Ervin Fodor

University of Oxford, Oxford, England, United Kingdom

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Publications (90)444.06 Total impact

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    ABSTRACT: Negative-sense RNA viruses, such as influenza, encode large, multidomain RNA-dependent RNA polymerases that can both transcribe and replicate the viral RNA genome. In influenza virus, the polymerase (FluPol) is composed of three polypeptides: PB1, PB2 and PA/P3. PB1 houses the polymerase active site, whereas PB2 and PA/P3 contain, respectively, cap-binding and endonuclease domains required for transcription initiation by cap-snatching. Replication occurs through de novo initiation and involves a complementary RNA intermediate. Currently available structures of the influenza A and B virus polymerases include promoter RNA (the 5' and 3' termini of viral genome segments), showing FluPol in transcription pre-initiation states. Here we report the structure of apo-FluPol from an influenza C virus, solved by X-ray crystallography to 3.9 Å, revealing a new 'closed' conformation. The apo-FluPol forms a compact particle with PB1 at its centre, capped on one face by PB2 and clamped between the two globular domains of P3. Notably, this structure is radically different from those of promoter-bound FluPols. The endonuclease domain of P3 and the domains within the carboxy-terminal two-thirds of PB2 are completely rearranged. The cap-binding site is occluded by PB2, resulting in a conformation that is incompatible with transcription initiation. Thus, our structure captures FluPol in a closed, transcription pre-activation state. This reveals the conformation of newly made apo-FluPol in an infected cell, but may also apply to FluPol in the context of a non-transcribing ribonucleoprotein complex. Comparison of the apo-FluPol structure with those of promoter-bound FluPols allows us to propose a mechanism for FluPol activation. Our study demonstrates the remarkable flexibility of influenza virus RNA polymerase, and aids our understanding of the mechanisms controlling transcription and genome replication.
    No preview · Article · Oct 2015 · Nature
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    ABSTRACT: Viral drug resistance is believed to occur less likely if compounds are directed against cellular rather than viral proteins. In this study, we analyzed the feasibility of a crucial viral replication factor, namely importin-α7, as a potential cellular drug target to combat pandemic influenza. Surprisingly, only five viral lung-to-lung passages were required to achieve 100% lethality in importin-α7(-/-) mice that are otherwise resistant. Viral escape from importin-α7 requirement was mediated by five mutations in the viral ribonucleoprotein complex and the surface glycoproteins. Moreover, the importin-α7(-/-) mouse-adapted strain became even more virulent for wild-type mice compared to the parental strain. These studies show that targeting host proteins may still result in viral escape by alternative pathways eventually giving rise to even more virulent virus strains. Thus, therapeutic intervention strategies should consider a multi-target approach to reduce viral drug resistance. Here, we investigated the longstanding hypothesis based on in vitro studies that viral drug resistance occurs less likely if compounds are directed against cellular rather than viral proteins. Here, we challenged this hypothesis by analyzing in an in vivo animal model the feasibility of targeting the cellular factor importin-α7 - that is crucial for human influenza virus replication and pathogenesis - as an efficient antiviral strategy against pandemic influenza viruses. In summary, our studies suggest that resistance against cellular factors is possible in vivo and the emergence of even more virulent viral escape variants calls for particular caution. Thus, therapeutic intervention strategies should consider a multi-target approach using compounds against viral as well as cellular factors to reduce the risk of viral drug resistance and potentially increased virulence. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
    Full-text · Article · Jun 2015 · Journal of Virology
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    ABSTRACT: The host interferon (IFN) response represents one of the first barriers that influenza viruses must surmount in order to establish an infection. Many advances have been made in recent years in the understanding of the interactions between influenza viruses and the interferon system. In this review, we summarise recent work regarding activation of the type I IFN response by influenza viruses, including attempts to identify the viral RNA responsible for IFN induction, the stage of the virus life cycle at which it is generated and the role of defective viruses in this process. Copyright © 2015. Published by Elsevier B.V.
    Preview · Article · Feb 2015 · Virus Research
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    Full-text · Article · Feb 2015 · Nature Communications
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    ABSTRACT: In the influenza virus ribonucleoprotein complex, the oligomerization of the nucleoprotein is mediated by an interaction between the tail-loop of one molecule and the groove of the neighboring molecule. In this study, we show that phosphorylation of a serine residue (S165) within the groove of influenza A virus nucleoprotein inhibits oligomerization and, consequently, ribonucleoprotein activity and viral growth. We propose that nucleoprotein oligomerization in infected cells is regulated by reversible phosphorylation.
    Full-text · Article · Oct 2014 · Journal of Virology
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    ABSTRACT: Viruses use virions to spread between hosts, and virion composition is therefore the primary determinant of viral transmissibility and immunogenicity. However, the virions of many viruses are complex and pleomorphic, making them difficult to analyse in detail. Here we address this by identifying and quantifying virion proteins with mass spectrometry, producing a complete and quantified model of the hundreds of host-encoded and viral proteins that make up the pleomorphic virions of influenza viruses. We show that a conserved influenza virion architecture is maintained across diverse combinations of virus and host. This 'core' architecture, which includes substantial quantities of host proteins as well as the viral protein NS1, is elaborated with abundant host-dependent features. As a result, influenza virions produced by mammalian and avian hosts have distinct protein compositions. Finally, we note that influenza virions share an underlying protein composition with exosomes, suggesting that influenza virions form by subverting microvesicle production.
    Full-text · Article · Sep 2014 · Nature Communications
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    Ashley York · Edward C Hutchinson · Ervin Fodor
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    ABSTRACT: Unlabelled: The negative-sense RNA genome of influenza A virus is transcribed and replicated by the viral RNA-dependent RNA polymerase (RdRP). The viral RdRP is an important host range determinant, indicating that its function is affected by interactions with cellular factors. However, the identities and the roles of most of these factors remain unknown. Here, we employed affinity purification followed by mass spectrometry to identify cellular proteins that interact with the influenza A virus RdRP in infected human cells. We purified RdRPs using a recombinant influenza virus in which the PB2 subunit of the RdRP is fused to a Strep-tag. When this tagged subunit was purified from infected cells, copurifying proteins included the other RdRP subunits (PB1 and PA) and the viral nucleoprotein and neuraminidase, as well as 171 cellular proteins. Label-free quantitative mass spectrometry revealed that the most abundant of these host proteins were chaperones, cytoskeletal proteins, importins, proteins involved in ubiquitination, kinases and phosphatases, and mitochondrial and ribosomal proteins. Among the phosphatases, we identified three subunits of the cellular serine/threonine protein phosphatase 6 (PP6), including the catalytic subunit PPP6C and regulatory subunits PPP6R1 and PPP6R3. PP6 was found to interact directly with the PB1 and PB2 subunits of the viral RdRP, and small interfering RNA (siRNA)-mediated knockdown of the catalytic subunit of PP6 in infected cells resulted in the reduction of viral RNA accumulation and the attenuation of virus growth. These results suggest that PP6 interacts with and positively regulates the activity of the influenza virus RdRP. Importance: Influenza A viruses are serious clinical and veterinary pathogens, causing substantial health and economic impacts. In addition to annual seasonal epidemics, occasional global pandemics occur when viral strains adapt to humans from other species. To replicate efficiently and cause disease, influenza viruses must interact with a large number of host factors. The reliance of the viral RNA-dependent RNA polymerase (RdRP) on host factors makes it a major host range determinant. This study describes and quantifies host proteins that interact, directly or indirectly, with a subunit of the RdRP. It increases our understanding of the role of host proteins in viral replication and identifies a large number of potential barriers to pandemic emergence. Identifying host factors allows their importance for viral replication to be tested. Here, we demonstrate a role for the cellular phosphatase PP6 in promoting viral replication, contributing to our emerging knowledge of regulatory phosphorylation in influenza virus biology.
    Full-text · Article · Sep 2014 · Journal of Virology
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    Edward C Hutchinson · Ervin Fodor
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    ABSTRACT: It is often useful to concentrate and purify the pleomorphic, enveloped virions of influenza viruses prior to analysis. This protocol describes a standard method for purifying ‘spherical’ influenza virions, the most common morphology of laboratory-adapted strains, by rate-zonal ultracentrifugation through a discontinuous density gradient. The protocol, which takes around eight hours to complete, is designed for virions shed into the growth media of infected cells, and would also be suitable for concentrating virions from the allantoic fluid of embryonated chicken eggs. Two different density gradients are described: a conventional gradient of sucrose and a gradient of iodixanol (OptiPrep™ density gradient medium). A high degree of virion purity can be achieved, but small quantities of contaminants will remain. These can be removed by an optional haemadsorption-elution (HAd) step, which takes an additional one and half hours. This allows extremely efficient removal of the original contaminants, but introduces other, distinct contaminants at low levels.
    Full-text · Article · Aug 2014
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    ABSTRACT: The influenza virus is a major human and animal pathogen responsible for seasonal epidemics and occasional pandemics. The genome of the influenza A virus comprises eight segments of single-stranded, negative-sense RNA with highly conserved 5' and 3' termini. These termini interact to form a double-stranded promoter structure that is recognized and bound by the viral RNA-dependent RNA polymerase (RNAP); however, no 3D structural information for the influenza polymerase-bound promoter exists. Functional studies have led to the proposal of several 2D models for the secondary structure of the bound promoter, including a corkscrew model in which the 5' and 3' termini form short hairpins. We have taken advantage of an insect-cell system to prepare large amounts of active recombinant influenza virus RNAP, and used this to develop a highly sensitive single-molecule FRET assay to measure distances between fluorescent dyes located on the promoter and map its structure both with and without the polymerase bound. These advances enabled the direct analysis of the influenza promoter structure in complex with the viral RNAP, and provided 3D structural information that is in agreement with the corkscrew model for the influenza virus promoter RNA. Our data provide insights into the mechanisms of promoter binding by the influenza RNAP and have implications for the understanding of the regulatory mechanisms involved in the transcription of viral genes and replication of the viral RNA genome. In addition, the simplicity of this system should translate readily to the study of any virus polymerase-promoter interaction.
    Preview · Article · Jul 2014 · Proceedings of the National Academy of Sciences
  • Gülsah Gabriel · Ervin Fodor
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    ABSTRACT: Viral pathogenesis involves numerous interactions between viral and cellular factors. In recent years, the influenza virus polymerase complex has emerged as a major determinant of interspecies transmission and pathogenicity. The viral RNA-dependent RNA polymerase, in concert with the nucleoprotein, mediates transcription and replication of the viral RNA genome in the nucleus of the infected cell. The activity by which the viral polymerase complex performs these processes in mammalian cells is considered to be a major contributor to viral pathogenicity in mammals. In this chapter, we summarise our current understanding on the pathogenicity determinants in the viral polymerase complex and highlight some of its cellular interaction partners. We particularly discuss the role of importin-α isoforms in host adaptation and pathogenesis as well as the role of the viral polymerase in regulating cellular responses to viral infection.
    No preview · Article · Jul 2014 · Current topics in microbiology and immunology
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    Full-text · Dataset · Mar 2014
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    Full-text · Dataset · Mar 2014
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    Full-text · Dataset · Mar 2014
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    ABSTRACT: Reassortment of influenza viral RNA (vRNA) segments in co-infected cells can lead to the emergence of viruses with pandemic potential. Replication of influenza vRNA occurs in the nucleus of infected cells, while progeny virions bud from the plasma membrane. However, the intracellular mechanics of vRNA assembly into progeny virions is not well understood. Here we used recent advances in microscopy to explore vRNA assembly and transport during a productive infection. We visualized four distinct vRNA segments within a single cell using fluorescent in situ hybridization (FISH) and observed that foci containing more than one vRNA segment were found at the external nuclear periphery, suggesting that vRNA segments are not exported to the cytoplasm individually. Although many cytoplasmic foci contain multiple vRNA segments, not all vRNA species are present in every focus, indicating that assembly of all eight vRNA segments does not occur prior to export from the nucleus. To extend the observations made in fixed cells, we used a virus that encodes GFP fused to the viral polymerase acidic (PA) protein (WSN PA-GFP) to explore the dynamics of vRNA assembly in live cells during a productive infection. Since WSN PA-GFP colocalizes with viral nucleoprotein and influenza vRNA segments, we used it as a surrogate for visualizing vRNA transport in 3D and at high speed by inverted selective-plane illumination microscopy. We observed cytoplasmic PA-GFP foci colocalizing and traveling together en route to the plasma membrane. Our data strongly support a model in which vRNA segments are exported from the nucleus as complexes that assemble en route to the plasma membrane through dynamic colocalization events in the cytoplasm.
    Full-text · Article · Mar 2014 · PLoS Pathogens
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    ABSTRACT: Most avian influenza viruses do not replicate efficiently in human cells. This is partly due to the low activity of the RNA polymerase of avian influenza viruses in mammalian cells. Nevertheless, this impediment can be overcome through an E→K adaptive mutation at residue 627 of the PB2 subunit of the polymerase. Accordingly, viral ribonucleoprotein (RNP) reconstitution assays show that a viral polymerase containing PB2 627E has impaired activity in mammalian cells compared to a viral polymerase that contains PB2 627K, characteristic of mammalian-adapted influenza viruses. In contrast, purified viral polymerases containing either PB2 627E or PB2 627K show comparable levels of activity in transcription assays that require no RNP assembly. We sought to reconcile these conflicting observations by using an NP-independent cell-based transcription/replication assay to assess viral polymerase activity. We found that PB2 627E polymerase restriction in mammalian cells is independent of NP expression but is dependent on the length of the viral RNA template. In addition, restriction of PB2 627E polymerase was overcome by mutations specific to the viral RNA template promoter sequence. Consequently, we propose that PB2 627E affects recruitment of the viral RNA promoter by the viral polymerase in mammalian cells.
    Full-text · Article · Oct 2013 · Journal of Virology
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    ABSTRACT: Negative-strand RNA viruses represent a significant class of important pathogens that cause substantial morbidity and mortality in human and animal hosts worldwide. A defining feature of these viruses is that their single-stranded RNA genomes are of opposite polarity to messenger RNA and are replicated through a positive-sense intermediate. The replicative intermediate is thought to exist as a complementary ribonucleoprotein (cRNP) complex. However, isolation of such complexes from infected cells has never been accomplished. Here we report the development of an RNA-based affinity-purification strategy for the isolation of cRNPs of influenza A virus from infected cells. This technological advance enabled the structural and functional characterization of this elusive but essential component of the viral RNA replication machine. The cRNP exhibits a filamentous double-helical organization with defined termini, containing the viral RNA-dependent RNA polymerase (RdRp) at one end and a loop structure at the other end. In vitro characterization of cRNP activity yielded mechanistic insights into the workings of this RNA synthesis machine. In particular, we found that cRNPs show activity in vitro only in the presence of added RdRp. Intriguingly, a replication-inactive RdRp mutant was also able to activate cRNP-templated viral RNA synthesis. We propose a model of influenza virus genome replication that relies on the trans-activation of the cRNP-associated RdRp. The described purification strategy should be applicable to other negative-strand RNA viruses and will promote studies into their replication mechanisms.
    No preview · Article · Oct 2013 · Proceedings of the National Academy of Sciences
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    Edward C Hutchinson · Ervin Fodor
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    ABSTRACT: The segmented genome of an influenza virus is encapsidated into ribonucleoprotein complexes (RNPs). Unusually among RNA viruses, influenza viruses replicate in the nucleus of an infected cell, and their RNPs must therefore recruit host factors to ensure transport across a number of cellular compartments during the course of an infection. Recent studies have shed new light on many of these processes, including the regulation of nuclear export, genome packaging, mechanisms of virion assembly and viral entry and, in particular, the identification of Rab11 on recycling endosomes as a key mediator of RNP transport and genome assembly. This review uses these recent gains in understanding to describe in detail the journey of an influenza A virus RNP from its synthesis in the nucleus through to its entry into the nucleus of a new host cell.
    Full-text · Article · Oct 2013 · Viruses
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    ABSTRACT: Transcription and replication of the influenza A virus RNA genome are mediated by the viral RNA polymerase from a promoter consisting of the partially base-paired 3′ and 5′ termini of viral genome segments. Here we show that transcription and replication can be uncoupled by mutation of an unpaired adenosine in the 5′ strand of the promoter. This residue is important for transcription but not replication by being essential for the cap-binding activity of the RNA polymerase.
    Full-text · Article · Jul 2013 · Journal of Virology
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    Ashley York · Ervin Fodor
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    ABSTRACT: The flow of genetic information from sites of transcription within the nucleus to the cytoplasmic translational machinery of eukaryotic cells is obstructed by a physical blockade, the nuclear double membrane, which must be overcome in order to adhere to the central dogma of molecular biology, DNA makes RNA makes protein. Advancement in the field of cellular and molecular biology has painted a detailed picture of the molecular mechanisms from transcription of genes to mRNAs and their processing that is closely coupled to export from the nucleus. The rules that govern delivering messenger transcripts from the nucleus must be obeyed by influenza A virus, a member of the Orthomyxoviridae that has adopted a nuclear replication cycle. The negative-sense genome of influenza A virus is segmented into eight individual viral ribonucleoprotein (vRNP) complexes containing the viral RNA-dependent RNA polymerase and single-stranded RNA encapsidated in viral nucleoprotein. Influenza A virus mRNAs fall into three major categories, intronless, intron-containing unspliced and spliced. During evolutionary history, influenza A virus has conceived a way of negotiating the passage of viral transcripts from the nucleus to cytoplasmic sites of protein synthesis. The major mRNA nuclear export NXF1 pathway is increasingly implicated in viral mRNA export and this review considers and discusses the current understanding of how influenza A virus exploits the host mRNA export pathway for replication.
    Preview · Article · Jun 2013 · RNA biology
  • E Fodor
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    ABSTRACT: The influenza A virus RNA genome segments are packaged in ribonucleoprotein complexes containing RNA polymerase and nucleoprotein. The ribonucleoprotein is involved in the transcription of viral genes and replication of the viral RNA genome in the nucleus of the infected cells, and represents the minimal transcriptional and replicative machinery of an influenza virus. During transcription, the viral RNA polymerase synthesizes capped and polyadenylated mRNA using 5΄ capped RNA primers. During replication, the viral RNA polymerase generates a complementary RNA (cRNA) replication intermediate, a full-length complement of the vRNA that serves as a template for the synthesis of new copies of vRNA. The nucleoprotein is also an essential component of the viral transcriptional machinery. The molecular determinants of the transcriptional and replicative activities of the viral RNA polymerase are not fully understood, but recent data suggest that transcription is performed by a cis-acting RNA polymerase, forming part of the ribonucleoprotein complex, while replication might be carried out by a trans-acting RNA polymerase. Viral as well as cellular factors are known to be involved in the regulation of the activities of the RNA polymerase, e.g. the viral nuclear export protein has been shown to regulate the accumulation of viral transcription and replication products. The viral transcriptional machinery represents an attractive target for the development of antiviral drugs and lead compounds targeting nucleoprotein and the PA endonuclease domain of the RNA polymerase have already been identified. Keywords: influenza virus; RNA polymerase; nucleoprotein; transcription; replication.
    No preview · Article · Apr 2013 · Acta virologica

Publication Stats

4k Citations
444.06 Total Impact Points

Institutions

  • 1993-2015
    • University of Oxford
      • Sir William Dunn School of Pathology
      Oxford, England, United Kingdom
  • 2011
    • University of Freiburg
      • Department of Virology
      Freiburg, Baden-Württemberg, Germany
  • 1998
    • Icahn School of Medicine at Mount Sinai
      • Department of Microbiology
      Borough of Manhattan, New York, United States