Biology subjects

Pelosse, M.

Publications and source records attributed to Pelosse, M..

9 recordsLinked to original sources

bioRxiv2026

Structures of the Tilapia Lake Virus replication complex show that ANP32 is a host factor for replication across the Articulavirales order

Tilapia Lake Virus (TiLV) is a species in the Amnoonviridae family within the Articulavirales order of nuclear-replicating, segmented, negative-strand RNA viruses. This order also encompasses the well-known, but phylogenetically distant Orthomyxoviridae family that includes influenza and Thogoto viruses. Influenza virus replication, but not Thogoto virus, essentially relies on the host acidic nuclear phosphoprotein 32 (ANP32) to form a functional genome replication complex, which comprises an asymmetric viral polymerase dimer composed of a replicase bridged by ANP32 to an encapsidase. However, remains unclear whether this dependency is conserved in divergent viruses from the same order. Using in vitro reconstitution, biophysical and biochemical analyses and high-resolution structure determination by single-particle cryo-electron microscopy, we investigate the complexes formed between the TiLV RNA-dependent RNA polymerase (TiLV-Pol) and either tilapia (ti) or human (hu) ANP32A. We show that the tiANP32 leucine-rich repeat domain acts as a scaffold that stabilizes apo-TiLV-Pol in an encapsidase conformation. The tiANP32-encapsidase complex is then able to recruit and stabilise a second TiLV-Pol in a replicase conformation. The tiANP32-stabilised asymmetric TiLV-Pol dimer has an architecture that is remarkably similar to its orthomyxovirus replication complex counterpart, despite the 40% smaller size of TiLV-Pol. We also demonstrate that the ANP32 low-complexity acidic region interacts with the TiLV nucleoprotein, consistent with a conserved role in co-ordinating ribonucleoprotein assembly. Overall, our findings suggest that ANP32 is an ancient and likely broadly conserved host factor for viral genome replication and encapsidation across the Articulavirales order. Furthermore, TiLV is an emerging fish pathogen causing high mortality in aquaculture and our results provide insight into tilapia ANP32 modifications that could be used to engineer TiLV-resistant tilapia.

microbiology↗

bioRxiv2024

Structure of the Tilapia lake virus nucleoprotein bound to RNA

Tilapia Lake virus (TiLV) belongs to the Amnoonviridae family within the Articulavirales order of segmented negative-strand RNA viruses and is highly diverged from more familiar orthomyxoviruses, such as influenza. The viral nucleoprotein (NP), a key component of the replication machinery, packages the viral genome into protective ribonucleoprotein particles. Here we describe the electron cryo-microscopy (cryo-EM) structure of TiLV-NP bound to RNA within in vitro reconstituted, small ring-like, pseudo-symmetrical oligomers. Although TiLV-NP is considerably smaller than its influenza counterpart and unrelated in sequence, it maintains the same topology and domain organisation. This comprises a head and body domain between which is a negatively-charged groove where single-stranded RNA binds. In addition, an oligomerisation loop inserts into a hydrophobic pocket in the neighbouring NP, the flexible hinges of which allow considerable variability orientation of neighbouring NPs. Focussed cryo-EM maps (up to 2.9 [A] resolution) unambiguously define the 5' to 3' direction of the bound RNA, confirmed by double stranded, A-form RNA regions that extrude out from some of the NP-NP interfaces. This is the first description of how RNA binds to an articulaviral NP and superposition with orthomyxoviral NPs suggest that the mode of RNA binding is likely conserved across the Articulavirales order.

molecular biology↗

bioRxiv2024

Structure of AcMNPV nucleocapsid reveals DNA portal organization and packaging apparatus of circular dsDNA baculovirus.

Baculoviruses are large DNA viruses found in nature propagating amongst insects and lepidoptera in particular. Their viral cycle has been studied and characterized over decades and they are nowadays considered as invaluable biotechnology tools widely used as biopesticides, recombinant expression systems or delivery vehicle for gene therapy. However, very little is known about the baculovirus nucleocapsid assembly at a molecular level. Here, we solved the whole structure of the nucleocapsid of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) by applying cryo-electron microscopy (CryoEM) combined to de novo modelling and Alphafold2 predictions. Our structure completes prior observations from the helical sheath and the basal structure and elucidate the intricate architecture of the apical cap, unravelling for the first time the organization of a baculoviral DNA portal featuring intriguing symmetry mismatches between its core and vertex. The core, closing the capsid at the apex, holds two antiparallel DNA helices of the covalently closed circular (ccc) DNA genome tethered to Ac54 proteins. Different symmetry components at the apical cap and basal structure are constituted of the same building block, made of Ac101/Ac144, proving the versatility of this modular pair. The crown forming the portal vertex displays a C21 symmetry and contains, amongst others, the motor-like protein Ac66. Our findings support the viral portal to be involved in DNA packaging, most probably in conjunction with other parts of a larger DNA packaging apparatus that are not bound to mature virions and await further elucidation.

biophysics↗

bioRxiv2024

biGMamAct: efficient CRISPR/Cas9-mediated docking of large functional DNA cargoes at the ACTB locus

Recent advances in molecular and cell biology and imaging have unprecedentedly enabled multi-scale structure-functional studies of entire metabolic pathways from atomic to micrometer resolution, and the visualization of macromolecular complexes in situ, especially if these molecules are expressed with appropriately-engineered and easily-detectable tags. However, genome editing in eukaryotic cells is challenging when generating stable cell lines loaded with large DNA cargoes. To address this limitation, here, we have conceived biGMamAct, a system that allows the straightforward assembly of a multitude of genetic modules and their subsequent integration in the genome at the ACTB locus with high efficacy, through standardized cloning steps. Our technology encompasses a set of modular plasmids for mammalian expression, which can be efficiently docked into the genome in tandem with a validated Cas9/sgRNA pair through homologous-independent targeted insertion (HITI). As a proof of concept, we have generated a stable cell line loaded with an 18.3-kilobase-long DNA cargo to express 6 fluorescently-tagged proteins and simultaneously visualize 5 different subcellular compartments. Our protocol leads from the in-silico design to the genetic and functional characterization of single clones within 6 weeks and can be implemented by any researcher with familiarity with molecular biology and access to mammalian cell culturing infrastructure.

cell biology↗

bioRxiv2024

Structures of influenza A and B replication complexes explain avian to human host adaption and reveal a role of ANP32 as an electrostatic chaperone for the apo-polymerase.

Replication of influenza viral RNA depends on at least two viral polymerases, a parental replicase and an encapsidase, and cellular factor ANP32. ANP32 comprises an LRR domain and a long C-terminal low complexity acidic region (LCAR). Here we show that ANP32 is recruited to the replication complex (replicase-ANP32-encapsidase) by first acting as an electrostatic chaperone to stabilise the encapsidase moiety within apo-polymerase symmetric dimers that are distinct for influenza A and B polymerases. The encapsidase, with ANP32, then forms an asymmetric complex with the replicase. Cryo-EM structures of the influenza A and B replication complexes give new insight into the mutations known to adapt avian strain polymerases to use the distinct ANP32 in mammalian cells. The cryo-EM map of the FluPolB complex shows extra density attributable to the ANP32 LCAR wrapping around and stabilising the apo-encapsidase conformation. These results suggest a functional requirement for three polymerases for replication.

molecular biology↗

bioRxiv2023

FRET-based sensor for measuring adenine nucleotide binding to AMPK

AMP-activated protein kinase (AMPK) has evolved to detect a critical increase in cellular AMP/ATP and ADP/ATP concentration ratios as a signal for limiting energy supply. Such energy stress then leads to AMPK activation and downstream events that maintain cellular energy homeostasis. AMPK activation by AMP, ADP or pharmacological activators involves a conformational switch within the AMPK heterotrimeric complex. We have engineered an AMPK-based sensor, AMPfret, which translates the activating conformational switch into a fluorescence signal, based on increased fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores. Here we describe how this sensor can be used to analyze direct AMPK activation by small molecules in vitro using a fluorimeter, or to estimate changes in the energy state of cells using standard fluorescence or confocal microscopy.

biochemistry↗

bioRxiv2023

Structural and functional analysis of the minimal orthomyxovirus-like polymerase of Tilapia Lake Virus from the highly diverged Amnoonviridae family

Tilapia Lake Virus (TiLV), a recently discovered pathogen of tilapia fish, belongs to the Amnoonviridae family from the Articulavirales order. Its ten genome segments have characteristic conserved ends and encode proteins with no known homologues, apart from the segment 1, which encodes an orthomyxo-like RNA-dependent-RNA polymerase core subunit. Here we show that segments 1-3 encode respectively the PB1, PB2 and PA-like subunits of an active heterotrimeric polymerase that maintains all domains found in the distantly related influenza polymerase, despite an unprecedented overall size reduction of 40%. Multiple high-resolution cryo-EM structures of TiLV polymerase in pre-initiation, initiation and active elongation states, show how it binds the vRNA and cRNA promoters and performs RNA synthesis, with both transcriptase and replicase configurations being characterised. However, the highly truncated endonuclease-like domain appears inactive and the putative cap-binding domain is autoinhibited, emphasising that many functional aspects of TiLV polymerase remain to be elucidated.

microbiology↗

bioRxiv2021

Resolving phylogenetic and biochemical barriers to functional expression of heterologous iron-sulphur cluster enzymes

Many of the most promising applications of synthetic biology, including engineering of microbes for renewable chemical production, relies upon the ability of genetically-tractable hosts to express heterologous enzymes from foreign species. While countless methods for facilitating heterologous enzyme expression have been developed, comparable tools for facilitating heterologous enzyme activity are generally lacking. Such tools are needed to fully exploit the biosynthetic potential of the natural world. Here, using the model bacterium Escherichia coli, we investigate why iron-sulphur (Fe-S) enzymes are often inactive when heterologously expressed. By applying a simple growth complementation assay with collections of Fe-S enzyme orthologs from a wide range of prokaryotic diversity, we uncover a striking correlation between phylogenetic distance and probability of functional expression. Moreover, co-expression of a heterologous Fe-S biogenesis pathway increases the phylogenetic range of orthologs that can be functionally expressed. On the other hand, we find that heterologous Fe-S enzymes that require specific electron carrier proteins within their natural host are rarely functionally expressed unless their specific reducing partners are identified and co-expressed. We demonstrate in vitro that such selectivity in part derives from a need for low-potential electron donors. Our results clarify how phylogenetic distance and electron transfer biochemistry each separately impact functional heterologous expression and provide insight into how these barriers can be overcome for successful microbial engineering involving Fe-S enzymes.

synthetic biology↗

bioRxiv2020

Baculovirus-vectored precision delivery of large DNA cargoes in human genomes

Precise gene editing and genome engineering by CRISPR technology requires simultaneous delivery of multiple DNA-encoded components into living cells rapidly exceeding the cargo capacity of currently utilized viral vector systems. Here we exploit the unmatched heterologous DNA cargo capacity of baculovirus to resolve this bottleneck. We implement hybrid DNA techniques (MultiMate) for rapid and error-free assembly of currently up to 25 functional DNA modules in a single baculoviral vector enabling CRISPR-based genome engineering. Utilizing homology-independent targeted integration (HITI), we achieve up to 30% correct genome interventions in human cells, including precision docking of large DNA payloads in the ACTB locus. We demonstrate baculovirus-vectored delivery of prime-editing toolkits for seamless DNA search-and-replace interventions achieving, with a single vector, highly efficient cleavage-free trinucleotide insertion in the HEK3 locus without any detectable indels. Our approach thus unlocks a wide range of editing and engineering applications in human cell genomes.

synthetic biology↗