systems such as human bodies and brains. In this paper, these developments are read and assessed from a psychoanalytical perspective. Three key concepts from psychoanalysis are used to come to terms with what is happening in research laboratories today. After assessing the general profile of the current revolution in this manner, I will focus on a particular case study, a line of research that may serve as exemplification of the vicissitudes of contemporary technosciences, namely viral biomaterials. Viral life forms can be genetically modified (their genomes can be rewritten) in such a manner that they may be inserted in human bodies in order to produce substances at specific sites such as hormones (testosterone), neurotransmitters (dopamine), enzymes (insulin) or bone and muscle tissue. Notably, certain target groups such as top athletes, soldiers or patients suffering from degenerative diseases may become the pioneers serving as research subjects for novel applications. The same technologies can be used for various purposes ranging from therapy up to prevention and enhancement

The object of regulation: tending the tensions of food safety

“I’m struggling to see what it actually is,” says Alison, peering into a colander of defrosting meat. What “it” is, we propose in this paper, is helpfully thought of as “the object of regulation” in at least three senses, which together signal both our inheritance of a Foucauldian problematic and our departure from it. Our suggestion is that much of even the best work on biopolitics, biopower, and biosecurity that has been inspired and informed by these writings has replicated Foucault’s own struggle to get to grips with the complexity of matters that he variously refers to “natural” or “artificial” “givens”. By following science and technology studies (STS) scholars in using broadly ethnographic techniques to explore objects as and at the intersection of practices, we redress this balance somewhat by thinking through an empirical study of the securing of food safety, specifically Alison’s inspection of a restaurant kitchen. What we find is that the securing of meat as a material object of regulation is primarily done by involving multiple versions of the future, something which requires a great deal of usually under-recognised, under-valued, and under-theorised articulation work. With risk based regulation, cost sharing, and public sector cuts in the UK set to redefine the ways in which Alison and her colleagues engage with food business operators, we conclude by arguing for a greater appreciation of the skilful work of tending the tensions of food safety, as well as recognition of its limitation

When does the co-evolution of technology and science overturn into technoscience?

In this paper, the relations between science and technology, intervention and representation, the natural and the artificial are analysed on the background of the formation of modern science in the sixteenth century. Due to the fact that technique has been essential for modern science from its early beginning, modern science is characterised by a hybridisation of knowledge and intervention. The manipulation of nature in order to measure its properties has steadily increased until artificial things have been produced, such as laser beams, chemical compounds, elementary particles. Furthermore, the structural bracing of natural science, technological development, and industrial exploitation of nature go also back to the foundation of modern science. In order to strengthen the debate on technoscience against this background, the specific characteristics of technoscientific objects have to be clarified as have the specific characteristics of the social organisation of technoscience and its performance

Host Factors interacting with the Pestivirus N terminal protease, Npro are Components of the Ribonucleoprotein Complex

The viral N-terminal protease N(pro) of pestiviruses counteracts cellular antiviral defenses through inhibition of IRF3. Here we used mass spectrometry to identify a new role for N(pro) through its interaction with over 55 associated proteins, mainly ribosomal proteins and ribonucleoproteins, including RNA helicase A (DHX9), Y-box binding protein (YBX1), DDX3, DDX5, eIF3, IGF2BP1, multiple myeloma tumor protein 2, interleukin enhancer binding factor 3 (IEBP3), guanine nucleotide binding protein 3, and polyadenylate-binding protein 1 (PABP-1). These are components of the translation machinery, ribonucleoprotein particles (RNPs), and stress granules. Significantly, we found that stress granule formation was inhibited in MDBK cells infected with a noncytopathic bovine viral diarrhea virus (BVDV) strain, Kyle. However, ribonucleoproteins binding to N(pro) did not inhibit these proteins from aggregating into stress granules. N(pro) interacted with YBX1 though its TRASH domain, since the mutant C112R protein with an inactive TRASH domain no longer redistributed to stress granules. Interestingly, RNA helicase A and La autoantigen relocated from a nuclear location to form cytoplasmic granules with N(pro). To address a proviral role for N(pro) in RNP granules, we investigated whether N(pro) affected RNA interference (RNAi), since interacting proteins are involved in RISC function during RNA silencing. Using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) silencing with small interfering RNAs (siRNAs) followed by Northern blotting of GAPDH, expression of N(pro) had no effect on RNAi silencing activity, contrasting with other viral suppressors of interferon. We propose that N(pro) is involved with virus RNA translation in the cytoplasm for virus particle production, and when translation is inhibited following stress, it redistributes to the replication complex. IMPORTANCE Although the pestivirus N-terminal protease, N(pro), has been shown to have an important role in degrading IRF3 to prevent apoptosis and interferon production during infection, the function of this unique viral protease in the pestivirus life cycle remains to be elucidated. We used proteomic mass spectrometry to identify novel interacting proteins and have shown that N(pro) is present in ribosomal and ribonucleoprotein particles (RNPs), indicating a translational role in virus particle production. The virus itself can prevent stress granule assembly from these complexes, but this inhibition is not due to N(pro). A proviral role to subvert RNA silencing through binding of these host RNP proteins was not identified for this viral suppressor of interferon