Viruses in motion: maturation of an insect virus-like particle – a nanomachine

Virus-like particles (VLPs) are particles that lack the infectious genome of the parent virus but retain the ability to self-assemble. Transient expression of viral coat proteins in a variety of systems can be used to synthesize VLPs that are morphologically indistinguishable from the virus from which they have been derived. In this thesis, I have used VLPs to study virus maturation mechanisms. The main focus of this thesis has been the study of the maturation of Nudaurelia capensis omega virus (NωV), an insect virus. NωV has a pH-controlled maturation mechanism that involves a precise autocatalytic cleavage within the capsid protein that is accompanied by a dramatic decrease in the diameter of the particles. I used insect cell and plant-based transient expression of the NωV coat protein to produce procapsids and showed that reduction in pH results in cleavage of the coat protein and the concomitant decrease in particle diameter. Mature VLPs purified from plants had a structure indistinguishable from the authentic virus at 2.7 A resolution, as determined by cryoelectron microscopy (cryo-EM). However, the plant-produced VLPs showed the formation of strong interactions between the subunits of NωV, probably representing covalent crosslinks, that limited their dynamic properties. Given the fact that insect cells did not present this difficulty, we used them to make the VLPs for cryo-EM analysis of intermediates in the process of maturation. These analyses revealed the conformational changes in the structure of the particles that accompany maturation, such as the formation of the cleavage site and the closure of the pores of the particle. The culmination of this thesis is represented by a movie of the maturation process of NωV. This movie provides, for the first time, high-resolution information about the large conformational changes and protein rearrangements occurring during the maturation of this virus. Keywords: Nudaurelia capensis omega virus, virus maturation, autocatalytic cleavage, virus-like particle, cryo-electron microscopy Resum (abstract in Catalan) Les particules similiviriques (VLPs), acronim de l’angles Virus-Like Particles, son particules que no tenen el material genetic viral pero que conserven la capacitat d’autoassemblar-se. Per sintetitzar les VLPs es poden utilitzar diversos sistemes mitjancant els quals es produeixen les proteines de la capsida del virus. Aquestes particules son morfologicament identiques als virus originals dels quals han estat derivades. En aquesta tesi he utilitzat les VLPs per estudiar els mecanismes de maduracio dels virus. L’objectiu principal d’aquesta tesi ha estat l’estudi del proces de maduracio de Nudaurelia capensis omega virus (NωV), un virus d’insecte. NωV te un mecanisme de maduracio, que esta controlat pel pH, que implica una segmentacio autocatalitica de la proteina de la capsida del virus i va acompanyada d’una reduccio del diametre de les particules. En aquesta tesi vaig utilitzar cel・lules d’insectes i plantes per tal de produir la proteina de la capsida de NωV i generar particules, conegudes com a procapsides. Endemes vaig demostrar que la reduccio en el pH dona lloc a la segmentacio de la proteina de la capsida i a la reduccio del diametre de les particules. Mitjancant criomicroscopia electronica (cryo-EM) vaig obtenir, a una resolucio de 2.7 A, l’estructura de particules que havien estat madurades en plantes i vaig veure que tenien una estructura identica als virus autentics. Tot i aixo, les VLPs extretes de plantes tenien interaccions fortes entre les seves subunitats, probablement degut a enllacos covalents, i aixo va limitar les seves propietats dinamiques. Tenint en compte que les cel・lules d’insecte no presentaven aquesta dificultat, les vam utilitzar per obtenir les estructures dels intermedis en el proces de maduracio mitjancant analisis amb cryo-EM. Aquestes analisis van revelar els grans canvis conformacionals en l’estructura de les particules que ocorren durant la maduracio, com ara la formacio del punt de segmentacio i el tancament dels porus de la particula. La conclusio d’aquesta tesi esta representada per una pel・licula del proces de maduracio de NωV. Aquesta pel・licula proporciona, per primera vegada, informacio d’alta resolucio sobre els grans canvis conformacionals i els reordenaments de proteines que es produeixen durant la maduracio d’aquest virus. Paraules clau: Nudaurelia capensis omega virus, maduracio viral, segmentacio autocatalitica, particules similiviriques, criomicroscopia electronic

Introduction of glucan synthase into the cytosol in wheat endosperm causes massive maltose accumulation and represses starch synthesis.

We expressed a bacterial glucan synthase (Agrobacterium GlgA) in the cytosol of developing endosperm cells in wheat grains, to discover whether it could generate a glucan from cytosolic ADP-glucose. Transgenic lines had high glucan synthase activity during grain filling, but did not accumulate glucan. Instead, grains accumulated very high concentrations of maltose. They had large volumes during development due to high water content, and very shrivelled grains at maturity. Starch synthesis was severely reduced. We propose that cytosolic glucan synthesized by the glucan synthase was immediately hydrolysed to maltose by cytosolic β-amylase(s). Maltose accumulation resulted in a high osmotic potential in developing grain, drawing in excess water that stretched the seed coat and pericarp. Loss of water during grain maturation then led to shrinkage when the grains matured. Maltose accumulation is likely to account for the reduced starch synthesis in transgenic grains, through signalling and toxic effects. Using bioinformatics, we identify an isoform of β-amylase likely to be responsible for maltose accumulation. Removal of this isoform through identification of TILLING mutants or genome editing, combined with co-expression of heterologous glucan synthase and a glucan branching enzyme, may in future enable elevated yields of carbohydrate through simultaneous accumulation of starch and cytosolic glucan

OnfD, an AraC-Type Transcriptional Regulator Encoded by Rhizobium tropici CIAT 899 and Involved in Nod Factor Synthesis and Symbiosis

Rhizobium tropici CIAT 899 is a broad-host-range rhizobial strain that establishes symbiotic interactions with legumes and tolerates different environmental stresses such as heat, acidity, or salinity. This rhizobial strain produces a wide variety of symbiotically active nodulation factors (NF) induced not only by the presence of plant-released flavonoids but also under osmotic stress conditions through the LysR-type transcriptional regulators NodD1 (flavonoids) and NodD2 (osmotic stress). However, the activation of NodD2 under high-osmotic-stress conditions remains elusive. Here, we have studied the role of a new AraC-type regulator (named as OnfD) in the symbiotic interaction of R. tropici CIAT 899 with Phaseolus vulgaris and Lotus plants. We determined that OnfD is required under salt stress conditions for the transcriptional activation of the nodulation genes and therefore the synthesis and export of NF, which are required for a successful symbiosis with P. vulgaris. Moreover, using bacterial two-hybrid analysis, we demonstrated that the OnfD and NodD2 proteins form homodimers and OnfD/NodD2 form heterodimers, which could be involved in the production of NF in the presence of osmotic stress conditions since both regulators are required for NF synthesis in the presence of salt. A structural model of OnfD is presented and discussed. IMPORTANCE The synthesis and export of rhizobial NF are mediated by a conserved group of LysR-type regulators, the NodD proteins. Here, we have demonstrated that a non-LysR-type regulator, an AraC-type protein, is required for the transcriptional activation of symbiotic genes and for the synthesis of symbiotically active NF under salt stress conditions.España Ministerio de Economía y Competitividad (project AGL2016-77163-R

Viruses in motion: exploring virus maturation stages with an insect virus, plants and cryo-electron microscopy

Trabajo presentado al Seminario del CRAG, celebrado el 7 de enero de 2020.Peer reviewe

X-ray crystal structure of a designed rigidified imaging scaffold in the ligand-free conformation.

Imaging scaffolds composed of designed protein cages fused to designed ankyrin repeat proteins (DARPins) have enabled the structure determination of small proteins by cryogenic electron microscopy (cryo-EM). One particularly well characterized scaffold type is a symmetric tetrahedral assembly composed of 24 subunits, 12 A and 12 B, which has three cargo-binding DARPins positioned on each vertex. Here, the X-ray crystal structure of a representative tetrahedral scaffold in the apo state is reported at 3.8 Å resolution. The X-ray crystal structure complements recent cryo-EM findings on a closely related scaffold, while also suggesting potential utility for crystallographic investigations. As observed in this crystal structure, one of the three DARPins, which serve as modular adaptors for binding diverse `cargo proteins, present on each of the vertices is oriented towards a large solvent channel. The crystal lattice is unusually porous, suggesting that it may be possible to soak crystals of the scaffold with small (≤30 kDa) protein cargo ligands and subsequently determine cage-cargo structures via X-ray crystallography. The results suggest the possibility that cryo-EM scaffolds may be repurposed for structure determination by X-ray crystallography, thus extending the utility of electron-microscopy scaffold designs for alternative structural biology applications

NRC4 Gene Cluster Is Not Essential for Bacterial Flagellin-Triggered Immunity

Plants utilise cell surface pattern recognition receptors (PRRs) and intracellular nucleotide-binding domain leucine-rich repeat containing receptors (NLRs) to fend off invading pathogens (Dodds and Rathjen, 2010; Win et al., 2012). Both types of immune receptors detect pathogen molecules directly or indirectly to activate complex immune responses and disease resistance (Kourelis and van der Hoorn, 2018). Although PRR- and NLR-triggered immunity are generally thought to activate distinct pathways, they can induce similar outputs such as production of reactive oxygen species (ROS) and hypersensitive cell death (Peng et al., 2018). Both PRR-and NLR-activated pathways involve calcium-dependent protein kinases, mitogen-activate protein kinases (MAPKs), phytohormone signalling, and transcriptional reprogramming (Peng et al., 2018). However, whether these two pathways converge at some point to potentiate and strengthen the immune response remains unclear. A recent study suggested that the tomato NLR helper NRC4 positively regulates the ROS burst induced by the bacterial flagellin peptide flg22 (Leibman-Markus et al. 2018b). We took advantage of the CRISPR/Cas9 system to knock out multiple NRC genes in tomato and Nicotiana benthamiana. Although these mutants failed to respond to the NRC-dependent NLRs, they remained unaltered in flg22-induced responses. We conclude that the NRC genes are not essential for flg22-induced responses in tomato and N. benthamiana

OnfD, an AraC-Type Transcriptional Regulator Encoded by Rhizobium tropici CIAT 899 and Involved in Nod Factor Synthesis and Symbiosis.

Rhizobium tropici CIAT 899 is a broad-host-range rhizobial strain that establishes symbiotic interactions with legumes and tolerates different environmental stresses such as heat, acidity, or salinity. This rhizobial strain produces a wide variety of symbiotically active nodulation factors (NF) induced not only by the presence of plant-released flavonoids but also under osmotic stress conditions through the LysR-type transcriptional regulators NodD1 (flavonoids) and NodD2 (osmotic stress). However, the activation of NodD2 under high-osmotic-stress conditions remains elusive. Here, we have studied the role of a new AraC-type regulator (named as OnfD) in the symbiotic interaction of R. tropici CIAT 899 with Phaseolus vulgaris and Lotus plants. We determined that OnfD is required under salt stress conditions for the transcriptional activation of the nodulation genes and therefore the synthesis and export of NF, which are required for a successful symbiosis with P. vulgaris Moreover, using bacterial two-hybrid analysis, we demonstrated that the OnfD and NodD2 proteins form homodimers and OnfD/NodD2 form heterodimers, which could be involved in the production of NF in the presence of osmotic stress conditions since both regulators are required for NF synthesis in the presence of salt. A structural model of OnfD is presented and discussed.IMPORTANCE The synthesis and export of rhizobial NF are mediated by a conserved group of LysR-type regulators, the NodD proteins. Here, we have demonstrated that a non-LysR-type regulator, an AraC-type protein, is required for the transcriptional activation of symbiotic genes and for the synthesis of symbiotically active NF under salt stress conditions