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The recent approval of vaccines and gene therapy products for human use produced in the Insect Cell-Baculovirus Expression Vector System (IC-BEVS) underlines the high potential and versatility of this platform. The interest in developing robust production processes emerges to cope with manufacturing pressure, as well as stringent product quality guidelines. Previously, we addressed the impact of the baculovirus infection on the physiology of insect host cell lines, identifying key cellular pathways enrolled in heterologous gene/protein expression. In the present work, this knowledge was applied to design tailored media supplementation schemes to boost IC-BEVS production yields and quality of enveloped viral particles: influenza VLPs (Inf-VLP) and baculovirus vectors (BV). The addition of reduced glutathione, antioxidants and polyamines increased the cell specific yields of baculovirus particles up to 3 fold. Cholesterol was identified as the most critical system booster, capable of improving 2.5 and 6-fold cell specific yields of BV and Inf-VLPs, respectively. Surprisingly, the combination of polyamines and cholesterol supplementation improved baculovirus stock quality, by preventing the accumulation of non-infectious particles during viral replication while selectively increasing infectious particles production. In addition, the specific yields of both enveloped viral particles, BVs and Inf-VLPs, were also increased. The correlation between supplement addition and systems productivity was extensively analyzed, providing a critical assessment on final product quantity and quality as drivers of bioprocess optimization efforts.

Multiprotein complexes catalyze vital biological functions in the cell. A paramount objective of the SPINE2 project was to address the structural molecular biology of these multiprotein complexes, by enlisting and developing enabling technologies for their study. An emerging key prerequisite for studying complex biological specimens is their recombinant overproduction. Novel reagents and streamlined protocols for rapidly assembling co-expression constructs for this purpose have been designed and validated. The high-throughput pipeline implemented at IGBMC Strasbourg and the ACEMBL platform at the EMBL Grenoble utilize recombinant overexpression systems for heterologous expression of proteins and their complexes. Extension of the ACEMBL platform technology to include eukaryotic hosts such as insect and mammalian cells has been achieved. Efficient production of large multicomponent protein complexes for structural studies using the baculovirus/insect cell system can be hampered by a stoichiometric imbalance of the subunits produced. A polyprotein strategy has been developed to overcome this bottleneck and has been successfully implemented in our MultiBac baculovirus expression system for producing multiprotein complexes.

International audience; The yeast protein Dom34 has been described to play a critical role in a newly identified mRNA decay pathway called No-Go decay. This pathway clears cells from mRNAs inducing translational stalls through endonucleolytic cleavage. Dom34 is related to the translation termination factor eRF1 and physically interacts with Hbs1, which is itself related to eRF3. We have solved the 2.5-Å resolution crystal structure of Saccharomyces cerevisiae Dom34. This protein is organized in three domains with the central and C-terminal domains structurally homologous to those from eRF1. The N-terminal domain of Dom34 is different from eRF1. It adopts a Sm-fold that is often involved in the recognition of mRNA stem loops or in the recruitment of mRNA degradation machinery. The comparison of eRF1 and Dom34 domains proposed to interact directly with eRF3 and Hbs1, respectively, highlights striking structural similarities with eRF1 motifs identified to be crucial for the binding to eRF3. In addition, as observed for eRF1 that enhances eRF3 binding to GTP, the interaction of Dom34 with Hbs1 results in an increase in the affinity constant of Hbs1 for GTP but not GDP. Taken together, these results emphasize that eukaryotic cells have evolved two structurally related complexes able to interact with ribosomes either paused at a stop codon or stalled in translation by the presence of a stable stem loop and to trigger ribosome release by catalyzing chemical bond hydrolysis.

Graphical abstract, Highlights • Structures have been determined for natural and recombinant polyproteins. • Native HIV Gag polyprotein architecture was revealed by cryo-EM of immature capsids. • Recombinant polyprotein technology has resolved sample preparation bottlenecks. • The high-resolution structure of influenza polymerase has been solved. • Single-molecule analysis of polyproteins revealed their folding characteristics., Polyproteins are chains of covalently conjoined smaller proteins that occur in nature as versatile means to organize the proteome of viruses including HIV. During maturation, viral polyproteins are typically cleaved into the constituent proteins with different biological functions by highly specific proteases, and structural analyses at defined stages of this maturation process can provide clues for antiviral intervention strategies. Recombinant polyproteins that use similar mechanisms are emerging as powerful tools for producing hitherto inaccessible protein targets such as the influenza polymerase, for high-resolution structure determination by X-ray crystallography. Conversely, covalent linking of individual protein subunits into single polypeptide chains are exploited to overcome sample preparation bottlenecks. Moreover, synthetic polyproteins provide a promising tool to dissect dynamic folding of polypeptide chains into three-dimensional architectures in single-molecule structure analysis by atomic force microscopy (AFM). The recent use of natural and synthetic polyproteins in structural biology and major achievements are highlighted in this contribution.

The initiation of gene transcription by RNA polymerase II is regulated by a plethora of proteins in human cells. The first general transcription factor to bind gene promoters is transcription factor IID (TFIID). TFIID triggers pre-initiation complex formation, functions as a coactivator by interacting with transcriptional activators and reads epigenetic marks. TFIID is a megadalton-sized multiprotein complex composed of TATA-box-binding protein (TBP) and 13 TBP-associated factors (TAFs). Despite its crucial role, the detailed architecture and assembly mechanism of TFIID remain elusive. Histone fold domains are prevalent in TAFs, and histone-like tetramer and octamer structures have been proposed in TFIID. A functional core-TFIID subcomplex was revealed in Drosophila nuclei, consisting of a subset of TAFs (TAF4, TAF5, TAF6, TAF9 and TAF12). These core subunits are thought to be present in two copies in holo-TFIID, in contrast to TBP and other TAFs that are present in a single copy, conveying a transition from symmetry to asymmetry in the TFIID assembly pathway. Here we present the structure of human core-TFIID determined by cryo-electron microscopy at 11.6 A resolution. Our structure reveals a two-fold symmetric, interlaced architecture, with pronounced protrusions, that accommodates all conserved structural features of the TAFs including the histone folds. We further demonstrate that binding of one TAF8-TAF10 complex breaks the original symmetry of core-TFIID. We propose that the resulting asymmetric structure serves as a functional scaffold to nucleate holo-TFIID assembly, by accreting one copy each of the remaining TAFs and TBP.

SummaryLoss of N7-methylguanosine (m7G) modification is involved in the recently discovered rapid tRNA degradation pathway. In yeast, this modification is catalyzed by the heterodimeric complex composed of a catalytic subunit Trm8 and a noncatalytic subunit Trm82. We have solved the crystal structure of Trm8 alone and in complex with Trm82. Trm8 undergoes subtle conformational changes upon Trm82 binding which explains the requirement of Trm82 for activity. Cocrystallization with the S-adenosyl-methionine methyl donor defines the putative catalytic site and a guanine binding pocket. Small-angle X-ray scattering in solution of the Trm8-Trm82 heterodimer in complex with tRNAPhe has enabled us to propose a low-resolution structure of the ternary complex which defines the tRNA binding mode of Trm8-Trm82 and the structural elements contributing to specificity.

Both dengue and West Nile virus infections are an increasing risk to humans, not only in tropical and subtropical areas, but also in North America and parts of Europe. These viral infections are generally transmitted by mosquitoes, but may also be tick-borne. Infection usually results in mild flu-like symptoms, but can also cause encephalitis and fatalities. Approximately 2799 severe West Nile virus cases were reported this year in the United States, resulting in 102 fatalities. With this alarming increase in the number of West Nile virus infections in western countries and the fact that dengue virus already affects millions of people per year in tropical and subtropical climates, there is a real need for effective medicines. A possible therapeutic target to combat these viruses is the protease, which is essential for virus replication. In order to provide structural information to help to guide a lead identification and optimization program, crystallizations of the NS2B–NS3 protease complexes from both dengue and West Nile viruses have been initiated. Crystals that diffract to high resolution, suitable for three-dimensional structure determinations, have been obtained.