Biology subjects

Arragain, B.

Publications and source records attributed to Arragain, B..

8 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

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

Structural characterization of the oligomerization of full-length Hantaan virus polymerase into symmetric dimers and hexamers

Hantaan virus is a dangerous human pathogen whose segmented negative-stranded RNA genome is replicated and transcribed by a virally-encoded multi-functional polymerase. Here we describe the complete cryo-electron microscopy structure of Hantaan virus polymerase in several oligomeric forms. Apo polymerase protomers can adopt two drastically different conformations, which assemble into two distinct homodimers, that can themselves gather to form hexamers. Polymerase dimerization induces the stabilization of most polymerase domains, including the C-terminal region that notably contains a C-terminal domain that contribute the most to dimers interface, along with a lariat region that participates to the polymerase steadying. Binding to viral RNA induces significant conformational changes resulting in oligomer disruption, suggesting the possible involvement of multimers as protecting systems that would stabilize the otherwise flexible C-terminal domains. Overall, these results provide new insights into the multimerization capability of Hantavirus polymerase and may help to define antiviral compounds to counteract these life-threatening viruses.

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↗

bioRxiv2023

The host RNA polymerase II C-terminal domain is the anchor for replication of the influenza virus genome

The current model is that the influenza virus polymerase (FluPol) binds either to host RNA polymerase II (RNAP II) or to the acidic nuclear phosphoprotein 32 (ANP32), which drives its conformation and activity towards transcription or replication of the viral genome, respectively. Here, we provide evidence that the FluPol-RNAP II binding interface has a so far overlooked function for replication of the viral genome. Using a combination of cell-based and in vitro approaches, we show that the RNAP II C-terminal-domain, jointly with ANP32, enhances FluPol replication activity and we propose a model in which the host RNAP II is the anchor for transcription and replication of the viral genome. Our data open new perspectives on the spatial coupling of viral transcription and replication and the coordinated balance between these two activities.

microbiology↗

bioRxiv2021

Structural snapshots of La Crosse virus polymerase reveal the mechanisms underlying Peribunyaviridae replication and transcription

Segmented negative-strand RNA bunyaviruses encode a multi-functional polymerase that performs genome replication and transcription. Here, we establish conditions for in vitro activity of La Crosse virus polymerase and visualize by cryo-electron microscopy its conformational dynamics, unveiling the precise molecular mechanics underlying its essential activities. Replication initiation is coupled to distal duplex promoter formation, endonuclease movement, prime-and-realign loop extension and closure of the polymerase core that direct the template towards the active site. Transcription initiation depends on C-terminal region closure and endonuclease movements that firstly prompt primer cleavage and secondly promote primer entry in the active site. Product realignment after priming, observed in replication and transcription, is triggered by the prime-and-realign loop. Switch to elongation results in polymerase reorganization and core region opening to facilitate template-product duplex formation in the active site cavity. The detailed uncovered mechanics will be crucial for future design of antivirals counteracting bunyaviral life-threatening pathogens.

microbiology↗

bioRxiv2020

Pre-initiation and elongation structures of full-length La Crosse virus polymerase reveal functionally important conformational changes

Bunyavirales is an order of segmented negative stranded RNA viruses comprising several life-threatening pathogens such as Lassa fever virus (Arenaviridae), Rift Valley Fever virus (Phenuiviridae) and La Crosse virus (LACV, Peribunyaviridae) against which neither specific treatment nor licenced vaccine is available. Replication and transcription of Bunyavirales genome constitute essential reactions of their viral cycle that are catalysed by the virally encoded RNA-dependent RNA polymerase or L protein. Here we describe the complete high-resolution cryo-EM structure of the full-length (FL) LACV-L protein. It reveals the presence of key C-terminal domains, notably the cap-binding domain that undergoes large movements related to its role in transcription initiation and a zinc-binding domain that displays a fold not previously observed. We capture the structure of LACV-L FL in two functionally relevant states, pre-initiation and elongation, that reveal large conformational changes inherent to its function. We uncover the coordinated movement of the polymerase priming loop, lid domain and C-terminal region required for the establishment of a ten-base-pair template-product RNA duplex before strand separation into respective exit tunnels. The revealed structural details and dynamics of functional elements will be instrumental for structure-based development of compounds that inhibit RNA synthesis by the polymerase.

biophysics↗