In situ Alphavirus Assembly and Budding Mechanism Revealed by Cellular CryoET

Chikungunya virus (CHIKV) is an alphavirus and the etiological agent for debilitating arthritogenic disease in humans. Previous studies with purified virions or budding mutants have not resolved the structural mechanism of alphavirus assembly in situ. Here we used cryogenic electron tomography (cryoET) imaging of CHIKV-infected human cells and subvolume classification to resolve distinct assembly intermediate conformations. These structures revealed that particle formation is driven by the spike envelope layer. Additionally, we showed that asymmetric immature nucleocapsids (NCs) provide scaffolds to trigger assembly of the icosahedral spike lattice, which progressively transforms immature NCs into icosahedral cores during virus budding. Further, cryoET of the infected cells treated with neutralizing antibodies (NAbs) showed that NAb-induced blockage of CHIKV assembly was achieved by preventing spike-spike lateral interactions that are required to bend the plasma membrane around NC cores. These findings provide molecular mechanisms for designing antivirals targeting spike-driven assembly/budding of viruses.

Properties of Photosystem II lacking the PsbJ subunit

Photosystem II (PSII), the oxygen-evolving enzyme, consists of 17 trans-membrane and 3 extrinsic membrane proteins. Other subunits bind to PSII during assembly, like Psb27, Psb28, Tsl0063. The presence of Psb27 has been proposed (Zabret et al. 2021; Huang et al. 2021; Xiao et al. 2021) to prevent the binding of PsbJ, a single transmembrane α-helix close to the quinone QB binding site. Consequently, a PSII rid of Psb27, Psb28 and Tsl0034 prior to the binding of PsbJ would logically correspond to an assembly intermediate. The present work describes experiments aiming at further characterizing such a ΔPsbJ-PSII, purified from the thermophilic Thermosynechococcus elongatus, by means of MALDI-TOF spectroscopy, Thermoluminescence, EPR spectroscopy and UV-visible time-resolved spectroscopy. In the purified ΔPsbJ-PSII, an active Mn4CaO5 cluster is present in 60-70 % of the centers. In these centers, although the forward electron transfer seems not affected, the Em of the QB/QB- couple increases by ≈120 mV thus disfavoring the electron coming back on QA. The increase of the energy gap between QA/QA- and QB/QB- could contribute in a protection against the charge recombination between the donor side and QB-, identified at the origin of photoinhibition under low light (Keren et al. 1997), and possibly during the slow photoactivation process.

Structural insights into how Prp5 proofreads the pre-mRNA branch site

During the splicing of introns from precursor messenger RNAs (pre-mRNAs), the U2 small nuclear ribonucleoprotein (snRNP) must undergo stable integration into the spliceosomal A complex—a poorly understood, multistep process that is facilitated by the DEAD-box helicase Prp5 (refs. 1–4). During this process, the U2 small nuclear RNA (snRNA) forms an RNA duplex with the pre-mRNA branch site (the U2–BS helix), which is proofread by Prp5 at this stage through an unclear mechanism5. Here, by deleting the branch-site adenosine (BS-A) or mutating the branch-site sequence of an actin pre-mRNA, we stall the assembly of spliceosomes in extracts from the yeast Saccharomyces cerevisiae directly before the A complex is formed. We then determine the three-dimensional structure of this newly identified assembly intermediate by cryo-electron microscopy. Our structure indicates that the U2–BS helix has formed in this pre-A complex, but is not yet clamped by the HEAT domain of the Hsh155 protein (Hsh155HEAT), which exhibits an open conformation. The structure further reveals a large-scale remodelling/repositioning of the U1 and U2 snRNPs during the formation of the A complex that is required to allow subsequent binding of the U4/U6.U5 tri-snRNP, but that this repositioning is blocked in the pre-A complex by the presence of Prp5. Our data suggest that binding of Hsh155HEAT to the bulged BS-A of the U2–BS helix triggers closure of Hsh155HEAT, which in turn destabilizes Prp5 binding. Thus, Prp5 proofreads the branch site indirectly, hindering spliceosome assembly if branch-site mutations prevent the remodelling of Hsh155HEAT. Our data provide structural insights into how a spliceosomal helicase enhances the fidelity of pre-mRNA splicing.

Tropomyosin isoforms segregate into distinct clusters on single actin filaments

Tropomyosins (Tpm) are rod-shaped proteins that interact head-to-tail to form a continuous polymer along both sides of most cellular actin filaments. Head-to-tail interaction between adjacent Tpm molecules and the formation of an overlap complex between them leads to the assembly of actin filaments with one type of Tpm isoform in time and space. Variations in the affinity of tropomyosin isoforms for different actin structures are proposed as a potential sorting mechanism. However, the detailed mechanisms of spatio-temporal sorting of Tpms remain elusive. In this study, we investigated the early intermediates during actin-tropomyosin filament assembly, using skeletal/cardiac Tpm isoform (Tpm1.1) and a cytoskeletal isoform (Tpm1.6) that differ only in the last 27 amino acids. We investigated how the muscle isoform Tpm1.1 and the cytoskeletal isoform Tpm1.6 nucleate domains on the actin filament and tested whether (1) recruitment is affected by the actin isoform (muscle vs cytoskeletal) and (2) whether there is specificity in recruiting the same isoform to a domain at these early stages. To address these questions, actin filaments were exposed to low concentrations of fluorescent tropomyosins in solution. The filaments were immobilized onto glass coverslips and the pattern of decoration was visualized by TIRF microscopy. We show that at the early assembly stage, tropomyosins formed multiple distinct fluorescent domains (here termed "cluster") on the actin filaments. An automated image analysis algorithm was developed and validated to identify clusters and estimate the number of tropomyosins in each cluster. The analysis showed that tropomyosin isoform sorting onto an actin filament is unlikely to be driven by a preference for nucleating on the corresponding muscle or cytoskeletal actin isoforms but rather is facilitated by a higher probability of incorporating the same tropomyosin isoforms into an early assembly intermediate. We showed that the 27 amino acids at the end of each tropomyosin seem to provide enough molecular information for attachment of the same tropomyosin isoforms adjacent to each other on an actin filament. This results in the formation of homogeneous clusters composed of the same isoform rather than clusters with mixed isoforms.

OJIP chlorophyll fluorescence induction profiles and plastoquinone binding affinity of the Photosystem II assembly intermediate PSII-I from Thermosynechococcus elongatus

Binding of Psb28 to the photosystem II assembly intermediate PSII-I induces conformational changes to the PSII acceptor side that impact charge recombination and reduce the in situ production of singlet oxygen (Zabret et al. 2021, Nat. Plants 7, 524-538). A detailed fluorometric analysis of the PSII-I assembly intermediate compared with OEC-disrupted and Mn-depleted PSII complexes showed differences between their variable (OJIP) chlorophyll fluorescence induction profiles. These revealed a distinct destabilisation of the QA- state in the PSII-I assembly intermediate and inactivated PSII samples related to an increased rate of direct and safe charge recombination. Furthermore, inactivation or removal of the OEC increases the binding affinity for plastoquinone analogues like DCBQ to the different PSII complexes. These results might indicate a mechanism that further contributes to the protection of PSII during biogenesis or repair.

An Engineered Maturation Cleavage Provides a Recombinant Mimic of Foot-and-Mouth Disease Virus Capsid Assembly-Disassembly

Picornavirus capsids are assembled from 60 copies of a capsid precursor via a pentameric assembly intermediate or ‘pentamer’. Upon completion of virion assembly, a maturation event induces a final cleavage of the capsid precursor to create the capsid protein VP4, which is essential for capsid stability and entry into new cells. For the picornavirus foot-and-mouth disease virus (FMDV), intact capsids are temperature and acid-labile and can disassemble into pentamers. During disassembly, capsid protein VP4 is lost, presumably altering the structure and properties of the resulting pentamers. The purpose of this study was to compare the characteristics of recombinant “assembly” and “disassembly” pentamers. We generated recombinant versions of these different pentamers containing an engineered cleavage site to mimic the maturation cleavage. We compared the sedimentation and antigenic characteristics of these pentamers using sucrose density gradients and reactivity with an antibody panel. Pentamers mimicking the assembly pathway sedimented faster than those on the disassembly pathway suggesting that for FMDV, in common with other picornaviruses, assembly pentamers sediment at 14S whereas only pentamers on the disassembly pathway sediment at 12S. The reactivity with anti-VP4 antibodies was reduced for the 12S pentamers, consistent with the predicted loss of VP4. Reactivity with other antibodies was similar for both pentamers suggesting that major antigenic features may be preserved between the VP4 containing assembly pentamers and the disassembly pentamers lacking VP4.

Edge strand of Escherichia coli BepA interacts with immature LptD on the β-barrel assembly machine to direct it to on- and off-pathways

The outer membrane (OM) of gram-negative bacteria is crucial for maintenance of cell viability as it functions as a selective permeability barrier. Escherichia coli periplasmic Zn-metallopeptidase BepA contributes to the maintenance of OM integrity through its involvement in the biogenesis and degradation of an essential OM protein, LptD, a β-barrel component of the lipopolysaccharide translocon. We have previously shown that BepA either promotes the maturation of LptD when it is on the normal assembly pathway (on-pathway) or degrades it when its assembly is compromised (off-pathway). BepA performs these functions possibly on the β‐barrel assembly machinery (BAM) complex. However, the mechanistic details of how BepA recognizes and directs the LptD assembly intermediates to different pathways remains unclear. Here, we performed site-directed mutagenesis and crosslinking experiments to explore the interactions among BepA, LptD, and the BAM complex. We found that the interaction of the BepA edge strand located adjacent to the active site with LptD was crucial not only for proteolysis but also for assembly promotion of LptD. Site-directed crosslinking analysis indicated that the unstructured N-terminal half of the β-barrel-forming domain of an LptD assembly intermediate directly contacts with the BepA edge strand. Furthermore, the C-terminal region of the β-barrel-forming domain of the BepA-bound LptD intermediate interacted with a 'seam' strand of BamA, suggesting that BepA recognized LptD assembling on the BAM complex. Our findings provide important insights into the involvement of BepA in the maintenance of OM structure and function, which can be helpful in developing OM-targeted novel drugs.

The state of oligomerization of Rubisco controls the rate of synthesis of the Rubisco large subunit in Chlamydomonas reinhardtii

Ribulose 1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) is present in all photosynthetic organisms and is a key enzyme for photosynthesis-driven life on Earth. Its most prominent form is a hetero-oligomer in which small subunits (SSU) stabilize the core of the enzyme built from large subunits (LSU), yielding, after a chaperone-assisted multistep assembly process, an LSU8SSU8 hexadecameric holoenzyme. Here we use Chlamydomonas reinhardtii and a combination of site-directed mutants to dissect the multistep biogenesis pathway of Rubisco in vivo. We identify assembly intermediates, in two of which LSU are associated with the RAF1 chaperone. Using genetic and biochemical approaches we further unravel a major regulation process during Rubisco biogenesis, in which LSU translation is controlled by its ability to assemble with the SSU, via the mechanism of Control by Epistasy of Synthesis (CES). Altogether this leads us to propose a model whereby the last assembly intermediate, an LSU8-RAF1 complex, provides the platform for SSU binding to form the Rubisco enzyme, and when SSU is not available, converts to a key regulatory form that exerts negative feed-back on the initiation of LSU translation.

Dual function of GTPBP6 in biogenesis and recycling of human mitochondrial ribosomes

Translation and ribosome biogenesis in mitochondria require auxiliary factors that ensure rapid and accurate synthesis of mitochondrial proteins. Defects in translation are associated with oxidative phosphorylation deficiency and cause severe human diseases, but the exact roles of mitochondrial translation-associated factors are not known. Here we identify the functions of GTPBP6, a homolog of the bacterial ribosome-recycling factor HflX, in human mitochondria. Similarly to HflX, GTPBP6 facilitates the dissociation of ribosomes in vitro and in vivo. In contrast to HflX, GTPBP6 is also required for the assembly of mitochondrial ribosomes. GTPBP6 ablation leads to accumulation of late assembly intermediate(s) of the large ribosomal subunit containing ribosome biogenesis factors MTERF4, NSUN4, MALSU1 and the GTPases GTPBP5, GTPBP7 and GTPBP10. Our data show that GTPBP6 has a dual function acting in ribosome recycling and biogenesis. These findings contribute to our understanding of large ribosomal subunit assembly as well as ribosome recycling pathway in mitochondria.

Structure of the mature kinetoplastids mitoribosome and insights into its large subunit biogenesis

Kinetoplastids are unicellular eukaryotic parasites responsible for such human pathologies as Chagas disease, sleeping sickness, and leishmaniasis. They have a single large mitochondrion, essential for the parasite survival. In kinetoplastid mitochondria, most of the molecular machineries and gene expression processes have significantly diverged and specialized, with an extreme example being their mitochondrial ribosomes. These large complexes are in charge of translating the few essential mRNAs encoded by mitochondrial genomes. Structural studies performed inTrypanosoma bruceialready highlighted the numerous peculiarities of these mitoribosomes and the maturation of their small subunit. However, several important aspects mainly related to the large subunit (LSU) remain elusive, such as the structure and maturation of its ribosomal RNA. Here we present a cryo-electron microscopy study of the protozoansLeishmania tarentolaeandTrypanosoma cruzimitoribosomes. For both species, we obtained the structure of their mature mitoribosomes, complete rRNA of the LSU, as well as previously unidentified ribosomal proteins. In addition, we introduce the structure of an LSU assembly intermediate in the presence of 16 identified maturation factors. These maturation factors act on both the intersubunit and the solvent sides of the LSU, where they refold and chemically modify the rRNA and prevent early translation before full maturation of the LSU.