Enteric bacteria such as Escherichia coli, Salmonella typhimirium and Vibrio cholera possess a wide array of acid stress response systems to counteract the extreme acidity encountered when invading the host’s digestive or urinary tracts. This project aims to analyse structure-functional relationships of the inducible lysine decarboxylase LdcI system to deeper understand its mechanism of action and its role in acid resistance, and to explore its newly described link with the stringent response. Indeed, we recently discovered that LdcI tightly binds to the procaryotic stringent response regulator alarmone. In addition, we identified LdcI as interacting with a novel AAA+ ATPase RavA that appears to have been specifically evolved to target LdcI. Moreover, we found that RavA reduces the inhibition of LdcI activity by the alarmone in vitro as well as in vivo. We solved Xray crystal structures of the LdcI decamer and of the RavA monomer, and obtained three-dimensional reconstructions of LdcI, the RavA-ADP hexamer and the LdcI-RavA-ADP complex by electron microscopy. The latter complex revealed itself as a suprising cage structure composed of two LdcI decamers linked together by five RavA hexamers. A combination of the Xray crystal structures with the low resolution electron microscopy maps enabled us to propose an atomic model of the RavA hexamer and get first insights into the basis of its interaction with LdcI. Furthermore, we have evidence for a ternary complex formation between LdcI, RavA and a novel metal-binding VWA domain protein ViaA. Our consortium will continue to explore the interplay between LdcI, RavA, ViaA, nucleotides as an energy source for RavA and alarmone as a stress signal molecule, by an intergated multi-level approach envolving structural, molecular and in vivo cell biology. In the scope of this grant application, we will focus on providing structural insights into this crucial enteric bacterial acid stress response system by high resolution cryoelectron microscopy. Unraveling these interactions is of particular interest for two reasons. First, this knowledge should be profitable for future clinical use, since bacterial infectivity correlates with their ability to withstand acid stress. Thus, blocking or regulating the LdcI interaction with its partners is a potential target for pharmaceutical intervention. Second, understanding the interaction in such an intriguing supramolecular complex as LdcI-RavA will greatly add to our knowledge of the basic principles of specific protein-protein recognition, which play central roles in all biological processes.

The Archaea constitutes one of three domains of life on Earth, along with Eukarya and Bacteria. Archaea comprise three major phyla Euryarchaeota, Crenarcheaota and Thaumarchaeota, that exhibit some critical differences in major molecular mechanisms, such as DNA replication and cell division. Thus, Euryarchaeota and Thaumarchaeota division employs a bacterial-like FtsZ, apparatus whereas for Crenarchaeota, where FtsZ is lacking, cytokinesis relies on a newly discovered Cdv (for Cell division) machinery. Importantly, two of the three Cdv proteins, CdvB and CdvC are homologous of eukaryotic proteins belonging to the “endosomal sorting complex required for transport” (ESCRT) machinery, which catalyzes vesicle budding during endosomal protein sorting, budding of some enveloped viruses and cytokinesis. CdvB is a homologue of ESCRT-III, implicated in membrane fission and CdvC is a homologue of the AAA-type ATPase Vps4, required for ESCRT-III disassembly. The third member of the Cdv cluster, namely CdvA, is only present in Archaea. Our own experiments support the hypothesis that CdvA could constitute an ancient cytoskeleton protein involved in cell division. Several data reinforce the concept that archaeal Cdv proteins are closely related to the eukaryotic ESCRT-III counterparts including the presence of a MIT domain interacting motif (MIM) in the C-terminus of CdvB which is employed to interact with CdvC. This region of CdvB also contains a domain responsible for its interaction with CdvA, the CdvA/CdvB and CdvC/CdvB interactions being not mutually exclusive. Although interactions of Cdv proteins encoded by the Cdv gene cluster were established, nothing is known about the CdvB paralogs present in Crenarcheal genomes. Moreover, we lack functional, mechanistic and structural details showing how these proteins catalyze cytokinesis. These findings raise fascinating questions regarding the evolutionary history of cell division in Archaea and, on a more global extend, the mechanisms responsible for membrane remodeling. In order to understand the structural basis of membrane remodeling by archaeal ESCRT-III we will pursue five main objectives. First, we will solve the crystal structure of full length CdvB. This will serve as a basis to dissect the structure and provide insight into its activation and polymerization properties. Secondly, we will dissect the polymerization propensity of CdvB and determine the structure of the repeating subunit of the polymer, which will provide important insight into membrane interaction and remodeling. Aims 1 and 2 will be extended to the CdvB homologues and characterized with regard to their interactions with CdvB and/or CdvC. The third aim is to study the mechanism of ESCRT-III disassembly by CdvC. Our main goal is to determine the crystal structure of the CdvC dodecamer in the presence or absence of nucleotides or analogues. This will ultimately allow to determine the conformational state of each protomer within the dodecamer and will thus provide essential new insight into the mechanism of AAA-type ATPases and ESCRT-III disassembly. A fourth goal is to study the effect of CdvB on membrane structure in collaboration with the Bassereau’s lab (Institut Curie, Paris) in order to directly visualize changes in membrane structure employing GUV-membrane tubes. Finally, insights from the structural studies will be complemented and validated by mutational studies in vivo to obtain further insight into the mechanism of archaeal cytokinesis (coll R. Bernander, Uppsala, Sweden). This project will provide a comprehensive view of the structural basis of the archaeal membrane remodeling machinery leading to cytokinesis. Because of the importance of ESCRT-III during diverse eukaryotic processes, the archaeal work is performed in parallel to studies on the eukaryotic ESCRT machinery in the Weissenhorn’s lab and will thus add important complementary insight into ESCRT-III driven membrane remodeling processes.

Smallpox has been for centuries a major disease before it was eradicated in 1979. Still the risks of its use in bioterrorism and a reintroduction of related poxviruses from animal reservoirs persist. Poxviruses are unique in terms of their cytoplasmic replication, which relies fully on virally encoded proteins. Their genome forms a linear double-stranded DNA, which is circularized at its extremities by hairpin structures. Such genomic organization is also found in some phycodnaviruses and afsarviruses. All these viruses also share the same type of DNA polymerase, helicase-primase, and some other specificities. They have been grouped in the Nucleo-Cytoplasmic Large DNA Viruses (NCLDV) clade. In the beginning of the 80s, several models of poxvirus replication have been proposed based on nick formation, then unidirectional strand elongation followed by strand rearrangement. But the identification of a primase and the first results on the 3D structure of the replication complex obtained recently by our teams challenge these established models. We showed that the helicase-primase D5 is hexameric as other SF3 helicases. This finding suggests rather a mechanism involving a replication fork. We propose to push further the structural and functional investigations of the replication machinery involving, on one hand, the complex formed by the DNA polymerase E9, the uracil-DNA glycosylase D4, and the processivity factor A20, and on the other hand, the hexameric helicase-primase D5. Using recombinant expression of proteins from vaccinia virus, which is a safe model system 97 % identical to smallpox virus, we are able to produce milligram amounts of the four components in insect cells. Some constructs of D4 and A20 have also been expressed in bacteria. Protein crystallography and electron microscopy will be used to continue our structural work in order to determine the details of the molecular interactions within these complexes. A reconstitution of the replication machinery in vitro is also in reach and will greatly facilitate our functional studies. As we revealed recently the considerable size of the replication complex, we would like to revisit the role of D4 and the primase activity of D5 using this in vitro model. The helicase activity of D5 has still to be shown experimentally and may require the identification and inclusion of additional partner proteins. We are confident that a breakthrough in the understanding of poxvirus replication is in reach, with strong implications for other viruses within the NCLDV clade and a potential application in the design of antivirals, in particular of compounds targeting the interaction surfaces within the complex.

Understanding the molecular mechanisms of viral replication and of the interactions between viruses and their host-cells is a major undertaking that will shed light on the evolution of viruses, on host-virus adaptations and that will give us new tools to fight against current and emerging pathogens. In this project, we focus on the non-segmented negative strand RNA viruses (NNV). Some of these viruses are highly prevalent in humans (measles virus, mumps virus, parainfluenza viruses, respiratory syncytial virus, metapneumovirus) while others continue to emerge and cause serious diseases (rabies virus, Ebola virus, Nipah virus …). Although vaccines are available against some NNV, we are still lacking an efficient drug to fight these pathogens. Because all NNV share a common organization of their genome and similar mechanisms of transcription and replication, we believe that deciphering the structure and the molecular mechanisms involved in the replication of a model virus such as vesicular stomatitis virus (VSV) will help in understanding the replication machine of other NNV and will point to Achilles’ heels that might then be specifically targeted for structural studies and the development of antiviral therapies. NNV carry their own machinery for transcribing the viral genome into mRNAs and for replication of this genome. This machinery is specific and thus represents a target for developing inhibitors of viral replication. It consists of a large ribonucleoprotein complex made of the RNA genome coated by the nucleoprotein (N), the phosphoprotein (P) and the large subunit (L) of the RNA-dependent RNA polymerase. The mechanism of gene expression of NNV has been studied for more than 40 years, but many details of action and regulation of this viral machine remain the subject of speculation and controversy. In particular, we are still lacking detailed structural information about the polymerase and we still do not know whether a unique complex catalyzes both types of RNA synthesis or whether complexes of different composition are involved. Also, we lack a clear description of the mechanisms by which the polymerase switches between its two activities, by which it gains access to the N-coated RNA and by which it moves along this template. With this project we aim to understand the fundamental mechanisms of the viral machine that transcribes and replicates the RNA genome of a model NNV, the vesicular stomatitis virus (VSV). The project relies on structures of several essential components of this machine that we solved recently, in particular of the N0-P complex. These structures brought new insights and raised new hypotheses about the functioning of these complex processes. It also relies on preliminary results obtained in our laboratories. In particular, we are now able to produce all highly purified components, including the L protein, and we have been able to reconstitute transcription activity by mixing them together. Also, for the first time, we have obtained a preliminary 3D reconstruction of the L protein by electron microscopy. By a multidisciplinary approach that combines in vitro and in vivo studies, we propose to pursue our structural and functional characterization of the VSV polymerase complex. We propose to determine the structure of the L protein at the highest possible resolution and to characterize the organization and structure of the polymerase machine in complex with its partners. By reconstituting the transcription and replication processes in vitro and in vivo, we propose to answer specific questions about the mechanisms of these reactions. Finally, we will also seek to validate molecular targets for the development of antiviral inhibitors and to determine their structures at high resolution.