Elongin C Contributes to RNA Polymerase II Degradation by the Interferon Antagonist NSs of La Crosse Orthobunyavirus.

Mosquito-borne La Crosse virus (LACV; genus Orthobunyavirus, family Peribunyaviridae, order Bunyavirales) causes up to 100 annual cases of severe meningoencephalitis in children and young adults in the United States. A major virulence factor of LACV is the nonstructural protein NSs, which inhibits host cell mRNA synthesis to prevent the induction of antiviral type I interferons (IFN-α/ß). To achieve this host transcriptional shutoff, LACV NSs drives the proteasomal degradation of RPB1, the large subunit of mammalian RNA polymerase II. Here, we show that NSs acts in a surprisingly rapid manner, as RPB1 degradation was commencing already at 1 h postinfection. The RPB1 degradation was partially dependent on the cellular E3 ubiquitin ligase subunit Elongin C. Consequently, removal of Elongin C, but also of the subunits Elongin A or B by siRNA transfection partially rescued general RNAP II transcription and IFN-beta mRNA synthesis from the blockade by NSs. In line with these results, LACV NSs was found to trigger the redistribution of Elongin C out of nucleolar speckles, which, however, is an epiphenomenon rather than part of the NSs mechanism. Our study also shows that the molecular phenotype of LACV NSs is different from RNA polymerase II inhibitors like α-amanitin or Rift Valley fever virus NSs, indicating that LACV is unique in involving the Elongin complex to shut off host transcription and IFN response.IMPORTANCE The mosquito-borne La Crosse virus (LACV; genus Orthobunyavirus, family Peribunyaviridae, order Bunyavirales) is prevalent in the United States and can cause severe childhood meningoencephalitis. Its main virulence factor, the nonstructural protein NSs, is a strong inhibitor of the antiviral type I interferon (IFN) system. NSs acts by imposing a global host mRNA synthesis shutoff, mediated by NSs-driven proteasomal degradation of the RPB1 subunit of RNA polymerase II. Here, we show that RPB1 degradation commences as early as 1 h postinfection, and identify the E3 ubiquitin ligase subunit Elongin C (and its binding partners Elongins A and B) as an NSs cofactor involved in RPB1 degradation and in suppression of global as well as IFN-related mRNA synthesis.

Robustness of DNA repair through collective rate control.

DNA repair and other chromatin-associated processes are carried out by enzymatic macromolecular complexes that assemble at specific sites on the chromatin fiber. How the rate of these molecular machineries is regulated by their constituent parts is poorly understood. Here we quantify nucleotide-excision DNA repair in mammalian cells and find that, despite the pathways' molecular complexity, repair effectively obeys slow first-order kinetics. Theoretical analysis and data-based modeling indicate that these kinetics are not due to a singular rate-limiting step. Rather, first-order kinetics emerge from the interplay of rapidly and reversibly assembling repair proteins, stochastically distributing DNA lesion repair over a broad time period. Based on this mechanism, the model predicts that the repair proteins collectively control the repair rate. Exploiting natural cell-to-cell variability, we corroborate this prediction for the lesion-recognition factor XPC and the downstream factor XPA. Our findings provide a rationale for the emergence of slow time scales in chromatin-associated processes from fast molecular steps and suggest that collective rate control might be a widespread mode of robust regulation in DNA repair and transcription.

Interferon antagonist NSs of La Crosse virus triggers a DNA damage response-like degradation of transcribing RNA polymerase II.

La Crosse encephalitis virus (LACV) is a mosquito-borne member of the negative-strand RNA virus family Bunyaviridae. We have previously shown that the virulence factor NSs of LACV is an efficient inhibitor of the antiviral type I interferon system. A recombinant virus unable to express NSs (rLACVdelNSs) strongly induced interferon transcription, whereas the corresponding wt virus (rLACV) suppressed it. Here, we show that interferon induction by rLACVdelNSs mainly occurs through the signaling pathway leading from the pattern recognition receptor RIG-I to the transcription factor IRF-3. NSs expressed by rLACV, however, acts downstream of IRF-3 by specifically blocking RNA polymerase II-dependent transcription. Further investigations revealed that NSs induces proteasomal degradation of the mammalian RNA polymerase II subunit RPB1. NSs thereby selectively targets RPB1 molecules of elongating RNA polymerase II complexes, the so-called IIo form. This phenotype has similarities to the cellular DNA damage response, and NSs was indeed found to transactivate the DNA damage response gene pak6. Moreover, NSs expressed by rLACV boosted serine 139 phosphorylation of histone H2A.X, one of the earliest cellular reactions to damaged DNA. However, other DNA damage response markers such as up-regulation and serine 15 phosphorylation of p53 or serine 1524 phosphorylation of BRCA1 were not triggered by LACV infection. Collectively, our data indicate that the strong suppression of interferon induction by LACV NSs is based on a shutdown of RNA polymerase II transcription and that NSs achieves this by exploiting parts of the cellular DNA damage response pathway to degrade IIo-borne RPB1 subunits.

Simulation of Stimulation: Cytokine Dosage and Cell Cycle Crosstalk Driving Timing-Dependent T Cell Differentiation.

Triggering an appropriate protective response against invading agents is crucial to the effectiveness of human innate and adaptive immunity. Pathogen recognition and elimination requires integration of a myriad of signals from many different immune cells. For example, T cell functioning is not qualitatively, but quantitatively determined by cellular and humoral signals. Tipping the balance of signals, such that one of these is favored or gains advantage on another one, may impact the plasticity of T cells. This may lead to switching their phenotypes and, ultimately, modulating the balance between proliferating and memory T cells to sustain an appropriate immune response. We hypothesize that, similar to other intracellular processes such as the cell cycle, the process of T cell differentiation is the result of: (i) pleiotropy (pattern) and (ii) magnitude (dosage/concentration) of input signals, as well as (iii) their timing and duration. That is, a flexible, yet robust immune response upon recognition of the pathogen may result from the integration of signals at the right dosage and timing. To investigate and understand how system's properties such as T cell plasticity and T cell-mediated robust response arise from the interplay between these signals, the use of experimental toolboxes that modulate immune proteins may be explored. Currently available methodologies to engineer T cells and a recently devised strategy to measure protein dosage may be employed to precisely determine, for example, the expression of transcription factors responsible for T cell differentiation into various subtypes. Thus, the immune response may be systematically investigated quantitatively. Here, we provide a perspective of how pattern, dosage and timing of specific signals, called interleukins, may influence T cell activation and differentiation during the course of the immune response. We further propose that interleukins alone cannot explain the phenotype variability observed in T cells. Specifically, we provide evidence that the dosage of intercellular components of both the immune system and the cell cycle regulating cell proliferation may contribute to T cell activation, differentiation, as well as T cell memory formation and maintenance. Altogether, we envision that a qualitative (pattern) and quantitative (dosage) crosstalk between the extracellular milieu and intracellular proteins leads to T cell plasticity and robustness. The understanding of this complex interplay is crucial to predict and prevent scenarios where tipping the balance of signals may be compromised, such as in autoimmunity.

Quantitative Systems Biology to decipher design principles of a dynamic cell cycle network: the "Maximum Allowable mammalian Trade-Off-Weight" (MAmTOW).

Network complexity is required to lend cellular processes flexibility to respond timely to a variety of dynamic signals, while simultaneously warranting robustness to protect cellular integrity against perturbations. The cell cycle serves as a paradigm for such processes; it maintains its frequency and temporal structure (although these may differ among cell types) under the former, but accelerates under the latter. Cell cycle molecules act together in time and in different cellular compartments to execute cell type-specific programs. Strikingly, the timing at which molecular switches occur is controlled by abundance and stoichiometry of multiple proteins within complexes. However, traditional methods that investigate one effector at a time are insufficient to understand how modulation of protein complex dynamics at cell cycle transitions shapes responsiveness, yet preserving robustness. To overcome this shortcoming, we propose a multidisciplinary approach to gain a systems-level understanding of quantitative cell cycle dynamics in mammalian cells from a new perspective. By suggesting advanced experimental technologies and dedicated modeling approaches, we present innovative strategies (i) to measure absolute protein concentration in vivo, and (ii) to determine how protein dosage, e.g., altered protein abundance, and spatial (de)regulation may affect timing and robustness of phase transitions. We describe a method that we name "Maximum Allowable mammalian Trade-Off-Weight" (MAmTOW), which may be realized to determine the upper limit of gene copy numbers in mammalian cells. These aspects, not covered by current systems biology approaches, are essential requirements to generate precise computational models and identify (sub)network-centered nodes underlying a plethora of pathological conditions.

The CERAB technique: tips, tricks and results.

In this article we wanted to present an overview of the latest study results, in vitro and in vivo, of the Covered Endovascular Reconstruction of the Aortic Bifurcation or CERAB technique and the C-CERAB or Chimney CERAB for the endovascular treatment of either extensive occlusive aortoiliac or iuxtarenal disease with preservation of visceral arteries; in combination with tips and tricks to facilitate recanalization and revascularization. A review was performed of the literature of the last 5 years regarding the endovascular treatment of aortoiliac and iuxtarenal TASC II C& D lesions with covered balloon expandable stents. Furthermore we did a retrospective analysis of our most special techniques to achieve a successful interventional recanalization of these challenging lesions. Both the in vitro and the in vivo studies pointed out that there exists an important benefit of the CERAB configuration with excellent patency rates at one and two years in combination with very low mortality and morbidity, when compared to other techniques. Also the C-CERAB variant seems to be a feasible and safe option; 100% technical success; to deal with iuxtarenal or extensive aortic occlusive disease together with the safeguarding of visceral arteries. The CERAB and C-CERAB techniques may change the treatment algorithm of extensive aortoiliac occlusive disease in the near future and appears to be a safe and feasible alternative with promising results, together with a more anatomical and physiological reconstruction of the aortic bifurcation, being a valid alternative for surgery and/or kissing stents. A few critical issues still need to be solved, such as cost-effectiveness, patient selection, fine-tuning of the technique and defining the optimal medical support.

Of monkeys and men: a metabolomic analysis of static and dynamic urinary metabolic phenotypes in two species.

BACKGROUND: Metabolomics has attracted the interest of the medical community for its potential in predicting early derangements from a healthy to a diseased metabolic phenotype. One key issue is the diversity observed in metabolic profiles of different healthy individuals, commonly attributed to the variation of intrinsic (such as (epi)genetic variation, gut microbiota, etc.) and extrinsic factors (such as dietary habits, life-style and environmental conditions). Understanding the relative contributions of these factors is essential to establish the robustness of the healthy individual metabolic phenotype. METHODS: To assess the relative contribution of intrinsic and extrinsic factors we compared multilevel analysis results obtained from subjects of Homo sapiens and Macaca mulatta, the latter kept in a controlled environment with a standardized diet by making use of previously published data and results. RESULTS: We observed similarities for the two species and found the diversity of urinary metabolic phenotypes as identified by nuclear magnetic resonance (NMR) spectroscopy could be ascribed to the complex interplay of intrinsic factors and, to a lesser extent, of extrinsic factors in particular minimizing the role played by diet in shaping the metabolic phenotype. Moreover, we show that despite the standardization of diet as the most relevant extrinsic factor, a clear individual and discriminative metabolic fingerprint also exists for monkeys. We investigate the metabolic phenotype both at the static (i.e., at the level of the average metabolite concentration) and at the dynamic level (i.e., concerning their variation over time), and we show that these two components sum up to the overall phenotype with different relative contributions of about 1/4 and 3/4, respectively, for both species. Finally, we show that the great degree diversity observed in the urinary metabolic phenotype of both species can be attributed to differences in both the static and dynamic part of their phenotype.

The P gene of Newcastle disease virus does not encode an accessory X protein.

Many paramyxoviruses encode non-essential accessory proteins that are involved in the regulation of virus replication and inhibition of cellular antiviral responses. It has been suggested that the P gene mRNA of Newcastle disease virus (NDV) encodes an accessory protein - the so-called X protein - by translation initiation at a conserved in-frame AUG codon at position 120. Using a monoclonal antibody that specifically detected the P and X proteins, it was shown that an accessory X protein was not expressed in NDV-infected cells. Recombinant NDV strains in which the AUG was changed into a GCC (Ala) or GUC (Val) codon were viable but showed a reduction in virulence, probably because the amino acid change affected the function of the P and/or V protein.