Showing papers by "Unit of Virus Host Cell Interactions published in 2012"
TL;DR: MultiBac as discussed by the authors is a baculovirus expression vector system that is tailored for the production of eukaryotic multiprotein complexes and has been used to produce many important proteins and their complexes for their structural characterization.
190 citations
01 Feb 2012
TL;DR: A baculovirus expression vector system that is particularly tailored for the production of eukaryotic multiprotein complexes and has been used to produce many important proteins and their complexes for their structural characterization, revealing fundamental cellular mechanisms.
Abstract: Protein complexes composed of many subunits carry out most essential processes in cells and, therefore, have become the focus of intense research. However, deciphering the structure and function of these multiprotein assemblies imposes the challenging task of producing them in sufficient quality and quantity. To overcome this bottleneck, powerful recombinant expression technologies are being developed. In this review, we describe the use of one of these technologies, MultiBac, a baculovirus expression vector system that is particularly tailored for the production of eukaryotic multiprotein complexes. Among other applications, MultiBac has been used to produce many important proteins and their complexes for their structural characterization, revealing fundamental cellular mechanisms.
152 citations
TL;DR: High resolution X-ray co-crystal structures of the 2009 pandemic H1N1 PA endonuclease domain are described with a series of specific inhibitors, including four diketo compounds and a green tea catechin, all of which chelate the two critical manganese ions in the active site of the enzyme.
Abstract: It is generally recognised that novel antiviral drugs, less prone to resistance, would be a desirable alternative to current drug options in order to be able to treat potentially serious influenza infections. The viral polymerase, which performs transcription and replication of the RNA genome, is an attractive target for antiviral drugs since potent polymerase inhibitors could directly stop viral replication at an early stage. Recent structural studies on functional domains of the heterotrimeric polymerase, which comprises subunits PA, PB1 and PB2, open the way to a structure based approach to optimise inhibitors of viral replication. In particular, the unique cap-snatching mechanism of viral transcription can be inhibited by targeting either the PB2 cap-binding or PA endonuclease domains. Here we describe high resolution X-ray co-crystal structures of the 2009 pandemic H1N1 (pH1N1) PA endonuclease domain with a series of specific inhibitors, including four diketo compounds and a green tea catechin, all of which chelate the two critical manganese ions in the active site of the enzyme. Comparison of the binding mode of the different compounds and that of a mononucleotide phosphate highlights, firstly, how different substituent groups on the basic metal binding scaffold can be orientated to bind in distinct sub-pockets within the active site cavity, and secondly, the plasticity of certain structural elements of the active site cavity, which result in induced fit binding. These results will be important in optimising the design of more potent inhibitors targeting the cap-snatching endonuclease activity of influenza virus polymerase.
141 citations
TL;DR: Comparison of high-resolution crystal structures of Rig-I and RIG-I:dsRNA cocrystals provides considerable insight into how this innate immune pattern recognition receptor is activated upon detecting and binding a certain class of viral RNAs.
Abstract: A series of high-resolution crystal structures of RIG-I and RIG-I:dsRNA cocrystals has recently been reported. Comparison of these structures provides considerable insight into how this innate immune pattern recognition receptor is activated upon detecting and binding a certain class of viral RNAs.
139 citations
TL;DR: Crystal structures for two acyl acid amido synthetases from Arabidopsis suggest how the enzymes might discriminate between apolar and acidic amino acids and lend insight into the reaction chemistries that add functional diversity to hormone signaling pathways.
Abstract: Acyl acid amido synthetases of the GH3 family act as critical prereceptor modulators of plant hormone action; however, the molecular basis for their hormone selectivity is unclear. Here, we report the crystal structures of benzoate-specific Arabidopsis thaliana AtGH3.12/PBS3 and jasmonic acid–specific AtGH3.11/JAR1. These structures, combined with biochemical analysis, define features for the conjugation of amino acids to diverse acyl acid substrates and highlight the importance of conformational changes in the carboxyl-terminal domain for catalysis. We also identify residues forming the acyl acid binding site across the GH3 family and residues critical for amino acid recognition. Our results demonstrate how a highly adaptable three-dimensional scaffold is used for the evolution of promiscuous activity across an enzyme family for modulation of plant signaling molecules.
132 citations
TL;DR: A new family of soluble ABA receptors, named PYR/PYL/RCAR, has emerged as ABA sensors able to inhibit the activity of specific protein phosphatases type-2C (PP2Cs) in an ABA-dependent manner.
107 citations
TL;DR: PNRC2 acts in synergy with DCP1a to stimulate the decapping activity of DCP2 by bridging the interaction between Dcp1a and Dcp2, suggesting that PNRC 2 is a decapping coactivator in addition to its adaptor role in NMD.
62 citations
TL;DR: The first cell-penetrating peptide that activates protein phosphatase-1 by disrupting a subset of PP1 complexes in living cells has been developed and can be a novel approach to study PP1 function and to counteract Ser/Thr kinase activity under pathologically increased kinase signaling.
Abstract: The first cell-penetrating peptide
that activates protein phosphatase-1 (PP1) by disrupting a subset of PP1 complexes in living cells has been developed. Activated PP1 rapidly dephosphorylates its substrates, counteracting kinase activity inside cells. Activation of PP1 can thus be a novel approach to study PP1 function and to counteract Ser/Thr kinase activity under pathologically increased kinase signaling.
62 citations
TL;DR: The application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas indicates the potential application of these VHH as anti-HIV-1 microbicides.
Abstract: Many of the neutralising antibodies, isolated to date, display limited activities against the globally most prevalent HIV-1 subtypes A and C. Therefore, those subtypes are considered to be an important target for antibody-based therapy. Variable domains of llama heavy chain antibodies (VHH) have some superior properties compared with classical antibodies. Therefore we describe the application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas. A panel of VHH, which interfere with CD4 binding to HIV-1 Env were selected with use of panning. The results of binding and competition assays to various Env, including a variant with a stabilized CD4-binding state (gp120Ds2), cross-competition experiments, maturation analysis and neutralisation assays, enabled us to classify the selected VHH into three groups. The VHH of group I were efficient mainly against viruses of subtype A, C and B′/C. The VHH of group II resemble the broadly neutralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. A representative of the third group, 2E7, had an even higher neutralization breadth, neutralizing 21 out of the 26 tested strains belonging to the A, A/G, B, B/C and C subtypes. To evaluate the contribution of certain amino acids to the potency of the VHH a small set of the mutants were constructed. Surprisingly this yielded one mutant with slightly improved neutralisation potency against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). These findings and the well-known stability of VHH indicate the potential application of these VHH as anti-HIV-1 microbicides.
60 citations
TL;DR: It is shown that CC2D1A is a novel regulator of ESCRT-III CHMP4B function and interacts directly with CC2d1A and CC2B with nanomolar affinity by forming a 1:1 complex.
57 citations
TL;DR: The split-GFP based virus enabled us to obtain a live-cell based confirmation for the model of vRNP trafficking which assumes accumulation of v RNP in recycling endosomes through a direct interaction of PB2 with Rab11, and subsequent transport across the cytoplasm involving microtubules and actin filaments.
TL;DR: Cryo-EM structures of Escherichia coli DegQ in its 12- and 24-mer states in complex with model substrates are described, providing a structural model of HtrA chaperone action.
Abstract: The HtrA protein family combines chaperone and protease activities and is essential for protein quality control in many organisms. Whereas the mechanisms underlying the proteolytic function of HtrA proteins are well characterized, their chaperone activity remains poorly understood. Here we describe cryo-EM structures of Escherichia coli DegQ in its 12- and 24-mer states in complex with model substrates, providing a structural model of HtrA chaperone action. Up to six lysozyme substrates bind inside the DegQ 12-mer cage and are visualized in a close-to-native state. An asymmetric reconstruction reveals the binding of a well-ordered lysozyme to four DegQ protomers. DegQ PDZ domains are located adjacent to substrate density and their presence is required for chaperone activity. The substrate-interacting regions appear conserved in 12- and 24-mer cages, suggesting a common mechanism of chaperone function.
TL;DR: Overall, the results show that TDRD1 can accommodate different peptides from different proteins, and can therefore act as a scaffold protein for complex assembly in the piRNA pathway.
Abstract: Piwi-interacting RNAs (piRNAs) are small noncoding RNAs expressed in the germline of animals. They associate with Argonaute proteins of the Piwi subfamily, forming ribonucleoprotein complexes that are involved in maintaining genome integrity. The N-terminal region of some Piwi proteins contains symmetrically dimethylated arginines. This modification is thought to enable recruitment of Tudor domain-containing proteins (TDRDs), which might serve as platforms mediating interactions between various proteins in the piRNA pathway. We measured the binding affinity of the four individual extended Tudor domains (TDs) of murine TDRD1 protein for three different methylarginine-containing peptides from murine Piwi protein MILI. The results show a preference of TD2 and TD3 for consecutive MILI peptides, whereas TD4 and TD1 have, respectively, lower and very weak affinity for any peptide. The affinity of TD1 for methylarginine peptides can be restored by a single-point mutation back to the consensus aromatic cage sequence. These observations were confirmed by pull-down experiments with endogenous Piwi and Piwi-associated proteins. The crystal structure of TD3 bound to a methylated MILI peptide shows an unexpected orientation of the bound peptide, with additional contacts of nonmethylated residues being made outside of the aromatic cage, consistent with solution NMR titration experiments. Finally, the molecular envelope of the four tandem Tudor domains of TDRD1, derived from small angle scattering data, reveals a flexible, elongated shape for the protein. Overall, the results show that TDRD1 can accommodate different peptides from different proteins, and can therefore act as a scaffold protein for complex assembly in the piRNA pathway.
TL;DR: It is proposed that Msl1-mediated dimerization of the entire MSL complex is required for Msl2 binding, X chromosome recognition, and spreading along the X chromosome.
TL;DR: A powerful and easy-to-use workflow environment has been developed at the ESRF for combining experiment control with online data analysis on synchrotron beamlines by accessing defined beamline services.
Abstract: The automation of beam delivery, sample handling and data analysis, together with increasing photon flux, diminishing focal spot size and the appearance of fast-readout detectors on synchrotron beamlines, have changed the way that many macromolecular crystallography experiments are planned and executed. Screening for the best diffracting crystal, or even the best diffracting part of a selected crystal, has been enabled by the development of microfocus beams, precise goniometers and fast-readout detectors that all require rapid feedback from the initial processing of images in order to be effective. All of these advances require the coupling of data feedback to the experimental control system and depend on immediate online data-analysis results during the experiment. To facilitate this, a Data Analysis WorkBench (DAWB) for the flexible creation of complex automated protocols has been developed. Here, example workflows designed and implemented using DAWB are presented for enhanced multi-step crystal characterizations, experiments involving crystal reorientation with kappa goniometers, crystal-burning experiments for empirically determining the radiation sensitivity of a crystal system and the application of mesh scans to find the best location of a crystal to obtain the highest diffraction quality. Beamline users interact with the prepared workflows through a specific brick within the beamline-control GUI MXCuBE.
TL;DR: Both oligomerization routes the authors characterized could be exploited by cellular or viral factors to modulate or control viral RNA encapsidation by NP and bind directly as oligomers to RNA with a higher affinity than that of the monomers.
TL;DR: The HpDnaB double-hexamer architecture supports an alternative strategy to load bacterial helicases onto forks in the absence of helicase loaders.
TL;DR: A combination of nuclear magnetic resonance spectroscopy and small-angle X-ray scattering is used to depict VSV P as an ensemble of continuously exchanging conformers that captures the dynamic character of this protein.
TL;DR: A computationally identified five key residues that are crucial for folding and secretion in variable domains of llama heavy-chain antibodies, and validated their importance with systematic site-directed mutations.
Abstract: Variable domains of llama heavy-chain antibodies (VHH) are becoming a potent tool for a wide range of biotechnological and medical applications. Because of structural features typical of their single-domain nature, they are relatively easy to produce in lower eukaryotes, but it is not uncommon that some molecules have poor secretion efficiency. We therefore set out to study the production yield of VHH. We computationally identified five key residues that are crucial for folding and secretion, and we validated their importance with systematic site-directed mutations. The observation that all key residues were localised in the V segment, in proximity of the J segment of VHH, led us to study the importance of J segment in secretion efficiency. Intriguingly, we found that the use of specific J segments in VHH could strongly influence the production yield. Sequence analysis and expression experiments strongly suggested that interactions with chaperones, especially with the J segment, are a crucial aspect of the production yield of VHH.
TL;DR: It is shown that BGLF5 can degrade mRNAs of both cellular and viral origin, irrespective of polyadenylation, and that the flexible “bridge” that crosses the active-site canyon of B GLF5 appears to contribute to the interaction with RNA substrates.
Abstract: Replication of the human herpesvirus Epstein-Barr virus drastically impairs cellular protein synthesis. This shutoff phenotype results from mRNA degradation upon expression of the early lytic-phase protein BGLF5. Interestingly, BGLF5 is the viral DNase, or alkaline exonuclease, homologues of which are present throughout the herpesvirus family. During productive infection, this DNase is essential for processing and packaging of the viral genome. In contrast to this widely conserved DNase activity, shutoff is only mediated by the alkaline exonucleases of the subfamily of gammaherpesviruses. Here, we show that BGLF5 can degrade mRNAs of both cellular and viral origin, irrespective of polyadenylation. Furthermore, shutoff by BGLF5 induces nuclear relocalization of the cytosolic poly(A) binding protein. Guided by the recently resolved BGLF5 structure, mutants were generated and analyzed for functional consequences on DNase and shutoff activities. On the one hand, a point mutation destroying DNase activity also blocks RNase function, implying that both activities share a catalytic site. On the other hand, other mutations are more selective, having a more pronounced effect on either DNA degradation or shutoff. The latter results are indicative of an oligonucleotide-binding site that is partially shared by DNA and RNA. For this, the flexible "bridge" that crosses the active-site canyon of BGLF5 appears to contribute to the interaction with RNA substrates. These findings extend our understanding of the molecular basis for the shutoff function of BGLF5 that is conserved in gammaherpesviruses but not in alpha- and betaherpesviruses.
TL;DR: This study investigated the relationship between high blood EBV DNA loads and subsequent progression to ARL and found patients with high EBV burden are more at risk of developing ARL.
Abstract: Background
AIDS-related lymphoma (ARL) remains the main cause of AIDS-related deaths in the combined antiretroviral therapy (cART) era. Although most ARLs are associated with the Epstein–Barr virus (EBV), whether patients with high EBV burden are more at risk of developing ARL is unknown. This study investigated the relationship between high blood EBV DNA loads and subsequent progression to ARL.
Methods
We identified 43 cases of ARL diagnosed between 1988 and 2007 within two cohorts (ANRS SEROCO/HEMOCO and PRIMO) and for which stored serum and peripheral blood mononuclear cell (PBMC) samples were available within 3 years before ARL diagnosis. For each case, two controls matched for the cohort and CD4 cell count in the year of ARL diagnosis were selected. EBV DNA was measured in PBMCs and serum samples with a commercial kit.
Results
High levels of EBV DNA in PBMCs collected a median of 10 months before diagnosis were associated with an increased risk of developing systemic B lymphoma (adjusted odds ratio 2.47; 95% confidence interval 1.15; 5.32 for each 1 log copies/106 PBMC increase in EBV load) but not with primary brain lymphoma.
Conclusion
In this study, HIV-infected patients with undetectable EBV DNA in PBMCs did not develop ARL in the following 3 years, while high levels of EBV DNA in PBMCs predicted subsequent progression to systemic B lymphoma. Clinicians should be aware of the increased risk of developing systemic B lymphoma in HIV-infected patients with a high blood EBV DNA load.
TL;DR: The cEIA using FSB specimens collected on DBSs preserved in appropriate storage conditions was a reliable alternative, equivalent to the POC assay, for HCV testing without venipuncture.
TL;DR: Large architectural variation in the binding sites, both for the acyl donor and for the acceptor, although their overall protein folds are structurally conserved point to the independent, divergent evolution of plant HXXXD acyltransferases in different species.
Abstract: Plant HXXXD acyltransferase-catalyzed malonylation is an important modification reaction in elaborating the structural diversity of flavonoids and anthocyanins, and a universal adaptive mechanism to detoxify xenobiotics. Nicotiana tabacum malonyltransferase 1 (NtMaT1) is a member of anthocyanin acyltransferase subfamily that uses malonyl-CoA (MLC) as donor catalyzing transacylation in a range of flavonoid and naphthol glucosides. To gain insights into the molecular basis underlying its catalytic mechanism and versatile substrate specificity, we resolved the X-ray crystal structure of NtMaT1 to 3.1 A resolution. The structure comprises two α/β mixed subdomains, as typically found in the HXXXD acyltransferases. The partial electron density map of malonyl-CoA allowed us to reliably dock the entire molecule into the solvent channel and subsequently define the binding sites for both donor and acceptor substrates. MLC bound to the NtMaT1 occupies one end of the long solvent channel between two subdomains. On superimposing and comparing the structure of NtMaT1 with that of an enzyme from anthocyanin acyltransferase subfamily from red chrysanthemum (Dm3Mat3) revealed large architectural variation in the binding sites, both for the acyl donor and for the acceptor, although their overall protein folds are structurally conserved. Consequently, the shape and the interactions of malonyl-CoA with the binding sites’ amino acid residues differ substantially. These major local architectural disparities point to the independent, divergent evolution of plant HXXXD acyltransferases in different species. The structural flexibility of the enzyme and the amendable binding pattern of the substrates provide a basis for the evolution of the distinct, versatile substrate specificity of plant HXXXD acyltransferases.
TL;DR: This work shows that the first 47 residues of Nup50 bind to the C terminus of importin α5 like a “clip,” stabilizing the closed conformation of ARM 10, the first evidence for a nucleoporin modulating the import reaction by directly altering the three-dimensional structure of an import adaptor.
TL;DR: Well-balanced: 19F NMR spectroscopy defined a trifluoromagnesate complex for protein kinase A with adenosine diphosphate, the MgF3− ion, and the SP20 peptide substrate, which embraces all catalytic components and much of theSP20 substrate.
Abstract: Well-balanced: 19F NMR spectroscopy defined a trifluoromagnesate complex for protein kinase A (multicolored) with adenosine diphosphate (black), the MgF3− ion (green), and the SP20 peptide substrate (purple with dots). A sphere (cyan) centered on the MgF3− ion embraces all catalytic components and much of the SP20 substrate. The content of the sphere is uncharged conforming to the charge balance hypothesis.
01 Jun 2012
TL;DR: The current status of SP methods for the structure determination pipeline, including target selection, isolation, expression, purification, crystallization, diffraction data collection, structure solution, refinement and functional annotation are reviewed.
Abstract: A broad working definition of structural proteomics (SP) is that it is the process of the high-throughput characterization of the three-dimensional structures of biological macromolecules. Recently, the process for protein structure determination has become highly automated and SP platforms have been established around the globe, utilizing X-ray crystallography as a tool. Although protein structures often provide clues about the biological function of a target, once the three-dimensional structures have been determined, bioinformatics and proteomics-driven strategies can be employed to derive their biological activities and physiological roles. This article reviews the current status of SP methods for the structure determination pipeline, including target selection, isolation, expression, purification, crystallization, diffraction data collection, structure solution, refinement and functional annotation.
TL;DR: This review summarizes current structural, dynamic and functional data of this peculiar molecular machinery and provides a unified model of how it can function, and illuminates the overall common strategies and the subtle variations in the different viruses.
Abstract: Viruses with a non-segmented negative-sense RNA genome, or Mononegavirales, are important pathogens for plants, animals and humans with major socio-economic and health impacts. Among them are well-known human pathogens such as measles, mumps and respiratory syncytial virus. Moreover, animal reservoirs appear much larger than previously thought, hence broadening the risk of emergence of life-threatening zoonotic viruses such as Rabies, Ebola, Marburg, Nipah or Hendra related viruses. These viruses have peculiar transcription and replication machinery that make them unique in the living world. Indeed, their genomic RNA, when naked, is non-infectious because it can be neither transcribed nor translated, and the L RNA-dependent RNA-polymerase is at best able to initiate the synthesis of an RNA copy of a few of tens of nucleotides in length. To serve as a template, the genomic RNA must be encapsidated in a helicoidal homopolymer made of a regular and continuous array of docked N protomers in which the ribose-phosphate backbone is fully embedded. This complex, or nucleocapsid, is recognized by the L polymerase thanks to its cofactor, the P protein, to sequentially transcribe the five genes into five processed mRNAs for the simplest viruses. Subsequently, a switch occurs and the polymerase replicates a full copy of antigenomic RNA that is concurrently encapsidated. This new template is then used for the production of new infectious genomic nucleocapsids. This review summarizes current structural, dynamic and functional data of this peculiar molecular machinery and provides a unified model of how it can function. It illuminates the overall common strategies and the subtle variations in the different viruses, along with the key role of the dual ordered/disordered structure of the protein components in the dynamics of the viral polymerase machinery.
TL;DR: Results suggest that Ubn-1 is a scaffold protein influencing protein subcellular localization and is involved in several processes such as cell-cell contact signalling or modulation of gene activity.
TL;DR: A method is presented in which a single large data set that has not been optimized in any way for radiation-damage-induced phasing (RIP) is segmented into multiple sub-data sets, which can then be used for RIP.
Abstract: Specific radiation damage can be used for the phasing of macromolecular crystal structures. In practice, however, the optimization of the X-ray dose used to `burn' the crystal to induce specific damage can be difficult. Here, a method is presented in which a single large data set that has not been optimized in any way for radiation-damage-induced phasing (RIP) is segmented into multiple sub-data sets, which can then be used for RIP. The efficacy of this method is demonstrated using two model systems and two test systems. A method to improve the success of this type of phasing experiment by varying the composition of the two sub-data sets with respect to their separation by image number, and hence by absorbed dose, as well as their individual completeness is illustrated.
TL;DR: Translation elongation factor eEF1A2 was purified to homogeneity from rabbit muscle by two consecutive ion-exchange column-chromatography steps and this mammalian eEF2 was successfully crystallized for the first time.
Abstract: Translation elongation factor eEF1A2 was purified to homogeneity from rabbit muscle by two consecutive ion-exchange column-chromatography steps and this mammalian eEF1A2 was successfully crystallized for the first time. Protein crystals obtained using ammonium sulfate as precipitant diffracted to 2.5 A resolution and belonged to space group P6(1)22 or P6(3)22 (unit-cell parameters a = b = 135.4, c = 304.6 A). A complete native data set was collected to 2.7 A resolution.