Jean-Paul Rolland

A supramolecular assembly formed by influenza A virus genomic RNA segments

The influenza A virus genome consists of eight viral RNAs (vRNAs) that form viral ribonucleoproteins (vRNPs). Even though evidence supporting segment-specific packaging of vRNAs is accumulating, the mechanism ensuring selective packaging of one copy of each vRNA into the viral particles remains largely unknown. We used electron tomography to show that the eight vRNPs emerge from a common ‘transition zone’ located underneath the matrix layer at the budding tip of the virions, where they appear to be interconnected and often form a star-like structure. This zone appears as a platform in 3D surface rendering and is thick enough to contain all known packaging signals. In vitro, all vRNA segments are involved in a single network of intermolecular interactions. The regions involved in the strongest interactions were identified and correspond to known packaging signals. A limited set of nucleotides in the 5′ region of vRNA 7 was shown to interact with vRNA 6 and to be crucial for packaging of the former vRNA. Collectively, our findings support a model in which the eight genomic RNA segments are selected and packaged as an organized supramolecular complex held together by direct base pairing of the packaging signals.

Supramolecular shuttle and protective agent: a multiple role of methylated cyclodextrins in the chemoselective hydrogenation of benzene derivatives with ruthenium nanoparticles

Efficient chemoselectivities have been obtained in the hydrogenation of benzene derivatives under biphasic liquid-liquid conditions using Ru(0) nanoparticles stabilized and controlled by the relevant choice of cavity and methylation degree of cyclodextrins.

Apo-Hsp90 coexists in two open conformational states in solution

Background information. Hsp90 (90 kDa heat‐shock protein) plays a key role in the folding and activation of many client proteins involved in signal transduction and cell cycle control. The cycle of Hsp90 has been intimately associated with large conformational rearrangements, which are nucleotide‐binding‐dependent. However, up to now, our understanding of Hsp90 conformational changes derives from structural information, which refers to the crystal states of either recombinant Hsp90 constructs or the prokaryotic homologue HtpG (Hsp90 prokaryotic homologue).

Functional characterization of a microbial aquaglyceroporin

The major intrinsic proteins (MIPs) constitute a widespread membrane channel family essential for osmotic cell equilibrium. The MIPs can be classified into three functional subgroups: aquaporins, glycerol facilitators and aquaglyceroporins. Bacterial MIP genes have been identified in archaea as well as in Gram-positive and Gram-negative eubacteria. However, with the exception of Escherichia coli, most bacterial MIPs have been analysed by sequence homology. Since no MIP has yet been functionally characterized in Gram-positive bacteria, we have studied one of these members from Lactococcus lactis. This MIP is shown to be permeable to glycerol, like E. coli GlpF, and to water, like E. coli AqpZ. This is the first characterization of a microbial MIP that has a mixed function. This result provides important insights to reconstruct the evolutionary history of the MIP family and to elucidate the molecular pathway of water and other solutes in these channels.

tmRNA–SmpB: a journey to the centre of the bacterial ribosome

Ribosomes mediate protein synthesis by decoding the information carried by messenger RNAs (mRNAs) and catalysing peptide bond formation between amino acids. When bacterial ribosomes stall on incomplete messages, the trans‐translation quality control mechanism is activated by the transfer‐messenger RNA bound to small protein B (tmRNA–SmpB ribonucleoprotein complex). Trans‐translation liberates the stalled ribosomes and triggers degradation of the incomplete proteins. Here, we present the cryo‐electron microscopy structures of tmRNA–SmpB accommodated or translocated into stalled ribosomes. Two atomic models for each state are proposed. This study reveals how tmRNA–SmpB crosses the ribosome and how, as the problematic mRNA is ejected, the tmRNA resume codon is placed onto the ribosomal decoding site by new contacts between SmpB and the nucleotides upstream of the tag‐encoding sequence. This provides a structural basis for the transit of the large tmRNA–SmpB complex through the ribosome and for the means by which the tmRNA internal frame is set for translation to resume.

Aquaglyceroporins, one channel for two molecules.

In the light of the recently published structure of GlpF and AQP1, we have analysed the nature of the residues which could be involved in the formation of the selectivity filter of aquaporins, glycerol facilitators and aquaglyceroporins. We demonstrate that the functional specificity for major intrinsic protein (MIP) channels can be explained on one side by analysing the polar environment of the residues that form the selective filter. On the other side, we show that the channel selectivity could be associated with the oligomeric state of the membrane protein. We conclude that a non-polar environment in the vicinity of the top of helix 5 could allow aquaglyceroporins and GlpF to exist as monomers within the hydrophobic environment of the membrane.

Accommodation of tmRNA-SmpB into stalled ribosomes: A cryo-EM study

In eubacteria, translation of defective messenger RNAs (mRNAs) produces truncated polypeptides that stall on the ribosome. A quality control mechanism referred to as trans-translation is performed by transfer-messenger RNA (tmRNA), a specialized RNA acting as both a tRNA and an mRNA, associated with small protein B (SmpB). So far, a clear view of the structural movements of both the protein and RNA necessary to perform accommodation is still lacking. By using a construct containing the tRNA-like domain as well as the extended helix H2 of tmRNA, we present a cryo-electron microscopy study of the process of accommodation. The structure suggests how tmRNA and SmpB move into the ribosome decoding site after the release of EF-Tu.GDP. While two SmpB molecules are bound per ribosome in a preaccommodated state, our results show that during accommodation the SmpB protein interacting with the small subunit decoding site stays in place while the one interacting with the large subunit moves away. Relative to canonical translation, an additional movement is observed due to the rotation of H2. This suggests that the larger movement required to resume translation on a tmRNA internal open reading frame starts during accommodation.

Importance of viral genomic composition in modulating glycoprotein content on the surface of influenza virus particles.

Despite progress in our knowledge of the internal organisation of influenza virus particles, little is known about the determinants of their morphology and, more particularly, of the actual abundance of structural proteins at the virion level. To address these issues, we used cryo-EM to focus on viral (and host) factors that might account for observed differences in virion morphology and characteristics such as size, shape and glycoprotein (GP) spike density. Twelve recombinant viruses were characterised in terms of their morphology, neuraminidase activity and virus growth. The genomic composition was shown to be important in determining the GP spike density. In particular, polymerase gene segments and especially PB1/PB2 were shown to have a prominent influence in addition to that for HA in determining GP spike density, a feature consistent with a functional link between these virus components important for virus fitness.

In vitro characterization of naturally occurring influenza H3NA− viruses lacking the NA gene segment: Toward a new mechanism of viral resistance?

Among a panel of 788 clinical influenza H3N2 isolates, two isolates were characterized by an oseltamivir-resistant phenotype linked to the absence of any detectable NA activity. Here, we established that the two H3NA- isolates lack any detectable full-length NA segment, and one of these could be rescued by reverse genetics in the absence of any NA segment sequence. We found that the absence of NA segment induced a moderate growth defect of the H3NA- viruses as on cultured cells. The glycoproteins density at the surface of H3NA- virions was unchanged as compared to H3N2 virions. The HA protein as well as residues 188 and 617 of the PB1 protein were shown to be strong determinants of the ability of H3NA- viruses to grow in the absence of the NA segment. The significance of these findings about naturally occurring seven-segment influenza A viruses is discussed.

Parainfluenza virus type 5 (PIV-5) morphology revealed by cryo-electron microscopy

The knowledge of parainfluenza type 5 (PIV-5) virion morphology is essentially based on the observation of negatively stained preparations in conventional transmission electron microscopy (CTEM). In this study, the ultrastructure of frozen-hydrated intact PIV-5 was examined by cryo-electron microscopy (cryo-EM). Cryo-EM revealed a majority of spherical virions (70%), with a lower pleiomorphy than originally observed in CTEM. Phospholipid bilayer thickness, spike length and glycoprotein spikes density were measured. About 2000 glycoprotein spikes were present in an average-sized spherical virion. Altogether, these data depict a more precise view of PIV-5 morphology.