Jean-Pierre Simorre

Kissing-Loop Interaction in the 3′ End of the Hepatitis C Virus Genome Essential for RNA Replication

ABSTRACT The hepatitis C virus (HCV) is a positive-strand RNA virus belonging to the Flaviviridae. Its genome carries at either end highly conserved nontranslated regions (NTRs) containing cis-acting RNA elements that are crucial for replication. In this study, we identified a novel RNA element within the NS5B coding sequence that is indispensable for replication. By using secondary structure prediction and nuclear magnetic resonance spectroscopy, we found that this RNA element, designated 5BSL3.2 by analogy to a recent report (S. You, D. D. Stump, A. D. Branch, and C. M. Rice, J. Virol. 78:1352-1366, 2004), consists of an 8-bp lower and a 6-bp upper stem, an 8-nucleotide-long bulge, and a 12-nucleotide-long upper loop. Mutational disruption of 5BSL3.2 structure blocked RNA replication, which could be restored when an intact copy of this RNA element was inserted into the 3′ NTR. By using this replicon design, we mapped the elements in 5BSL3.2 that are critical for RNA replication. Most importantly, we discovered a nucleotide sequence complementarity between the upper loop of this RNA element and the loop region of stem-loop 2 in the 3′ NTR. Mismatches introduced into the loops inhibited RNA replication, which could be rescued when complementarity was restored. These data provide strong evidence for a pseudoknot structure at the 3′ end of the HCV genome that is essential for replication.

Structure and nuclear import function of the C-terminal domain of influenza virus polymerase PB2 subunit.

The trimeric influenza virus polymerase, comprising subunits PA, PB1 and PB2, is responsible for transcription and replication of the segmented viral RNA genome. Using a novel library-based screening technique called expression of soluble proteins by random incremental truncation (ESPRIT), we identified an independently folded C-terminal domain from PB2 and determined its solution structure by NMR. Using green fluorescent protein fusions, we show that both the domain and the full-length PB2 subunit are efficiently imported into the nucleus dependent on a previously overlooked bipartite nuclear localization sequence (NLS). The crystal structure of the domain complexed with human importin α5 shows how the last 20 residues unfold to permit binding to the import factor. The domain contains three surface residues implicated in adaptation from avian to mammalian hosts. One of these tethers the NLS-containing peptide to the core of the domain in the unbound state.

Structural Basis of HIV-1 Tethering to Membranes by the BST-2/Tetherin Ectodomain

The restriction factor BST-2/tetherin contains two membrane anchors employed to retain some enveloped viruses, including HIV-1 tethered to the plasma membrane in the absence of virus-encoded antagonists. The 2.77 A crystal structure of the BST-2/tetherin extracellular core presented here reveals a parallel 90 A long disulfide-linked coiled-coil domain, while the complete extracellular domain forms an extended 170 A long rod-like structure based on small-angle X-ray scattering data. Mutagenesis analyses indicate that both the coiled coil and the N-terminal region are required for retention of HIV-1, suggesting that the elongated structure can function as a molecular ruler to bridge long distances. The structure reveals substantial irregularities and instabilities throughout the coiled coil, which contribute to its low stability in the absence of disulfide bonds. We propose that the irregular coiled coil provides conformational flexibility, ensuring that BST-2/tetherin anchoring both in the plasma membrane and in the newly formed virus membrane is maintained during virus budding.

NMR structure and ion channel activity of the p7 protein from hepatitis C virus

The small membrane protein p7 of hepatitis C virus forms oligomers and exhibits ion channel activity essential for virus infectivity. These viroporin features render p7 an attractive target for antiviral drug development. In this study, p7 from strain HCV-J (genotype 1b) was chemically synthesized and purified for ion channel activity measurements and structure analyses. p7 forms cation-selective ion channels in planar lipid bilayers and at the single-channel level by the patch clamp technique. Ion channel activity was shown to be inhibited by hexamethylene amiloride but not by amantadine. Circular dichroism analyses revealed that the structure of p7 is mainly α-helical, irrespective of the membrane mimetic medium (e.g. lysolipids, detergents, or organic solvent/water mixtures). The secondary structure elements of the monomeric form of p7 were determined by 1H and 13C NMR in trifluoroethanol/water mixtures. Molecular dynamics simulations in a model membrane were combined synergistically with structural data obtained from NMR experiments. This approach allowed us to determine the secondary structure elements of p7, which significantly differ from predictions, and to propose a three-dimensional model of the monomeric form of p7 associated with the phospholipid bilayer. These studies revealed the presence of a turn connecting an unexpected N-terminal α-helix to the first transmembrane helix, TM1, and a long cytosolic loop bearing the dibasic motif and connecting TM1 to TM2. These results provide the first detailed experimental structural framework for a better understanding of p7 processing, oligomerization, and ion channel gating mechanism.

Solid-state NMR on bacterial cells: selective cell wall signal enhancement and resolution improvement using dynamic nuclear polarization.

Dynamic nuclear polarization (DNP) enhanced solid-state nuclear magnetic resonance (NMR) has recently emerged as a powerful technique for the study of material surfaces. In this study, we demonstrate its potential to investigate cell surface in intact cells. Using Bacillus subtilis bacterial cells as an example, it is shown that the polarizing agent 1-(TEMPO-4-oxy)-3-(TEMPO-4-amino)propan-2-ol (TOTAPOL) has a strong binding affinity to cell wall polymers (peptidoglycan). This particular interaction is thoroughly investigated with a systematic study on extracted cell wall materials, disrupted cells, and entire cells, which proved that TOTAPOL is mainly accumulating in the cell wall. This property is used on one hand to selectively enhance or suppress cell wall signals by controlling radical concentrations and on the other hand to improve spectral resolution by means of a difference spectrum. Comparing DNP-enhanced and conventional solid-state NMR, an absolute sensitivity ratio of 24 was obtained on the entire cell sample. This important increase in sensitivity together with the possibility of enhancing specifically cell wall signals and improving resolution really opens new avenues for the use of DNP-enhanced solid-state NMR as an on-cell investigation tool.

A gp41 MPER-specific llama VHH requires a hydrophobic CDR3 for neutralization but not for antigen recognition.

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10.

Longitudinal-relaxation-enhanced NMR experiments for the study of nucleic acids in solution.

Atomic-resolution information on the structure and dynamics of nucleic acids is essential for a better understanding of the mechanistic basis of many cellular processes. NMR spectroscopy is a powerful method for studying the structure and dynamics of nucleic acids; however, solution NMR studies are currently limited to relatively small nucleic acids at high concentrations. Thus, technological and methodological improvements that increase the experimental sensitivity and spectral resolution of NMR spectroscopy are required for studies of larger nucleic acids or protein-nucleic acid complexes. Here we introduce a series of imino-proton-detected NMR experiments that yield an over 2-fold increase in sensitivity compared to conventional pulse schemes. These methods can be applied to the detection of base pair interactions, RNA-ligand titration experiments, measurement of residual dipolar (15)N-(1)H couplings, and direct measurements of conformational transitions. These NMR experiments employ longitudinal spin relaxation enhancement techniques that have proven useful in protein NMR spectroscopy. The performance of these new experiments is demonstrated for a 10 kDa TAR-TAR*(GA) RNA kissing complex and a 26 kDa tRNA.

Isolation and analysis of cell wall components from Streptococcus pneumoniae

The complex and heterogeneous cell wall of the pathogenic bacterium Streptococcus pneumoniae is composed of peptidoglycan and a covalently attached wall teichoic acid. The net-like peptidoglycan is formed by glycan chains that are crosslinked by short peptides. We have developed a method to purify the glycan chains, and we show that they are longer than approximately 25 disaccharide units. From purified peptidoglycan, we released 50 muropeptides that differ in the length of their peptides (tri-, tetra-, or pentapeptides with or without mono- or dipeptide branch), the degree of peptide crosslinking (monomer, dimer, or trimer), and the presence of modifications in the glycan chains (N-deacetylation, O-acetylation, or lack of GlcNAc or GlcNAc-MurNAc) or peptides (glutamic acid instead of glutamine). We also established a method to isolate wall teichoic acid chains and show that the most abundant chains have 6 or 7 repeating units. Finally, we obtained solid-state nuclear magnetic resonance spectra of whole insoluble cell walls. These novel tools will help to characterize mutant strains, cell wall-modifying enzymes, and protein-cell wall interactions.

Coordination of peptidoglycan synthesis and outer membrane constriction during Escherichia coli cell division

To maintain cellular structure and integrity during division, Gram-negative bacteria must carefully coordinate constriction of a tripartite cell envelope of inner membrane, peptidoglycan (PG), and outer membrane (OM). It has remained enigmatic how this is accomplished. Here, we show that envelope machines facilitating septal PG synthesis (PBP1B-LpoB complex) and OM constriction (Tol system) are physically and functionally coordinated via YbgF, renamed CpoB (Coordinator of PG synthesis and OM constriction, associated with PBP1B). CpoB localizes to the septum concurrent with PBP1B-LpoB and Tol at the onset of constriction, interacts with both complexes, and regulates PBP1B activity in response to Tol energy state. This coordination links PG synthesis with OM invagination and imparts a unique mode of bifunctional PG synthase regulation by selectively modulating PBP1B cross-linking activity. Coordination of the PBP1B and Tol machines by CpoB contributes to effective PBP1B function in vivo and maintenance of cell envelope integrity during division. DOI: http://dx.doi.org/10.7554/eLife.07118.001

Dynamics Characterization of Fully Hydrated Bacterial Cell Walls by Solid-State NMR: Evidence for Cooperative Binding of Metal Ions

The bacterial cell wall maintains a cells integrity while allowing growth and division. It is made up of peptidoglycan (PG), a biopolymer forming a multigigadalton bag-like structure, and, additionally in gram-positive bacteria, of covalently linked anionic polymers collectively called teichoic acids. These anionic polymers are thought to play important roles in host-cell adhesion, inflammation, and immune activation. In this Article, we compare the flexibility and the organization of peptidoglycans from gram-negative bacteria (E. coli) with its counterpart from different gram-positive bacteria using solid-state nuclear magnetic resonance spectroscopy (NMR) under magic-angle sample spinning (MAS). The NMR fingerprints suggest an identical local conformation of the PG in all of these bacterial species. Dynamics in the peptidoglycan network decreases from E. coli to B. subtilis and from B. subtilis to S. aureus and correlates mainly with the degree of peptide cross-linkage. For intact bacterial cells and isolated cell walls, we show that (31)P solid-state NMR is particularly well adapted to characterize and differentiate wall teichoic acids of different species. We have further observed complexation with divalent ions, highlighting an important structural aspect of gram-positive cell wall architecture. We propose a new model for the interaction of divalent cations with both wall teichoic acids and carbonyl groups of peptidoglycan.