Biology subjects

Krischuns, T.

Publications and source records attributed to Krischuns, T..

8 recordsLinked to original sources

bioRxiv2024

Mechanism of Co-Transcriptional Cap-Snatching by Influenza Polymerase

Influenza virus mRNA is stable and competent for nuclear export and translation because it receives a 5' cap(1) structure in a process called cap-snatching1. During cap-snatching, the viral RNA-dependent RNA polymerase (FluPol) binds to host RNA polymerase II (Pol II) and the emerging transcript2,3. The FluPol endonuclease then cleaves a capped RNA fragment that sub-sequently acts as a primer for the transcription of viral genes4,5. Here, we present the cryo-EM structure of FluPol bound to a transcribing Pol II in complex with the elongation factor DSIF in the pre-cleavage state. The structure shows that FluPol directly interacts with both Pol II and DSIF, which position the FluPol endonuclease domain near the RNA exit channel of Pol II. These interactions are important for the endonuclease activity of FluPol and FluPol activity in cells. A second structure trapped after cap-snatching shows that cleavage rearranges the capped RNA primer within the FluPol, directing the capped RNA 3'-end towards the FluPol polymerase active site for viral transcription initiation. Altogether, our results provide the molecular mechanisms of co-transcriptional cap-snatching by FluPol.

biochemistry↗

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

High-resolution structure of a replication-initiation like configuration of influenza polymerase active site visualises the essential role of a conserved dibasic motif in the PA subunit

Influenza polymerase, comprising subunits PA, PB1 and PB2, transcribes the negative-sense genomic viral RNA (vRNA) into mRNA or replicates it first into complementary RNA (cRNA) and then back to vRNA. Here we investigate the mechanism of de novo (unprimed) initiation of vRNA to cRNA replication. We present a high-resolution structure of A/little-yellow-shouldered-bat/H17N10 polymerase with the 3 end of the template in the RNA synthesis active site, both in the apo-state and after soaking with GTP and CTP. The priming GTP and incoming CTP are observed to base-pair to template nucleotides C2 and G3 at the -1 and +1 positions respectively, thus representing a replication initiation-like state. This configuration is stabilised by partial stacking of the tip of the priming loop on the GTP:C2 base-pair and the interaction of PB1/H649 and dibasic motif residues PA/R658 and K659 with the triphosphate of the priming GTP. The dibasic motif is universally conserved in orthomyxovirus PA subunits. Trans-complementation assays in cells using mutants of PA/K659 show that the dibasic motif is specifically essential for replication. These results shed light on the mechanism of replication initiation even though vRNA to cRNA replication is expected to be terminally initiated, with priming ATP and incoming GTP base-pairing to template nucleotides U1 and C2 at the -1 and +1 positions respectively, implying a different position of the template.

molecular biology↗

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↗

bioRxiv2023

Multivalent dynamic colocalization of avian influenza polymerase and nucleoprotein by intrinsically disordered ANP32A reveals the molecular basis of human adaptation

Adaptation of avian influenza RNA polymerase (FluPol) to human cells requires mutations on the 627-NLS domains of the PB2 subunit. The E627K adaptive mutation compensates a 33-amino-acid deletion in the acidic intrinsically disordered domain of the host transcription regulator ANP32A, a deletion that restricts FluPol activity in mammalian cells. The function of ANP32A in the replication transcription complex and in particular its role in host restriction remain poorly understood. Here we characterise ternary complexes formed between ANP32A, FluPol and the viral nucleoprotein, NP, supporting the putative role of ANP32A in shuttling NP to the replicase complex. We demonstrate that while FluPol and NP can simultaneously bind distinct linear motifs on avian ANP32A, the deletion in the shorter human ANP32A blocks this mode of colocalization. NMR reveals that NP and human-adapted Pol, containing the E627K mutation, simultaneously bind the identical extended linear motif on human ANP32A, in an electrostatically driven, highly dynamic and multivalent ternary complex. This study reveals a probable molecular mechanism underlying host adaptation, whereby E627K, which enhances the basic surface of the 627 domain, is selected to confer the necessary multivalent properties to allow ANP32A to colocalize NP and FluPol in human cells.

biochemistry↗

bioRxiv2023

The RBPome of influenza A virus mRNA reveals a role for TDP-43 in viral replication

Recent technical advances have significantly improved our understanding of the RNA-binding protein (RBP) repertoire present within eukaryotic cells, with a particular focus on the RBPs that interact with cellular polyadenylated mRNAs. However, recent studies utilising the same technologies have begun to tease apart the RBP interactome of viral mRNAs, notably SARS-CoV-2, revealing both similarities and differences between the RBP profiles of viral and cellular mRNAs. Herein, we comprehensively identified the RBPs that associate with the NP mRNA of an influenza A virus. Moreover, we provide evidence that the viral polymerase is essential for the recruitment of RPBs to viral mRNAs through direct polymerase-RBP interactions during transcription. We show that loss of TDP-43, which associates with the viral mRNAs, results in lower levels of viral mRNAs within infected cells, and a decreased yield of infectious viral particles. Overall, our results uncover an important role for TDP-43 in the influenza A virus replication cycle via a direct interaction with viral mRNAs, and point to a role of the viral polymerase in orchestrating the assembly of viral mRNPs.

microbiology↗

bioRxiv2022

Type B and Type A influenza polymerases have evolved distinct binding interfaces to recruit the RNA polymerase II CTD

During annual influenza epidemics, influenza B viruses (IBVs) co-circulate with influenza A viruses (IAVs), can become predominant and cause severe morbidity and mortality. Phylogenetic analyses suggest that IAVs (primarily avian viruses) and IBVs (primarily human viruses) have diverged over long time scales. Identifying their common and distinctive features is an effective approach to increase knowledge about the molecular details of influenza infection. The virus-encoded RNA-dependent RNA polymerases (FluPolB and FluPolA) are PB1-PB2-PA heterotrimers that perform transcription and replication of the viral genome in the nucleus of infected cells. Initiation of viral mRNA synthesis requires a direct association of FluPol with the host RNA polymerase II (RNAP II), in particular the repetitive C-terminal domain (CTD) of the major RNAP II subunit, to enable "cap-snatching" whereby 5-capped oligomers derived from nascent RNAP II transcripts are pirated to prime viral transcription. Here, we present the first high-resolution co-crystal structure of FluPolB bound to a CTD mimicking peptide at a binding site crossing from PA to PB2. By performing structure-based mutagenesis of FluPolB and FluPolA followed by a systematic investigation of FluPol-CTD binding, FluPol activity and viral phenotype, we demonstrate that IBVs and IAVs have evolved distinct binding interfaces to recruit the RNAP II CTD, despite the CTD sequence being highly conserved across host species. We find that the PB2 627 subdomain, a major determinant of FluPol-host cell interactions and IAV host-range, is involved in CTD-binding for IBVs but not for IAVs, and we show that FluPolB and FluPolA bind to the host RNAP II independently of the CTD. Altogether, our results strongly suggest that the CTD-binding modes of IAV and IBV represent avian- and human-optimized binding modes, respectively, and that their divergent evolution was shaped by the broader interaction network between the FluPol and the host transcriptional machinery. Authors summaryDuring seasonal influenza epidemics, influenza B viruses (IBVs) co-circulate with influenza A viruses (IAVs) and can cause severe outcomes. The influenza polymerase is a key drug target and it is therefore important to understand the common and distinctive molecular features of IBV and IAV polymerases. To achieve efficient transcription and replication in the nucleus of infected cells, influenza polymerases closely cooperate with the cellular RNA polymerase II (RNAP II) and interact with the repetitive C-terminal domain (CTD) of its major subunit. Here we gained new insights into the way IBV and IAV polymerases interact with the CTD of RNAP II. High-resolution structural data was used to perform structure-based mutagenesis of IBV and IAV polymerases followed by a systematic investigation of their interaction with RNAP II, transcription/replication activity and viral phenotype. Strikingly, we found that IBVs and IAVs have evolved distinct interfaces to interact with the host transcriptional machinery, in particular with the CTD of RNAP II. We provide evidence that these differences may have evolved as a consequence of the differences in IBV and IAV host range. Our findings are of significant importance with regard to the development of broad-spectrum antivirals that target the virus-host interface.

microbiology↗

bioRxiv2021

A highly sensitive cell-based luciferase assay for high-throughput automated screening of SARS-CoV-2 nsp5/3CLpro inhibitors

Effective drugs against SARS-CoV-2 are urgently needed to treat severe cases of infection and for prophylactic use. The main viral protease (nsp5 or 3CLpro) represents an attractive and possibly broad-spectrum target for drug development as it is essential to the virus life cycle and highly conserved among betacoronaviruses. Sensitive and efficient high-throughput screening methods are key for drug discovery. Here we report the development of a gain-of-signal, highly sensitive cell-based luciferase assay to monitor SARS-CoV-2 nsp5 activity and show that it is suitable for high-throughput screening of compounds in a 384-well format. A benefit of miniaturisation and automation is that screening can be performed in parallel on a wild-type and a catalytically inactive nsp5, which improves the selectivity of the assay. We performed molecular docking-based screening on a set of 14,468 compounds from an in-house chemical database, selected 359 candidate nsp5 inhibitors and tested them experimentally. We identified four molecules, including the broad-spectrum antiviral merimepodib/VX-497, which show anti-nsp5 activity and inhibit SARS-CoV-2 replication in A549-ACE2 cells with IC50 values in the 4-21 {micro}M range. The here described assay will allow the screening of large-scale compound libraries for SARS-CoV-2 nsp5 inhibitors. Moreover, we provide evidence that this assay can be adapted to other coronaviruses and viruses which rely on a viral protease.

microbiology↗