mRNA maturation in giant viruses: variation on a theme

International audienceGiant viruses from the Mimiviridae family replicate entirely in their host cytoplasm where their genes are transcribed by a viral transcription apparatus. mRNA polyadenylation uniquely occurs at hairpin-forming palindromic sequences terminating viral transcripts. Here we show that a conserved gene cluster both encode the enzyme responsible for the hairpin cleavage and the viral polyA polymerases (vPAP). Unexpectedly, the vPAPs are homodimeric and uniquely self-processive. The vPAP backbone structures exhibit a symmetrical architecture with two subdomains sharing a nucleotidyltransferase topology, suggesting that vPAPs originate from an ancestral duplication. A Poxvirus processivity factor homologue encoded by Megavirus chilensis displays a conserved 5'-GpppA 2'O methyltransferase activity but is also able to internally methylate the mRNAs' polyA tails. These findings elucidate how the arm wrestling between hosts and their viruses to access the translation machinery is taking place in Mimiviridae

The Leishmania ARL-1 and Golgi Traffic

We present here the characterisation of the Leishmania small G protein ADP-Ribosylation Factor-Like protein 1 (ARL-1). The ARL-1 gene is present in one copy per haploid genome and conserved among trypanosomatids. It encodes a protein of 20 kDa, which is equally expressed in the insect promastigote and mammalian amastigote forms of the parasite. ARL-1 localises to the Trans-Golgi Network (TGN); N-terminal myristoylation is essential for TGN localisation. In vivo expression of the LdARL-1/Q74L and LdARL-1/T51N mutants (GTP- and GDP-bound blocked forms respectively) shows that GDP/GTP cycling occurs entirely within the TGN. This is contrary to previous reports in yeast and mammals, where the mutant empty form devoid of nucleotide has been considered as the GDP-blocked form. The dominant-negative empty form mutant LdARL-1/T34N inhibits endocytosis and intracellular trafficking from the TGN to the Lysosome/Multivesicular Tubule and to the acidocalcisomes; these defects are probably related to a mislocalisation of the GRIP domain-containing vesicle tethering factors which cannot be recruited to the TGN by the cytoplasmic LdARL-1/T34N. Thus, besides the functional characterization of a new mutant and a better understanding of ARL-1 GDP/GTP cycling, this work shows that Leishmania ARL-1 is a key component of an essential pathway worth future study

Pandoravirus Celtis Illustrates the Microevolution Processes at Work in the Giant Pandoraviridae Genomes

With genomes of up to 2.7 Mb propagated in μm-long oblong particles and initially predicted to encode more than 2000 proteins, members of the Pandoraviridae family display the most extreme features of the known viral world. The mere existence of such giant viruses raises fundamental questions about their origin and the processes governing their evolution. A previous analysis of six newly available isolates, independently confirmed by a study including three others, established that the Pandoraviridae pan-genome is open, meaning that each new strain exhibits protein-coding genes not previously identified in other family members. With an average increment of about 60 proteins, the gene repertoire shows no sign of reaching a limit and remains largely coding for proteins without recognizable homologs in other viruses or cells (ORFans). To explain these results, we proposed that most new protein-coding genes were created de novo, from pre-existing non-coding regions of the G+C rich pandoravirus genomes. The comparison of the gene content of a new isolate, pandoravirus celtis, closely related (96% identical genome) to the previously described p. quercus is now used to test this hypothesis by studying genomic changes in a microevolution range. Our results confirm that the differences between these two similar gene contents mostly consist of protein-coding genes without known homologs, with statistical signatures close to that of intergenic regions. These newborn proteins are under slight negative selection, perhaps to maintain stable folds and prevent protein aggregation pending the eventual emergence of fitness-increasing functions. Our study also unraveled several insertion events mediated by a transposase of the hAT family, 3 copies of which are found in p. celtis and are presumably active. Members of the Pandoraviridae are presently the first viruses known to encode this type of transposase

Etudes structurales et fonctionnelles de protéines impliquées dans l'olfaction des insectes

La détection des phéromones a lieu dans les sensilles des insectes. Elles stimulent les récepteurs olfactifs, induisant des modifications comportementales. Deux classes de protéines solubles, les Chemosensory Proteins (CSP) et les Pheromone Binding Proteins (PBP), sont présentes à haute concentration dans la lymphe sensillaire Leur rôle reste sujet à discussion : solubilisation, spécificité, transport des molécules aux récepteurs ? 1 CSP et 3 PBPs ont été exprimées, purifiées, et la résolution de structure 3D par cristallographie des Rayons X ou RMN a été accompagnée d'études de fluorescence. Les structures de la CSP de Mamestra brassicae (apo et complexée), présentent un repliement nouveau en 6 hélices a, la fixation de ligand induisant un changement conformationnel important. Les 2 structures de PBPs présentées montrent un autre type de repliement en 6 hélices a ; .il semblerait que le mécanisme de fixation/relargage des ligands dépend de la longueur des PBPs.In insects, pheromones or odor detection takes place in sensilla. They induce behavioural modifications of an individual of the same species. Two different protein classes present in sensilliary lymph are the Chemosensory Protein (CSP) and the Pheromone Binding Proteins (PBP). To date, their precise function remain uncertain : solubilization, specificity, pheromone carrier to receptors. To bring some structural and functional elements, one CSP and three PBP were expressed in E. coli and purified. They were afterward studied by X-Ray crystallography, NMR and fluorescence. The structures of the apo and complexed CSP (Mamestra brassicae) reveal a new 6 a-helix fold. The protein interacts with aliphatic molecules which induce a large conformational change. PBPs of Leucophaea maderae et Apis mellifera display a different 6 a-helix fold. In this protein family, the mechanism for ligand binding and release may depend of their length.AIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

Giant Mimiviridae CsCl Purification Protocol

International audienceWhile different giant viruses' purification protocols are available, they are not fully described and they use sucrose gradient that does not reach an equilibrium. Here, we report a protocol for the purification of members of the Mimiviridae family virions resulting from Acanthamoeaba castellanii infections. Viruses are harvested after cell lysis and purified through a high density CsCl gradient to optimize the isolation of the virus from the cell debris or other potential contaminants. Due to the large size of the virion capsids, reaching half a micrometer diameter, the quality of the process can be monitored by light microscopy. The resulting purified particles can then be used to perform new infections, DNA extraction, structural studies, sugar composition analyses, sub-compartment characterization or proteomic experiments

Solution structure of a chemosensory protein from the moth Mamestra brassicae.

Chemosensory proteins (CSPs) are believed to be involved in chemical communication and perception. A number of such proteins, of molecular mass approximately 13 kDa, have been isolated from different sensory organs of a wide range of insect species. Several CSPs have been identified in the antennae and proboscis of the moth Mamestra brassicae. CSPMbraA6, a 112-amino-acid antennal protein, has been expressed in a soluble form in large quantities in the Escherichi coli periplasm. NMR structure determination of CSPMbraA6 has been performed with 1H- and 15N-labelled samples. The calculated structures present an average root mean square deviation about the mean structure of 0.63 A for backbone atoms and 1.27 A for all non-hydrogen atoms except the 12 N-terminal residues. The protein is well folded from residue 12 to residue 110, and consists of a non-bundle alpha-helical structure with six helices connected by alpha alpha loops. It has a globular shape, with overall dimensions of 32 A x 28 A x 24 A. A channel is visible in the hydrophobic core, with dimensions of 3 A x 9 A x 21 A. In some of the 20 solution structures calculated, this channel is closed either by Trp-94 at one end or by Tyr-26 at the other end; in some other solutions, this channel is closed at both ends. Binding experiments with 12-bromododecanol indicate that the CSPMbraA6 structure is modified upon ligand binding

Optimization of crystals from nanodrops: crystallization and preliminary crystallographic study of a pheromone-binding protein from the honeybee Apis mellifera L

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