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Arita M. An efficient trans complementation system for in vivo replication of defective poliovirus mutants. J Virol 2024; 98:e0052324. [PMID: 38837378 PMCID: PMC11265389 DOI: 10.1128/jvi.00523-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2024] [Accepted: 05/13/2024] [Indexed: 06/07/2024] Open
Abstract
The picornavirus genome encodes a large, single polyprotein that is processed by viral proteases to form an active replication complex. The replication complex is formed with the viral genome, host proteins, and viral proteins that are produced/translated directly from each of the viral genomes (viral proteins provided in cis). Efficient complementation in vivo of replication complex formation by viral proteins provided in trans, thus exogenous or ectopically expressed viral proteins, remains to be demonstrated. Here, we report an efficient trans complementation system for the replication of defective poliovirus (PV) mutants by a viral polyprotein precursor in HEK293 cells. Viral 3AB in the polyprotein, but not 2BC, was processed exclusively in cis. Replication of a defective PV replicon mutant, with a disrupted cleavage site for viral 3Cpro protease between 3Cpro and 3Dpol (3C/D[A/G] mutant) could be rescued by a viral polyprotein provided in trans. Only a defect of 3Dpol activity of the replicon could be rescued in trans; inactivating mutations in 2CATPase/hel, 3B, and 3Cpro of the replicon completely abrogated the trans-rescued replication. An intact N-terminus of the 3Cpro domain of the 3CDpro provided in trans was essential for the trans-active function. By using this trans complementation system, a high-titer defective PV pseudovirus (PVpv) (>107 infectious units per mL) could be produced with the defective mutants, whose replication was completely dependent on trans complementation. This work reveals potential roles of exogenous viral proteins in PV replication and offers insights into protein/protein interaction during picornavirus infection. IMPORTANCE Viral polyprotein processing is an elaborately controlled step by viral proteases encoded in the polyprotein; fully processed proteins and processing intermediates need to be correctly produced for replication, which can be detrimentally affected even by a small modification of the polyprotein. Purified/isolated viral proteins can retain their enzymatic activities required for viral replication, such as protease, helicase, polymerase, etc. However, when these proteins of picornavirus are exogenously provided (provided in trans) to the viral replication complex with a defective viral genome, replication is generally not rescued/complemented, suggesting the importance of viral proteins endogenously provided (provided in cis) to the replication complex. In this study, I discovered that only the viral polymerase activity of poliovirus (PV) (the typical member of picornavirus family) could be efficiently rescued by exogenously expressed viral proteins. The current study reveals potential roles for exogenous viral proteins in viral replication and offers insights into interactions during picornavirus infection.
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Affiliation(s)
- Minetaro Arita
- Department of Virology II, National Institute of Infectious Diseases, Musashimurayama-shi, Tokyo, Japan
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2
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Oh E, Choi SJ, Han S, Lee KH, Choi HJ. Highly Effective Salt-Activated Alcohol-Based Disinfectants with Enhanced Antimicrobial Activity. ACS NANO 2023; 17:17811-17825. [PMID: 37639494 DOI: 10.1021/acsnano.3c03315] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 08/31/2023]
Abstract
Surfaces contaminated with pathogens raise concerns about the increased risk of disease transmission and infection. To clean biocontaminated surfaces, alcohol-based disinfectants have been predominantly used for disinfecting high-touch areas in diverse settings. However, due to its limited antimicrobial activities and concern over the emergence of alcohol-tolerant strains, much effort has been made to develop highly efficient disinfectant formulations. In this study, we hypothesize that the addition of a physical pathogen inactivation mechanism by salt recrystallization (besides the existing chemical inactivation mechanism by alcohol in such formulations) can improve inactivation efficiency by preventing the emergence of alcohol tolerance. To this end, we employed the drying-induced salt recrystallization process to implement the concept of highly efficient alcohol-based disinfectant formulations. To identify the individual and combined effects of isopropyl alcohol (IPA) and NaCl, time-dependent morphological/structural changes of various IPA solutions containing NaCl have been characterized by optical microscopy/X-ray diffraction analysis. Their antimicrobial activities have been tested on surfaces (glass slide, polystyrene Petri dish, and stainless steel) contaminated with Gram-positive/negative bacteria (methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella enterica subsp. enterica Typhimurium) and viruses (A/PR8/34 H1N1 influenza virus and HCoV-OC43 human coronavirus). We found that additional salt crystallization during the drying of the alcohol solution facilitated stronger biocidal effects than IPA-only formulations, regardless of the types of solid surfaces and pathogens, including alcohol-tolerant strains adapted from wild-type Escherichia coli MG1655. Our findings can be useful in developing highly effective disinfectant formulations by minimizing the use of toxic antimicrobial substances to improve public health and safety.
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Affiliation(s)
- Euna Oh
- Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB T6G 1H9, Canada
| | - Seung Joon Choi
- Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB T6G 1H9, Canada
- Department of Materials Science and Engineering, Yonsei University, Seoul 03722, Republic of Korea
| | - Sumin Han
- Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB T6G 1H9, Canada
| | - Kyu Hyoung Lee
- Department of Materials Science and Engineering, Yonsei University, Seoul 03722, Republic of Korea
| | - Hyo-Jick Choi
- Department of Chemical and Materials Engineering, University of Alberta, Edmonton, AB T6G 1H9, Canada
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Meissner JM, Akhmetova K, Szul T, Viktorova EG, Sha B, Bhatt JM, Lee EJ, Kahn RA, Belov GA, Chesnokov I, Sztul E. The Arf-GEF GBF1 undergoes multi-domain structural shifts to activate Arf at the Golgi. Front Cell Dev Biol 2023; 11:1233272. [PMID: 37745300 PMCID: PMC10512945 DOI: 10.3389/fcell.2023.1233272] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/01/2023] [Accepted: 08/29/2023] [Indexed: 09/26/2023] Open
Abstract
Golgi homeostasis require the activation of Arf GTPases by the guanine-nucleotide exchange factor requires GBF1, whose recruitment to the Golgi represents a rate limiting step in the process. GBF1 contains a conserved, catalytic, Sec7 domain (Sec7d) and five additional (DCB, HUS, HDS1-3) domains. Herein, we identify the HDS3 domain as essential for GBF1 membrane association in mammalian cells and document the critical role of HDS3 during the development of Drosophila melanogaster. We show that upon binding to Golgi membranes, GBF1 undergoes conformational changes in regions bracketing the catalytic Sec7d. We illuminate GBF1 interdomain arrangements by negative staining electron microscopy of full-length human GBF1 to show that GBF1 forms an anti-parallel dimer held together by the paired central DCB-HUS core, with two sets of HDS1-3 arms extending outward in opposite directions. The catalytic Sec7d protrudes from the central core as a largely independent domain, but is closely opposed to a previously unassigned α-helix from the HDS1 domain. Based on our data, we propose models of GBF1 engagement on the membrane to provide a paradigm for understanding GBF1-mediated Arf activation required for cellular and organismal function.
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Affiliation(s)
- Justyna M. Meissner
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, United States
| | - Katarina Akhmetova
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, United States
| | - Tomasz Szul
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, United States
| | - Ekaterina G. Viktorova
- Department of Veterinary Medicine, Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, MD, United States
| | - Bingdong Sha
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, United States
| | - Jay M. Bhatt
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, United States
| | - Eunjoo J. Lee
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, United States
| | - Richard A. Kahn
- Department of Biochemistry, Emory University School of Medicine, Atlanta, GA, United States
| | - George A. Belov
- Department of Veterinary Medicine, Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, MD, United States
| | - Igor Chesnokov
- Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, United States
| | - Elizabeth Sztul
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL, United States
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Viktorova EG, Gabaglio S, Moghimi S, Zimina A, Wynn BG, Sztul E, Belov GA. The development of resistance to an inhibitor of a cellular protein reveals a critical interaction between the enterovirus protein 2C and a small GTPase Arf1. PLoS Pathog 2023; 19:e1011673. [PMID: 37721955 PMCID: PMC10538752 DOI: 10.1371/journal.ppat.1011673] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2023] [Revised: 09/28/2023] [Accepted: 09/08/2023] [Indexed: 09/20/2023] Open
Abstract
The cellular protein GBF1, an activator of Arf GTPases (ArfGEF: Arf guanine nucleotide exchange factor), is recruited to the replication organelles of enteroviruses through interaction with the viral protein 3A, and its ArfGEF activity is required for viral replication, however how GBF1-dependent Arf activation supports the infection remains enigmatic. Here, we investigated the development of resistance of poliovirus, a prototype enterovirus, to increasing concentrations of brefeldin A (BFA), an inhibitor of GBF1. High level of resistance required a gradual accumulation of multiple mutations in the viral protein 2C. The 2C mutations conferred BFA resistance even in the context of a 3A mutant previously shown to be defective in the recruitment of GBF1 to replication organelles, and in cells depleted of GBF1, suggesting a GBF1-independent replication mechanism. Still, activated Arfs accumulated on the replication organelles of this mutant even in the presence of BFA, its replication was inhibited by a pan-ArfGEF inhibitor LM11, and the BFA-resistant phenotype was compromised in Arf1-knockout cells. Importantly, the mutations strongly increased the interaction of 2C with the activated form of Arf1. Analysis of other enteroviruses revealed a particularly strong interaction of 2C of human rhinovirus 1A with activated Arf1. Accordingly, the replication of this virus was significantly less sensitive to BFA than that of poliovirus. Thus, our data demonstrate that enterovirus 2Cs may behave like Arf1 effector proteins and that GBF1 but not Arf activation can be dispensable for enterovirus replication. These findings have important implications for the development of host-targeted anti-viral therapeutics.
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Affiliation(s)
- Ekaterina G. Viktorova
- Department of Veterinary Medicine and Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
| | - Samuel Gabaglio
- Department of Veterinary Medicine and Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
| | - Seyedehmahsa Moghimi
- Department of Veterinary Medicine and Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
| | - Anna Zimina
- Department of Veterinary Medicine and Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
| | - Bridge G. Wynn
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham; Birmingham, Alabama, United States of America
| | - Elizabeth Sztul
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham; Birmingham, Alabama, United States of America
| | - George A. Belov
- Department of Veterinary Medicine and Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
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Walton K, Nawara TJ, Angermeier AR, Rosengrant H, Lee E, Wynn B, Victorova E, Belov G, Sztul E. Site-specific phosphorylations of the Arf activator GBF1 differentially regulate GBF1 function in Golgi homeostasis and secretion versus cytokinesis. Sci Rep 2023; 13:13609. [PMID: 37604968 PMCID: PMC10442430 DOI: 10.1038/s41598-023-40705-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2022] [Accepted: 08/16/2023] [Indexed: 08/23/2023] Open
Abstract
Diverse cellular processes, including membrane traffic, lipid homeostasis, cytokinesis, mitochondrial positioning, and cell motility are critically dependent on the Sec7 domain guanine nucleotide exchange factor GBF1. Yet, how the participation of GBF1 in a particular cellular function is regulated is unknown. Here, we show that the phosphorylation of specific highly conserved serine and tyrosine residues within the N-terminal domain of GBF1 differentially regulates its function in maintaining Golgi homeostasis and facilitating secretion versus its role in cytokinesis. Specifically, GBF1 mutants containing single amino acid substitutions that mimic a stably phosphorylated S233, S371, Y377, and Y515 or the S233A mutant that can't be phosphorylated are fully able to maintain Golgi architecture and support cargo traffic through the secretory pathway when assessed in multiple functional assays. However, the same mutants cause multi-nucleation when expressed in cells, and appear to inhibit the progression through mitosis and the resolution of cytokinetic bridges. Thus, GBF1 participates in distinct interactive networks when mediating Golgi homeostasis and secretion versus facilitating cytokinesis, and GBF1 integration into such networks is differentially regulated by the phosphorylation of specific GBF1 residues.
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Affiliation(s)
- Kendall Walton
- Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, 1918 University Boulevard, MCLM 668, Birmingham, AL, 35233-2008, USA.
| | - Tomasz J Nawara
- Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, 1918 University Boulevard, MCLM 668, Birmingham, AL, 35233-2008, USA
| | - Allyson R Angermeier
- Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, 1918 University Boulevard, MCLM 668, Birmingham, AL, 35233-2008, USA
| | - Hadley Rosengrant
- Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, 1918 University Boulevard, MCLM 668, Birmingham, AL, 35233-2008, USA
| | - Eunjoo Lee
- Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, 1918 University Boulevard, MCLM 668, Birmingham, AL, 35233-2008, USA
| | - Bridge Wynn
- Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, 1918 University Boulevard, MCLM 668, Birmingham, AL, 35233-2008, USA
| | - Ekaterina Victorova
- Department of Veterinary Medicine, Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, MD, 20742, USA
| | - George Belov
- Department of Veterinary Medicine, Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, MD, 20742, USA
| | - Elizabeth Sztul
- Department of Cell, Developmental, and Integrative Biology, University of Alabama at Birmingham, 1918 University Boulevard, MCLM 668, Birmingham, AL, 35233-2008, USA
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6
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Navare AT, Mast FD, Olivier JP, Bertomeu T, Neal ML, Carpp LN, Kaushansky A, Coulombe-Huntington J, Tyers M, Aitchison JD. Viral protein engagement of GBF1 induces host cell vulnerability through synthetic lethality. J Cell Biol 2022; 221:213618. [PMID: 36305789 PMCID: PMC9623979 DOI: 10.1083/jcb.202011050] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2020] [Revised: 06/15/2022] [Accepted: 08/26/2022] [Indexed: 12/14/2022] Open
Abstract
Viruses co-opt host proteins to carry out their lifecycle. Repurposed host proteins may thus become functionally compromised; a situation analogous to a loss-of-function mutation. We term such host proteins as viral-induced hypomorphs. Cells bearing cancer driver loss-of-function mutations have successfully been targeted with drugs perturbing proteins encoded by the synthetic lethal (SL) partners of cancer-specific mutations. Similarly, SL interactions of viral-induced hypomorphs can potentially be targeted as host-based antiviral therapeutics. Here, we use GBF1, which supports the infection of many RNA viruses, as a proof-of-concept. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. Screening for SL partners of GBF1 revealed ARF1 as the top hit, disruption of which selectively killed cells that synthesize 3A alone or in the context of a poliovirus replicon. Thus, viral protein interactions can induce hypomorphs that render host cells selectively vulnerable to perturbations that leave uninfected cells otherwise unscathed. Exploiting viral-induced vulnerabilities could lead to broad-spectrum antivirals for many viruses, including SARS-CoV-2.
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Affiliation(s)
- Arti T. Navare
- Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, WA
| | - Fred D. Mast
- Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, WA
| | - Jean Paul Olivier
- Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, WA
| | - Thierry Bertomeu
- Institute for Research in Immunology and Cancer, Université de Montréal, Montreal, Quebec, Canada
| | - Maxwell L. Neal
- Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, WA
| | | | - Alexis Kaushansky
- Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, WA,Department of Pediatrics, University of Washington, Seattle, WA
| | | | - Mike Tyers
- Institute for Research in Immunology and Cancer, Université de Montréal, Montreal, Quebec, Canada
| | - John D. Aitchison
- Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, WA,Department of Pediatrics, University of Washington, Seattle, WA,Department of Biochemistry, University of Washington, Seattle, WA,Correspondence to John D. Aitchison:
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7
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Wang Y, Ramos M, Jefferson M, Zhang W, Beraza N, Carding S, Powell PP, Stewart JP, Mayer U, Wileman T. Control of infection by LC3-associated phagocytosis, CASM, and detection of raised vacuolar pH by the V-ATPase-ATG16L1 axis. SCIENCE ADVANCES 2022; 8:eabn3298. [PMID: 36288298 PMCID: PMC9604538 DOI: 10.1126/sciadv.abn3298] [Citation(s) in RCA: 25] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/18/2021] [Accepted: 07/07/2022] [Indexed: 05/29/2023]
Abstract
The delivery of pathogens to lysosomes for degradation provides an important defense against infection. Degradation is enhanced when LC3 is conjugated to endosomes and phagosomes containing pathogens to facilitate fusion with lysosomes. In phagocytic cells, TLR signaling and Rubicon activate LC3-associated phagocytosis (LAP) where stabilization of the NADPH oxidase leads to sustained ROS production and raised vacuolar pH. Raised pH triggers the assembly of the vacuolar ATPase on the vacuole membrane where it binds ATG16L1 to recruit the core LC3 conjugation complex (ATG16L1:ATG5-12). This V-ATPase-ATG16L1 axis is also activated in nonphagocytic cells to conjugate LC3 to endosomes containing extracellular microbes. Pathogens provide additional signals for recruitment of LC3 when they raise vacuolar pH with pore-forming toxins and proteins, phospholipases, or specialized secretion systems. Many microbes secrete virulence factors to inhibit ROS production and/or the V-ATPase-ATG16L1 axis to slow LC3 recruitment and avoid degradation in lysosomes.
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Affiliation(s)
- Yingxue Wang
- Norwich Medical School, University of East Anglia, Norwich, UK
- Quadram Institute Bioscience, Norwich, UK
| | - Maria Ramos
- Norwich Medical School, University of East Anglia, Norwich, UK
- Quadram Institute Bioscience, Norwich, UK
| | | | - Weijiao Zhang
- Norwich Medical School, University of East Anglia, Norwich, UK
| | | | | | - Penny P. Powell
- Norwich Medical School, University of East Anglia, Norwich, UK
| | - James P. Stewart
- Department of Infection Biology, University of Liverpool, Liverpool, UK
| | - Ulrike Mayer
- School of Biological Sciences, University of East Anglia, Norwich, UK
| | - Thomas Wileman
- Norwich Medical School, University of East Anglia, Norwich, UK
- Quadram Institute Bioscience, Norwich, UK
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Moghimi S, Viktorova EG, Gabaglio S, Zimina A, Budnik B, Wynn BG, Sztul E, Belov GA. A Proximity biotinylation assay with a host protein bait reveals multiple factors modulating enterovirus replication. PLoS Pathog 2022; 18:e1010906. [PMID: 36306280 PMCID: PMC9645661 DOI: 10.1371/journal.ppat.1010906] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2022] [Revised: 11/09/2022] [Accepted: 09/30/2022] [Indexed: 11/07/2022] Open
Abstract
As ultimate parasites, viruses depend on host factors for every step of their life cycle. On the other hand, cells evolved multiple mechanisms of detecting and interfering with viral replication. Yet, our understanding of the complex ensembles of pro- and anti-viral factors is very limited in virtually every virus-cell system. Here we investigated the proteins recruited to the replication organelles of poliovirus, a representative of the genus Enterovirus of the Picornaviridae family. We took advantage of a strict dependence of enterovirus replication on a host protein GBF1, and established a stable cell line expressing a truncated GBF1 fused to APEX2 peroxidase that effectively supported viral replication upon inhibition of the endogenous GBF1. This construct biotinylated multiple host and viral proteins on the replication organelles. Among the viral proteins, the polyprotein cleavage intermediates were overrepresented, suggesting that the GBF1 environment is linked to viral polyprotein processing. The proteomics characterization of biotinylated host proteins identified multiple proteins previously associated with enterovirus replication, as well as more than 200 new factors recruited to the replication organelles. RNA metabolism proteins, many of which normally localize in the nucleus, constituted the largest group, underscoring the massive release of nuclear factors into the cytoplasm of infected cells and their involvement in viral replication. Functional analysis of several newly identified proteins revealed both pro- and anti-viral factors, including a novel component of infection-induced stress granules. Depletion of these proteins similarly affected the replication of diverse enteroviruses indicating broad conservation of the replication mechanisms. Thus, our data significantly expand the knowledge of the composition of enterovirus replication organelles, provide new insights into viral replication, and offer a novel resource for identifying targets for anti-viral interventions.
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Affiliation(s)
- Seyedehmahsa Moghimi
- Department of Veterinary Medicine and Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
| | - Ekaterina G. Viktorova
- Department of Veterinary Medicine and Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
| | - Samuel Gabaglio
- Department of Veterinary Medicine and Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
| | - Anna Zimina
- Department of Veterinary Medicine and Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
| | - Bogdan Budnik
- Mass Spectrometry and Proteomics Resource Laboratory (MSPRL), FAS Division of Science, Harvard University, Cambridge, Massachusetts, United States of America
| | - Bridge G. Wynn
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham; Birmingham, Alabama, United States of America
| | - Elizabeth Sztul
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham; Birmingham, Alabama, United States of America
| | - George A. Belov
- Department of Veterinary Medicine and Virginia-Maryland College of Veterinary Medicine, University of Maryland, College Park, Maryland, United States of America
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AG1478 Elicits a Novel Anti-Influenza Function via an EGFR-Independent, GBF1-Dependent Pathway. Int J Mol Sci 2022; 23:ijms23105557. [PMID: 35628375 PMCID: PMC9145774 DOI: 10.3390/ijms23105557] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2022] [Revised: 05/10/2022] [Accepted: 05/12/2022] [Indexed: 12/10/2022] Open
Abstract
Current options for preventing or treating influenza are still limited, and new treatments for influenza viral infection are urgently needed. In the present study, we serendipitously found that a small-molecule inhibitor (AG1478), previously used for epidermal growth factor receptor (EGFR) inhibition, demonstrated a potent activity against influenza both in vitro and in vivo. Surprisingly, the antiviral effect of AG1478 was not mediated by its EGFR inhibitory activity, as influenza virus was insensitive to EGFR blockade by other EGFR inhibitors or by siRNA knockdown of EGFR. Its antiviral activity was also interferon independent as demonstrated by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) knockout approach. Instead, AG1478 was found to target the Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1)–ADP-ribosylation factor 1 (ARF1) system by reversibly inhibiting GBF1 activity and disrupting its Golgi-cytoplasmic trafficking. Compared to known GBF1 inhibitors, AG1478 demonstrated lower cellular toxicity and better preservation of Golgi structure. Furthermore, GBF1 was found to interact with a specific set of viral proteins including M1, NP, and PA. Additionally, the alternation of GBF1 distribution induced by AG1478 treatment disrupted these interactions. Because targeting host factors, instead of the viral component, imposes a higher barrier for developing resistance, GBF1 modulation may be an effective approach to treat influenza infection.
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Abstract
Birnaviruses are members of the Birnaviridae family, responsible for major economic losses to poultry and aquaculture. The family is composed of non-enveloped viruses with a segmented double-stranded RNA (dsRNA) genome. Infectious bursal disease virus (IBDV), the prototypic family member, is the etiological agent of Gumboro disease, a highly contagious immunosuppressive disease in the poultry industry worldwide. We previously demonstrated that IBDV hijacks the endocytic pathway for establishing the viral replication complexes on endosomes associated with the Golgi complex (GC). In this work, we report that IBDV reorganizes the GC to localize the endosome-associated replication complexes without affecting its secretory functionality. Analyzing crucial proteins involved in the secretory pathway, we showed the essential requirement of Rab1b for viral replication. Rab1b comprises a key regulator of GC transport and we demonstrate that transfecting the negative mutant Rab1b N121I or knocking down Rab1b expression by RNA interference significantly reduces the yield of infectious viral progeny. Furthermore, we showed that the Rab1b downstream effector Golgi-specific BFA resistance factor 1 (GBF1), which activates the small GTPase ADP-ribosylation factor 1 (ARF1), is required for IBDV replication since inhibiting its activity by treatment with brefeldin A (BFA) or Golgicide A (GCA) significantly reduces the yield of infectious viral progeny. Finally, we show that ARF1 dominant negative-mutant T31N over-expression hampered the IBDV infection. Taken together, these results demonstrate that IBDV requires the function of the Rab1b-GBF1-ARF1 axis to promote its replication, making a substantial contribution to the field of birnaviruses-host cell interactions. IMPORTANCE Birnaviruses are unconventional members of the dsRNA viruses, being the lack of a transcriptionally active core the main differential feature. This structural trait, among others that resemble the plus single-stranded (+ssRNA) viruses features, suggests that birnaviruses might follow a different replication program from that conducted by prototypical dsRNA members and have argued the hypothesis that birnaviruses could be evolutionary links between +ssRNA and dsRNA viruses. Here, we present original data showing the IBDV-induced GC reorganization and the crosstalk between IBDV and the Rab1b-GBF1-ARF1 mediated intracellular trafficking pathway. The replication of several +ssRNA viruses depends on the cellular protein GBF1, but its role in the replication process is not clear. Thus, our findings make a substantial contribution to the field of birnaviruses-host cells and provide further evidence supporting the proposed evolutionary connection role of birnaviruses, an aspect which we consider especially relevant for researchers working in the virology field.
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Hassan Z, Kumar ND, Reggiori F, Khan G. How Viruses Hijack and Modify the Secretory Transport Pathway. Cells 2021; 10:2535. [PMID: 34685515 PMCID: PMC8534161 DOI: 10.3390/cells10102535] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2021] [Revised: 08/28/2021] [Accepted: 09/06/2021] [Indexed: 12/23/2022] Open
Abstract
Eukaryotic cells contain dynamic membrane-bound organelles that are constantly remodeled in response to physiological and environmental cues. Key organelles are the endoplasmic reticulum, the Golgi apparatus and the plasma membrane, which are interconnected by vesicular traffic through the secretory transport route. Numerous viruses, especially enveloped viruses, use and modify compartments of the secretory pathway to promote their replication, assembly and cell egression by hijacking the host cell machinery. In some cases, the subversion mechanism has been uncovered. In this review, we summarize our current understanding of how the secretory pathway is subverted and exploited by viruses belonging to Picornaviridae, Coronaviridae, Flaviviridae,Poxviridae, Parvoviridae and Herpesviridae families.
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Affiliation(s)
- Zubaida Hassan
- Department of Medical Microbiology and Immunology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain P.O. Box 17666, United Arab Emirates;
- Department of Microbiology, School of Life Sciences, Modibbo Adama University, Yola PMB 2076, Nigeria
| | - Nilima Dinesh Kumar
- Department of Biomedical Sciences of Cells and Systems, University of Groningen, University Medical Center Groningen, 9713 AV Groningen, The Netherlands; (N.D.K.); (F.R.)
- Department of Medical Microbiology and Infection Prevention, University of Groningen, University Medical Center Groningen, 9713 AV Groningen, The Netherlands
| | - Fulvio Reggiori
- Department of Biomedical Sciences of Cells and Systems, University of Groningen, University Medical Center Groningen, 9713 AV Groningen, The Netherlands; (N.D.K.); (F.R.)
| | - Gulfaraz Khan
- Department of Medical Microbiology and Immunology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain P.O. Box 17666, United Arab Emirates;
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12
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Secretory Carrier Membrane Protein 3 Interacts with 3A Viral Protein of Enterovirus and Participates in Viral Replication. Microbiol Spectr 2021; 9:e0047521. [PMID: 34378951 PMCID: PMC8552740 DOI: 10.1128/spectrum.00475-21] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022] Open
Abstract
Picornaviruses are a diverse and major cause of human disease, and their genomes replicate with intracellular membranes. The functionality of these replication organelles depends on the activities of both viral nonstructural proteins and co-opted host proteins. The mechanism by which viral-host interactions generate viral replication organelles and regulate viral RNA synthesis is unclear. To elucidate this mechanism, enterovirus A71 (EV-A71) was used here as a virus model to investigate how these replication organelles are formed and to identify the cellular components that are critical in this process. An immunoprecipitation assay was combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis to identify 172 cellular proteins and four viral proteins associating with viral 3A protein. Secretory carrier membrane protein 3 (SCAMP3) was one of the host proteins we selected for further investigation. Here, we demonstrate by immunoprecipitation assay that SCAMP3 associates with 3A protein and colocalizes with 3A protein during virus infection. SCAMP3 knockdown or knockout in infected cells decreases synthesis of EV-A71 viral RNA, viral proteins, and viral growth. Furthermore, the viral 3A protein associates with SCAMP3 and phosphatidylinositol-4-kinase type III β (PI4KIIIβ) as shown by immunoprecipitation assay and colocalizes to the replication complex. Upon infection of cells with a SCAMP3 knockout construct, PI4KIIIβ and phosphatidylinositol-4-phosphate (PI4P) colocalization with EV-A71 3A protein decreases; viral RNA synthesis also decreases. SCAMP3 is also involved in the extracellular signal-regulated kinase (ERK) signaling pathway to regulate viral replication. The 3A and SCAMP3 interaction is also important for the replication of coxsackievirus B3 (CVB3). SCAMP3 also associates with 3A protein of CVB3 and enhances viral replication but does not regulate dengue virus 2 (DENV2) replication. Taken together, the results suggest that enterovirus 3A protein, SCAMP3, PI4KIIIβ, and PI4P form a replication complex and positively regulate enterovirus replication. IMPORTANCE Virus-host interaction plays an important role in viral replication. 3A protein of enterovirus A71 (EV-A71) recruits other viral and host factors to form a replication complex, which is important for viral replication. In this investigation, we utilized immunoprecipitation combined with proteomics approaches to identify 3A-interacting factors. Our results demonstrate that secretory carrier membrane protein 3 (SCAMP3) is a novel host factor that associates with enterovirus 3A protein, phosphatidylinositol-4-kinase type III β (PI4KIIIβ), and phosphatidylinositol-4-phosphate (PI4P) to form a replication complex and positively regulates viral replication. SCAMP3 is also involved in the extracellular signal-regulated kinase (ERK) signaling pathway to regulate viral replication.
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Domanska A, Guryanov S, Butcher SJ. A comparative analysis of parechovirus protein structures with other picornaviruses. Open Biol 2021; 11:210008. [PMID: 34315275 PMCID: PMC8316810 DOI: 10.1098/rsob.210008] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2021] [Accepted: 07/01/2021] [Indexed: 12/26/2022] Open
Abstract
Parechoviruses belong to the genus Parechovirus within the family Picornaviridae and are non-enveloped icosahedral viruses with a single-stranded RNA genome. Parechoviruses include human and animal pathogens classified into six species. Those that infect humans belong to the Parechovirus A species and can cause infections ranging from mild gastrointestinal or respiratory illness to severe neonatal sepsis. There are no approved antivirals available to treat parechovirus (nor any other picornavirus) infections. In this parechovirus review, we focus on the cleaved protein products resulting from the polyprotein processing after translation comparing and contrasting their known or predicted structures and functions to those of other picornaviruses. The review also includes our original analysis from sequence and structure prediction. This review highlights significant structural differences between parechoviral and other picornaviral proteins, suggesting that parechovirus drug development should specifically be directed to parechoviral targets.
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Affiliation(s)
- Aušra Domanska
- Faculty of Biological and Environmental Sciences, Molecular and Integrative Bioscience Research Programme, and Helsinki Institute of Life Sciences–Institute of Biotechnology, University of Helsinki, FI-00014 Helsinki, Finland
| | - Sergey Guryanov
- Faculty of Biological and Environmental Sciences, Molecular and Integrative Bioscience Research Programme, and Helsinki Institute of Life Sciences–Institute of Biotechnology, University of Helsinki, FI-00014 Helsinki, Finland
| | - Sarah J. Butcher
- Faculty of Biological and Environmental Sciences, Molecular and Integrative Bioscience Research Programme, and Helsinki Institute of Life Sciences–Institute of Biotechnology, University of Helsinki, FI-00014 Helsinki, Finland
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14
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Jackson T, Belsham GJ. Picornaviruses: A View from 3A. Viruses 2021; 13:v13030456. [PMID: 33799649 PMCID: PMC7999760 DOI: 10.3390/v13030456] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2021] [Revised: 03/08/2021] [Accepted: 03/09/2021] [Indexed: 12/14/2022] Open
Abstract
Picornaviruses are comprised of a positive-sense RNA genome surrounded by a protein shell (or capsid). They are ubiquitous in vertebrates and cause a wide range of important human and animal diseases. The genome encodes a single large polyprotein that is processed to structural (capsid) and non-structural proteins. The non-structural proteins have key functions within the viral replication complex. Some, such as 3Dpol (the RNA dependent RNA polymerase) have conserved functions and participate directly in replicating the viral genome, whereas others, such as 3A, have accessory roles. The 3A proteins are highly divergent across the Picornaviridae and have specific roles both within and outside of the replication complex, which differ between the different genera. These roles include subverting host proteins to generate replication organelles and inhibition of cellular functions (such as protein secretion) to influence virus replication efficiency and the host response to infection. In addition, 3A proteins are associated with the determination of host range. However, recent observations have challenged some of the roles assigned to 3A and suggest that other viral proteins may carry them out. In this review, we revisit the roles of 3A in the picornavirus life cycle. The 3AB precursor and mature 3A have distinct functions during viral replication and, therefore, we have also included discussion of some of the roles assigned to 3AB.
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Affiliation(s)
- Terry Jackson
- The Pirbright Institute, Pirbright, Woking, Surrey GU24 0NF, UK;
| | - Graham J. Belsham
- Department of Veterinary and Animal Sciences, University of Copenhagen, 1870 Frederiksberg, Denmark
- Correspondence:
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15
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Enterovirus Infection Induces Massive Recruitment of All Isoforms of Small Cellular Arf GTPases to the Replication Organelles. J Virol 2020; 95:JVI.01629-20. [PMID: 33087467 DOI: 10.1128/jvi.01629-20] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/14/2020] [Accepted: 10/18/2020] [Indexed: 12/12/2022] Open
Abstract
Enterovirus replication requires the cellular protein GBF1, a guanine nucleotide exchange factor for small Arf GTPases. When activated, Arfs associate with membranes, where they regulate numerous steps of membrane homeostasis. The requirement for GBF1 implies that Arfs are important for replication, but which of the different Arfs function(s) during replication remains poorly understood. Here, we established cell lines expressing each of the human Arfs fused to a fluorescent tag and investigated their behavior during enterovirus infection. Arf1 was the first to be recruited to the replication organelles, where it strongly colocalized with the viral antigen 2B and mature virions but not double-stranded RNA. By the end of the infectious cycle, Arf3, Arf4, Arf5, and Arf6 were also concentrated on the replication organelles. Once on the replication membranes, all Arfs except Arf3 were no longer sensitive to inhibition of GBF1, suggesting that in infected cells they do not actively cycle between GTP- and GDP-bound states. Only the depletion of Arf1, but not other class 1 and 2 Arfs, significantly increased the sensitivity of replication to GBF1 inhibition. Surprisingly, depletion of Arf6, a class 3 Arf, normally implicated in plasma membrane events, also increased the sensitivity to GBF1 inhibition. Together, our results suggest that GBF1-dependent Arf1 activation directly supports the development and/or functioning of the replication complexes and that Arf6 plays a previously unappreciated role in viral replication. Our data reveal a complex pattern of Arf activation in enterovirus-infected cells that may contribute to the resilience of viral replication in different cellular environments.IMPORTANCE Enteroviruses include many known and emerging pathogens, such as poliovirus, enteroviruses 71 and D68, and others. However, licensed vaccines are available only against poliovirus and enterovirus 71, and specific anti-enterovirus therapeutics are lacking. Enterovirus infection induces the massive remodeling of intracellular membranes and the development of specialized domains harboring viral replication complexes, replication organelles. Here, we investigated the roles of small Arf GTPases during enterovirus infection. Arfs control distinct steps in intracellular membrane traffic, and one of the Arf-activating proteins, GBF1, is a cellular factor required for enterovirus replication. We found that all Arfs expressed in human cells, including Arf6, normally associated with the plasma membrane, are recruited to the replication organelles and that Arf1 appears to be the most important Arf for enterovirus replication. These results document the rewiring of the cellular membrane pathways in infected cells and may provide new ways of controlling enterovirus infections.
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16
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Navare AT, Mast FD, Olivier JP, Bertomeu T, Neal M, Carpp LN, Kaushansky A, Coulombe-Huntington J, Tyers M, Aitchison JD. Viral protein engagement of GBF1 induces host cell vulnerability through synthetic lethality. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2020; 221:2020.10.12.336487. [PMID: 33173868 PMCID: PMC7654857 DOI: 10.1101/2020.10.12.336487] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Viruses co-opt host proteins to carry out their lifecycle. Repurposed host proteins may thus become functionally compromised; a situation analogous to a loss-of-function mutation. We term such host proteins viral-induced hypomorphs. Cells bearing cancer driver loss-of-function mutations have successfully been targeted with drugs perturbing proteins encoded by the synthetic lethal partners of cancer-specific mutations. Synthetic lethal interactions of viral-induced hypomorphs have the potential to be similarly targeted for the development of host-based antiviral therapeutics. Here, we use GBF1, which supports the infection of many RNA viruses, as a proof-of-concept. GBF1 becomes a hypomorph upon interaction with the poliovirus protein 3A. Screening for synthetic lethal partners of GBF1 revealed ARF1 as the top hit, disruption of which, selectively killed cells that synthesize poliovirus 3A. Thus, viral protein interactions can induce hypomorphs that render host cells vulnerable to perturbations that leave uninfected cells intact. Exploiting viral-induced vulnerabilities could lead to broad-spectrum antivirals for many viruses, including SARS-CoV-2. SUMMARY Using a viral-induced hypomorph of GBF1, Navare et al., demonstrate that the principle of synthetic lethality is a mechanism to selectively kill virus-infected cells.
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Affiliation(s)
- Arti T Navare
- Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA
| | - Fred D Mast
- Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA
| | - Jean Paul Olivier
- Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA
| | - Thierry Bertomeu
- Institute for Research in Immunology and Cancer, Université de Montréal, Montreal, Quebec, Canada
| | - Maxwell Neal
- Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA
| | - Lindsay N Carpp
- Center for Infectious Disease Research, Seattle, Washington, USA
| | - Alexis Kaushansky
- Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA
- Department of Pediatrics, University of Washington, Seattle, Washington, USA
| | | | - Mike Tyers
- Institute for Research in Immunology and Cancer, Université de Montréal, Montreal, Quebec, Canada
| | - John D Aitchison
- Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, Washington, USA
- Department of Pediatrics, University of Washington, Seattle, Washington, USA
- Department of Biochemistry, University of Washington, Seattle, Washington, USA
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17
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Martínez JL, Arias CF. Role of the Guanine Nucleotide Exchange Factor GBF1 in the Replication of RNA Viruses. Viruses 2020; 12:E682. [PMID: 32599855 PMCID: PMC7354614 DOI: 10.3390/v12060682] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2020] [Revised: 04/06/2020] [Accepted: 04/06/2020] [Indexed: 12/12/2022] Open
Abstract
The guanine nucleotide exchange factor GBF1 is a well-known factor that can activate different ADP-ribosylation factor (Arf) proteins during the regulation of different cellular vesicular transport processes. In the last decade, it has become increasingly evident that GBF1 can also regulate different steps of the replication cycle of RNA viruses belonging to different virus families. GBF1 has been shown not only to facilitate the intracellular traffic of different viral and cellular elements during infection, but also to modulate the replication of viral RNA, the formation and maturation of viral replication complexes, and the processing of viral proteins through mechanisms that do not depend on its canonical role in intracellular transport. Here, we review the various roles that GBF1 plays during the replication of different RNA viruses.
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Affiliation(s)
| | - Carlos F. Arias
- Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca 4510, Morelos, Mexico;
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18
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Wolff G, Melia CE, Snijder EJ, Bárcena M. Double-Membrane Vesicles as Platforms for Viral Replication. Trends Microbiol 2020; 28:1022-1033. [PMID: 32536523 PMCID: PMC7289118 DOI: 10.1016/j.tim.2020.05.009] [Citation(s) in RCA: 226] [Impact Index Per Article: 45.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2020] [Revised: 05/09/2020] [Accepted: 05/14/2020] [Indexed: 12/12/2022]
Abstract
Viruses, as obligate intracellular parasites, exploit cellular pathways and resources in a variety of fascinating ways. A striking example of this is the remodelling of intracellular membranes into specialized structures that support the replication of positive-sense ssRNA (+RNA) viruses infecting eukaryotes. These distinct forms of virus-induced structures include double-membrane vesicles (DMVs), found during viral infections as diverse and notorious as those of coronaviruses, enteroviruses, noroviruses, or hepatitis C virus. Our understanding of these DMVs has evolved over the past 15 years thanks to advances in imaging techniques and modern molecular biology tools. In this article, we review contemporary understanding of the biogenesis, structure, and function of virus-induced DMVs as well as the open questions posed by these intriguing structures.
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Affiliation(s)
- Georg Wolff
- Section Electron Microscopy, Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands
| | - Charlotte E Melia
- Section Electron Microscopy, Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands
| | - Eric J Snijder
- Molecular Virology laboratory, Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands
| | - Montserrat Bárcena
- Section Electron Microscopy, Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, The Netherlands.
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19
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On the Host Side of the Hepatitis E Virus Life Cycle. Cells 2020; 9:cells9051294. [PMID: 32456000 PMCID: PMC7291229 DOI: 10.3390/cells9051294] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2020] [Revised: 05/20/2020] [Accepted: 05/21/2020] [Indexed: 12/12/2022] Open
Abstract
Hepatitis E virus (HEV) infection is one of the most common causes of acute hepatitis in the world. HEV is an enterically transmitted positive-strand RNA virus found as a non-enveloped particle in bile as well as stool and as a quasi-enveloped particle in blood. Current understanding of the molecular mechanisms and host factors involved in productive HEV infection is incomplete, but recently developed model systems have facilitated rapid progress in this area. Here, we provide an overview of the HEV life cycle with a focus on the host factors required for viral entry, RNA replication, assembly and release. Further developments of HEV model systems and novel technologies should yield a broader picture in the future.
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20
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Structural Biology of the Enterovirus Replication-Linked 5'-Cloverleaf RNA and Associated Virus Proteins. Microbiol Mol Biol Rev 2020; 84:84/2/e00062-19. [PMID: 32188627 DOI: 10.1128/mmbr.00062-19] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Although enteroviruses are associated with a wide variety of diseases and conditions, their mode of replication is well conserved. Their genome is carried as a single, positive-sense RNA strand. At the 5' end of the strand is an approximately 90-nucleotide self-complementary region called the 5' cloverleaf, or the oriL. This noncoding region serves as a platform upon which host and virus proteins, including the 3B, 3C, and 3D virus proteins, assemble in order to initiate replication of a negative-sense RNA strand. The negative strand in turn serves as a template for synthesis of multiple positive-sense RNA strands. Building on structural studies of individual RNA stem-loops, the structure of the intact 5' cloverleaf from rhinovirus has recently been determined via nuclear magnetic resonance/small-angle X-ray scattering (NMR/SAXS)-based methods, while structures have also been determined for enterovirus 3A, 3B, 3C, and 3D proteins. Analysis of these structures, together with structural and modeling studies of interactions between host and virus proteins and RNA, has begun to provide insight into the enterovirus replication mechanism and the potential to inhibit replication by blocking these interactions.
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21
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A Redundant Mechanism of Recruitment Underlies the Remarkable Plasticity of the Requirement of Poliovirus Replication for the Cellular ArfGEF GBF1. J Virol 2019; 93:JVI.00856-19. [PMID: 31375590 DOI: 10.1128/jvi.00856-19] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2019] [Accepted: 07/26/2019] [Indexed: 12/24/2022] Open
Abstract
The replication of many positive-strand RNA viruses [(+)RNA viruses] depends on the cellular protein GBF1, but its role in the replication process is not clear. In uninfected cells, GBF1 activates small GTPases of the Arf family and coordinates multiple steps of membrane metabolism, including functioning of the cellular secretory pathway. The nonstructural protein 3A of poliovirus and related viruses has been shown to directly interact with GBF1, likely mediating its recruitment to the replication complexes. Surprisingly, viral mutants with a severely reduced level of 3A-GBF1 interaction demonstrate minimal replication defects in cell culture. Here, we systematically investigated the conserved elements of GBF1 to understand which determinants are important to support poliovirus replication. We demonstrate that multiple GBF1 mutants inactive in cellular metabolism could still be fully functional in the replication complexes. Our results show that the Arf-activating property, but not the primary structure of the Sec7 domain, is indispensable for viral replication. They also suggest a redundant mechanism of recruitment of GBF1 to the replication sites, which is dependent not only on direct interaction of the protein with the viral protein 3A but also on determinants located in the noncatalytic C-terminal domains of GBF1. Such a double-targeting mechanism explains the previous observations of the remarkable tolerance of different levels of GBF1-3A interaction by the virus and likely constitutes an important element of the resilience of viral replication.IMPORTANCE Enteroviruses are a vast group of viruses associated with diverse human diseases, but only two of them could be controlled with vaccines, and effective antiviral therapeutics are lacking. Here, we investigated in detail the contribution of a cellular protein, GBF1, in the replication of poliovirus, a representative enterovirus. GBF1 supports the functioning of cellular membrane metabolism and is recruited to viral replication complexes upon infection. Our results demonstrate that the virus requires a limited subset of the normal GBF1 functions and reveal the elements of GBF1 essential to support viral replication under different conditions. Since diverse viruses often rely on the same cellular proteins for replication, understanding the mechanisms by which these proteins support infection is essential for the development of broad-spectrum antiviral therapeutics.
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22
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The Guanine Nucleotide Exchange Factor GBF1 Participates in Rotavirus Replication. J Virol 2019; 93:JVI.01062-19. [PMID: 31270230 DOI: 10.1128/jvi.01062-19] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2019] [Accepted: 07/01/2019] [Indexed: 01/06/2023] Open
Abstract
Cellular and viral factors participate in the replication cycle of rotavirus. We report that the guanine nucleotide exchange factor GBF1, which activates the small GTPase Arf1 to induce COPI transport processes, is required for rotavirus replication since knocking down GBF1 expression by RNA interference or inhibiting its activity by treatment with brefeldin A (BFA) or Golgicide A (GCA) significantly reduces the yield of infectious viral progeny. This reduction in virus yield was related to a block in virus assembly, since in the presence of either BFA or GCA, the assembly of infectious mature triple-layered virions was significantly prevented and only double-layered particles were detected. We report that the catalytic activity of GBF1, but not the activation of Arf1, is essential for the assembly of the outer capsid of rotavirus. We show that both BFA and GCA, as well as interfering with the synthesis of GBF1, alter the electrophoretic mobility of glycoproteins VP7 and NSP4 and block the trimerization of the virus surface protein VP7, a step required for its incorporation into virus particles. Although a posttranslational modification of VP7 (other than glycosylation) could be related to the lack of trimerization, we found that NSP4 might also be involved in this process, since knocking down its expression reduces VP7 trimerization. In support, recombinant VP7 protein overexpressed in transfected cells formed trimers only when cotransfected with NSP4.IMPORTANCE Rotavirus, a member of the family Reoviridae, is the major cause of severe diarrhea in children and young animals worldwide. Despite significant advances in the characterization of the biology of this virus, the mechanisms involved in morphogenesis of the virus particle are still poorly understood. In this work, we show that the guanine nucleotide exchange factor GBF1, relevant for COPI/Arf1-mediated cellular vesicular transport, participates in the replication cycle of the virus, influencing the correct processing of viral glycoproteins VP7 and NSP4 and the assembly of the virus surface proteins VP7 and VP4.
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23
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Flavivirus Replication Organelle Biogenesis in the Endoplasmic Reticulum: Comparison with Other Single-Stranded Positive-Sense RNA Viruses. Int J Mol Sci 2019; 20:ijms20092336. [PMID: 31083507 PMCID: PMC6539296 DOI: 10.3390/ijms20092336] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/11/2019] [Revised: 05/04/2019] [Accepted: 05/09/2019] [Indexed: 12/20/2022] Open
Abstract
Some single-stranded positive-sense RNA [ssRNA(+)] viruses, including Flavivirus, generate specific organelle-like structures in the host endoplasmic reticulum (ER). These structures are called virus replication organelles and consist of two distinct subdomains, the vesicle packets (VPs) and the convoluted membranes (CMs). The VPs are clusters of small vesicle compartments and are considered to be the site of viral genome replication. The CMs are electron-dense amorphous structures observed in proximity to the VPs, but the exact roles of CMs are mostly unknown. Several recent studies have revealed that flaviviruses recruit several host factors that are usually used for the biogenesis of other conventional organelles and usurp their function to generate virus replication organelles. In the current review, we summarize recent studies focusing on the role of host factors in the formation of virus replication organelles and discuss how these intricate membrane structures are organized.
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24
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Functional and Physical Interaction between the Arf Activator GBF1 and Hepatitis C Virus NS3 Protein. J Virol 2019; 93:JVI.01459-18. [PMID: 30567983 DOI: 10.1128/jvi.01459-18] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2018] [Accepted: 12/14/2018] [Indexed: 12/11/2022] Open
Abstract
GBF1 has emerged as a host factor required for the genome replication of RNA viruses of different families. During the hepatitis C virus (HCV) life cycle, GBF1 performs a critical function at the onset of genome replication but is dispensable when the replication is established. To better understand how GBF1 regulates HCV infection, we have looked for interactions between GBF1 and HCV proteins. NS3 was found to interact with GBF1 in yeast two-hybrid, coimmunoprecipitation, and proximity ligation assays and to interfere with GBF1 function and alter GBF1 intracellular localization in cells expressing NS3. The interaction was mapped to the Sec7 domain of GBF1 and the protease domain of NS3. A reverse yeast two-hybrid screen to identify mutations altering NS3-GBF1 interaction yielded an NS3 mutant (N77D, Con1 strain) that is nonreplicative despite conserved protease activity and does not interact with GBF1. The mutated residue is exposed at the surface of NS3, suggesting it is part of the domain of NS3 that interacts with GBF1. The corresponding mutation in strain JFH-1 (S77D) produces a similar phenotype. Our results provide evidence for an interaction between NS3 and GBF1 and suggest that an alteration of this interaction is detrimental to HCV genome replication.IMPORTANCE Single-stranded, positive-sense RNA viruses rely to a significant extent on host factors to achieve the replication of their genome. GBF1 is such a cellular protein that is required for the replication of several RNA viruses, but its mechanism of action during viral infections is not yet defined. In this study, we investigated potential interactions that GBF1 might engage in with proteins of HCV, a GBF1-dependent virus. We found that GBF1 interacts with NS3, a nonstructural protein involved in HCV genome replication, and our results suggest that this interaction is important for GBF1 function during HCV replication. Interestingly, GBF1 interaction with HCV appears different from its interaction with enteroviruses, another group of GBF1-dependent RNA viruses, in keeping with the fact that HCV and enteroviruses use different functions of GBF1.
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25
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Fernandez-Chamorro J, Francisco-Velilla R, Ramajo J, Martinez-Salas E. Rab1b and ARF5 are novel RNA-binding proteins involved in FMDV IRES-driven RNA localization. Life Sci Alliance 2019; 2:e201800131. [PMID: 30655362 PMCID: PMC6337736 DOI: 10.26508/lsa.201800131] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2018] [Revised: 01/08/2019] [Accepted: 01/09/2019] [Indexed: 01/07/2023] Open
Abstract
Internal ribosome entry site (IRES) elements are organized in domains that guide internal initiation of translation. Here, we have combined proteomic and imaging analysis to study novel foot-and-mouth disease virus IRES interactors recognizing specific RNA structural subdomains. Besides known picornavirus IRES-binding proteins, we identified novel factors belonging to networks involved in RNA and protein transport. Among those, Rab1b and ARF5, two components of the ER-Golgi, revealed direct binding to IRES transcripts. However, whereas Rab1b stimulated IRES function, ARF5 diminished IRES activity. RNA-FISH studies revealed novel features of the IRES element. First, IRES-RNA formed clusters within the cell cytoplasm, whereas cap-RNA displayed disperse punctate distribution. Second, the IRES-driven RNA localized in close proximity with ARF5 and Rab1b, but not with the dominant-negative of Rab1b that disorganizes the Golgi. Thus, our data suggest a role for domain 3 of the IRES in RNA localization around ER-Golgi, a ribosome-rich cellular compartment.
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Affiliation(s)
- Javier Fernandez-Chamorro
- Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain
| | - Rosario Francisco-Velilla
- Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain
| | - Jorge Ramajo
- Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain
| | - Encarnación Martinez-Salas
- Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Madrid, Spain
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Sager G, Gabaglio S, Sztul E, Belov GA. Role of Host Cell Secretory Machinery in Zika Virus Life Cycle. Viruses 2018; 10:E559. [PMID: 30326556 PMCID: PMC6213159 DOI: 10.3390/v10100559] [Citation(s) in RCA: 57] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2018] [Revised: 10/11/2018] [Accepted: 10/12/2018] [Indexed: 12/16/2022] Open
Abstract
The high human cost of Zika virus infections and the rapid establishment of virus circulation in novel areas, including the United States, present an urgent need for countermeasures against this emerging threat. The development of an effective vaccine against Zika virus may be problematic because of the cross reactivity of the antibodies with other flaviviruses leading to antibody-dependent enhancement of infection. Moreover, rapidly replicating positive strand RNA viruses, including Zika virus, generate large spectrum of mutant genomes (quasi species) every replication round, allowing rapid selection of variants resistant to drugs targeting virus-specific proteins. On the other hand, viruses are ultimate cellular parasites and rely on the host metabolism for every step of their life cycle, thus presenting an opportunity to manipulate host processes as an alternative approach to suppress virus replication and spread. Zika and other flaviviruses critically depend on the cellular secretory pathway, which transfers proteins and membranes from the ER through the Golgi to the plasma membrane, for virion assembly, maturation and release. In this review, we summarize the current knowledge of interactions of Zika and similar arthropod-borne flaviviruses with the cellular secretory machinery with a special emphasis on virus-specific changes of the secretory pathway. Identification of the regulatory networks and effector proteins required to accommodate the trafficking of virions, which represent a highly unusual cargo for the secretory pathway, may open an attractive and virtually untapped reservoir of alternative targets for the development of superior anti-viral drugs.
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Affiliation(s)
- Garrett Sager
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham AL 35294, UK.
| | - Samuel Gabaglio
- Department of Veterinary Medicine, Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, MD 20742, USA.
| | - Elizabeth Sztul
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham AL 35294, UK.
| | - George A Belov
- Department of Veterinary Medicine, Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, MD 20742, USA.
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27
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Phospholipid synthesis fueled by lipid droplets drives the structural development of poliovirus replication organelles. PLoS Pathog 2018; 14:e1007280. [PMID: 30148882 PMCID: PMC6128640 DOI: 10.1371/journal.ppat.1007280] [Citation(s) in RCA: 47] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2018] [Revised: 09/07/2018] [Accepted: 08/13/2018] [Indexed: 01/16/2023] Open
Abstract
Rapid development of complex membranous replication structures is a hallmark of picornavirus infections. However, neither the mechanisms underlying such dramatic reorganization of the cellular membrane architecture, nor the specific role of these membranes in the viral life cycle are sufficiently understood. Here we demonstrate that the cellular enzyme CCTα, responsible for the rate-limiting step in phosphatidylcholine synthesis, translocates from the nuclei to the cytoplasm upon infection and associates with the replication membranes, resulting in the rerouting of lipid synthesis from predominantly neutral lipids to phospholipids. The bulk supply of long chain fatty acids necessary to support the activated phospholipid synthesis in infected cells is provided by the hydrolysis of neutral lipids stored in lipid droplets. Such activation of phospholipid synthesis drives the massive membrane remodeling in infected cells. We also show that complex membranous scaffold of replication organelles is not essential for viral RNA replication but is required for protection of virus propagation from the cellular anti-viral response, especially during multi-cycle replication conditions. Inhibition of infection-specific phospholipid synthesis provides a new paradigm for controlling infection not by suppressing viral replication but by making it more visible to the immune system.
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Newcastle Disease Virus-Based Vectored Vaccine against Poliomyelitis. J Virol 2018; 92:JVI.00976-18. [PMID: 29925653 DOI: 10.1128/jvi.00976-18] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2018] [Accepted: 06/06/2018] [Indexed: 12/28/2022] Open
Abstract
The poliovirus eradication initiative has spawned global immunization infrastructure and dramatically decreased the prevalence of the disease, yet the original virus eradication goal has not been met. The suboptimal properties of the existing vaccines are among the major reasons why the program has repeatedly missed eradication deadlines. Oral live poliovirus vaccine (OPV), while affordable and effective, occasionally causes the disease in the primary recipients, and the attenuated viruses rapidly regain virulence and can cause poliomyelitis outbreaks. Inactivated poliovirus vaccine (IPV) is safe but expensive and does not induce the mucosal immunity necessary to interrupt virus transmission. While the need for a better vaccine is widely recognized, current efforts are focused largely on improvements to the OPV or IPV, which are still beset by the fundamental drawbacks of the original products. Here we demonstrate a different design of an antipoliovirus vaccine based on in situ production of virus-like particles (VLPs). The poliovirus capsid protein precursor, together with a protease required for its processing, are expressed from a Newcastle disease virus (NDV) vector, a negative-strand RNA virus with mucosal tropism. In this system, poliovirus VLPs are produced in the cells of vaccine recipients and are presented to their immune systems in the context of active replication of NDV, which serves as a natural adjuvant. Intranasal administration of the vectored vaccine to guinea pigs induced strong neutralizing systemic and mucosal antibody responses. Thus, the vectored poliovirus vaccine combines the affordability and efficiency of a live vaccine with absolute safety, since no full-length poliovirus genome is present at any stage of the vaccine life cycle.IMPORTANCE A new, safe, and effective vaccine against poliovirus is urgently needed not only to complete the eradication of the virus but also to be used in the future to prevent possible virus reemergence in a postpolio world. Currently, new formulations of the oral vaccine, as well as improvements to the inactivated vaccine, are being explored. In this study, we designed a viral vector with mucosal tropism that expresses poliovirus capsid proteins. Thus, poliovirus VLPs are produced in vivo, in the cells of a vaccine recipient, and are presented to the immune system in the context of vector virus replication, stimulating the development of systemic and mucosal immune responses. Such an approach allows the development of an affordable and safe vaccine that does not rely on the full-length poliovirus genome at any stage.
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Ferlin J, Farhat R, Belouzard S, Cocquerel L, Bertin A, Hober D, Dubuisson J, Rouillé Y. Investigation of the role of GBF1 in the replication of positive-sense single-stranded RNA viruses. J Gen Virol 2018; 99:1086-1096. [PMID: 29923822 DOI: 10.1099/jgv.0.001099] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
GBF1 has emerged as a host factor required for the replication of positive-sense single-stranded RNA viruses of different families, but its mechanism of action is still unknown. GBF1 is a guanine nucleotide exchange factor for Arf family members. Recently, we identified Arf4 and Arf5 (class II Arfs) as host factors required for the replication of hepatitis C virus (HCV), a GBF1-dependent virus. To assess whether a GBF1/class II Arf pathway is conserved among positive-sense single-stranded RNA viruses, we investigated yellow fever virus (YFV), Sindbis virus (SINV), coxsackievirus B4 (CVB4) and human coronavirus 229E (HCoV-229E). We found that GBF1 is involved in the replication of these viruses. However, using siRNA or CRISPR-Cas9 technologies, it was seen that the depletion of Arf1, Arf3, Arf4 or Arf5 had no impact on viral replication. In contrast, the depletion of Arf pairs suggested that class II Arfs could be involved in HCoV-229E, YFV and SINV infection, as for HCV, but not in CVB4 infection. In addition, another Arf pair, Arf1 and Arf4, appears to be essential for YFV and SINV infection, but not for infection by other viruses. Finally, CVB4 infection was not inhibited by any combination of Arf depletion. We conclude that the mechanism of action of GBF1 in viral replication appears not to be conserved, and that a subset of positive-sense single-stranded RNA viruses from different families might require class II Arfs for their replication.
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Affiliation(s)
- Juliette Ferlin
- 1Center for Infection and Immunity of Lille, Inserm U1019, CNRS UMR-8204, Institut Pasteur de Lille, Université de Lille, Lille, France
| | - Rayan Farhat
- 1Center for Infection and Immunity of Lille, Inserm U1019, CNRS UMR-8204, Institut Pasteur de Lille, Université de Lille, Lille, France.,†Present address: Inserm U1052, Cancer Research Center of Lyon (CRCL), Université de Lyon (UCBL1), CNRS UMR-5286, Centre Léon Bérard, Lyon, France
| | - Sandrine Belouzard
- 1Center for Infection and Immunity of Lille, Inserm U1019, CNRS UMR-8204, Institut Pasteur de Lille, Université de Lille, Lille, France
| | - Laurence Cocquerel
- 1Center for Infection and Immunity of Lille, Inserm U1019, CNRS UMR-8204, Institut Pasteur de Lille, Université de Lille, Lille, France
| | - Antoine Bertin
- 2Université de Lille, Faculté de Médecine, CHU Lille, Laboratoire de Virologie EA3610, Lille, France
| | - Didier Hober
- 2Université de Lille, Faculté de Médecine, CHU Lille, Laboratoire de Virologie EA3610, Lille, France
| | - Jean Dubuisson
- 1Center for Infection and Immunity of Lille, Inserm U1019, CNRS UMR-8204, Institut Pasteur de Lille, Université de Lille, Lille, France
| | - Yves Rouillé
- 1Center for Infection and Immunity of Lille, Inserm U1019, CNRS UMR-8204, Institut Pasteur de Lille, Université de Lille, Lille, France
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30
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Melia CE, van der Schaar HM, Lyoo H, Limpens RWAL, Feng Q, Wahedi M, Overheul GJ, van Rij RP, Snijder EJ, Koster AJ, Bárcena M, van Kuppeveld FJM. Escaping Host Factor PI4KB Inhibition: Enterovirus Genomic RNA Replication in the Absence of Replication Organelles. Cell Rep 2018; 21:587-599. [PMID: 29045829 PMCID: PMC5656745 DOI: 10.1016/j.celrep.2017.09.068] [Citation(s) in RCA: 43] [Impact Index Per Article: 6.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2017] [Revised: 08/25/2017] [Accepted: 09/20/2017] [Indexed: 01/15/2023] Open
Abstract
Enteroviruses reorganize cellular endomembranes into replication organelles (ROs) for genome replication. Although enterovirus replication depends on phosphatidylinositol 4-kinase type IIIβ (PI4KB), its role, and that of its product, phosphatidylinositol 4-phosphate (PI4P), is only partially understood. Exploiting a mutant coxsackievirus resistant to PI4KB inhibition, we show that PI4KB activity has distinct functions both in proteolytic processing of the viral polyprotein and in RO biogenesis. The escape mutation rectifies a proteolytic processing defect imposed by PI4KB inhibition, pointing to a possible escape mechanism. Remarkably, under PI4KB inhibition, the mutant virus could replicate its genome in the absence of ROs, using instead the Golgi apparatus. This impaired RO biogenesis provided an opportunity to investigate the proposed role of ROs in shielding enteroviral RNA from cellular sensors. Neither accelerated sensing of viral RNA nor enhanced innate immune responses was observed. Together, our findings challenge the notion that ROs are indispensable for enterovirus genome replication and immune evasion.
PI4KB activity expedites the formation of coxsackievirus replication organelles (ROs) PI4KB inhibition impairs polyprotein processing, which is rescued by a 3A mutation Upon PI4KB inhibition, this mutant replicates at the Golgi in the absence of ROs Innate immune responses are not enhanced when RO biogenesis is delayed
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Affiliation(s)
- Charlotte E Melia
- Department of Molecular Cell Biology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands
| | - Hilde M van der Schaar
- Department of Infectious Diseases & Immunology, Utrecht University, Utrecht 3584 CL, the Netherlands
| | - Heyrhyoung Lyoo
- Department of Infectious Diseases & Immunology, Utrecht University, Utrecht 3584 CL, the Netherlands
| | - Ronald W A L Limpens
- Department of Molecular Cell Biology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands
| | - Qian Feng
- Department of Infectious Diseases & Immunology, Utrecht University, Utrecht 3584 CL, the Netherlands
| | - Maryam Wahedi
- Department of Infectious Diseases & Immunology, Utrecht University, Utrecht 3584 CL, the Netherlands
| | - Gijs J Overheul
- Department of Medical Microbiology, Radboud Institute for Molecular Life Sciences, Nijmegen 6525 GA, the Netherlands
| | - Ronald P van Rij
- Department of Medical Microbiology, Radboud Institute for Molecular Life Sciences, Nijmegen 6525 GA, the Netherlands
| | - Eric J Snijder
- Department of Medical Microbiology, Leiden University Medical Center, Leiden 2333 ZA, the Netherlands
| | - Abraham J Koster
- Department of Molecular Cell Biology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands
| | - Montserrat Bárcena
- Department of Molecular Cell Biology, Leiden University Medical Center, Leiden 2333 ZC, the Netherlands.
| | - Frank J M van Kuppeveld
- Department of Infectious Diseases & Immunology, Utrecht University, Utrecht 3584 CL, the Netherlands.
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31
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Pocognoni CA, Viktorova EG, Wright J, Meissner JM, Sager G, Lee E, Belov GA, Sztul E. Highly conserved motifs within the large Sec7 ARF guanine nucleotide exchange factor GBF1 target it to the Golgi and are critical for GBF1 activity. Am J Physiol Cell Physiol 2018; 314:C675-C689. [PMID: 29443553 DOI: 10.1152/ajpcell.00221.2017] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Cellular life requires the activation of the ADP-ribosylation factors (ARFs) by Golgi brefeldin A-resistant factor 1 (GBF1), a guanine nucleotide exchange factor (GEF) with a highly conserved catalytic Sec7 domain (Sec7d). In addition to the Sec7d, GBF1 contains other conserved domains whose functions remain unclear. Here, we focus on HDS2 (homology downstream of Sec7d 2) domain because the L1246R substitution within the HDS2 α-helix 5 of the zebrafish GBF1 ortholog causes vascular hemorrhaging and embryonic lethality (13). To dissect the structure/function relationships within HDS2, we generated six variants, in which the most conserved residues within α-helices 1, 2, 4, and 6 were mutated to alanines. Each HDS2 mutant was assessed in a cell-based "replacement" assay for its ability to support cellular functions normally supported by GBF1, such as maintaining Golgi homeostasis, facilitating COPI recruitment, supporting secretion, and sustaining cellular viability. We show that cells treated with the pharmacological GBF1 inhibitor brefeldin A (BFA) and expressing a BFA-resistant GBF1 variant with alanine substitutions of RDR1168 or LF1266 are compromised in Golgi homeostasis, impaired in ARF activation, unable to sustain secretion, and defective in maintaining cellular viability. To gain insight into the molecular mechanism of this dysfunction, we assessed the ability of each GBF1 mutant to target to Golgi membranes and found that mutations in RDR1168 and LF1266 significantly decrease targeting efficiency. Thus, these residues within α-helix 2 and α-helix 6 of the HDS2 domain in GBF1 are novel regulatory determinants that support GBF1 cellular function by impacting the Golgi-specific membrane association of GBF1.
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Affiliation(s)
- Cristian A Pocognoni
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham , Birmingham, Alabama
| | - Ekaterina G Viktorova
- Department of Veterinary Medicine, Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland , College Park, Maryland
| | - John Wright
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham , Birmingham, Alabama
| | - Justyna M Meissner
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham , Birmingham, Alabama
| | - Garrett Sager
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham , Birmingham, Alabama
| | - Eunjoo Lee
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham , Birmingham, Alabama
| | - George A Belov
- Department of Veterinary Medicine, Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland , College Park, Maryland
| | - Elizabeth Sztul
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham , Birmingham, Alabama
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32
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Meissner JM, Bhatt JM, Lee E, Styers ML, Ivanova AA, Kahn RA, Sztul E. The ARF guanine nucleotide exchange factor GBF1 is targeted to Golgi membranes through a PIP-binding domain. J Cell Sci 2018; 131:jcs.210245. [PMID: 29361542 DOI: 10.1242/jcs.210245] [Citation(s) in RCA: 25] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2017] [Accepted: 12/15/2017] [Indexed: 11/20/2022] Open
Abstract
ADP-ribosylation factors (ARF) GTPases are activated by guanine nucleotide exchange factors (GEFs) to support cellular homeostasis. Key to understanding spatio-temporal regulation of ARF signaling is the mechanism of GEF recruitment to membranes. Small GEFs are recruited through phosphoinositide (PIP) binding by a pleckstrin homology (PH) domain downstream from the catalytic Sec7 domain (Sec7d). The large GEFs lack PH domains, and their recruitment mechanisms are poorly understood. We probed Golgi recruitment of GBF1, a GEF catalyzing ARF activation required for Golgi homeostasis. We show that the homology downstream of Sec7d-1 (HDS1) regulates Golgi recruitment of GBF1. We document that GBF1 binds phosphoinositides, preferentially PI3P, PI4P and PI(4,5)P2, and that lipid binding requires the HDS1 domain. Mutations within HDS1 that reduce GBF1 binding to specific PIPs in vitro inhibit GBF1 targeting to Golgi membranes in cells. Our data imply that HDS1 and PH domains are functionally analogous in that each uses lipid-based membrane information to regulate GEF recruitment. Lipid-based recruitment of GBF1 extends the paradigm of lipid regulation to small and large GEFs and suggests that lipid-based mechanisms evolved early during GEF diversification. This article has an associated First Person interview with the first author of the paper.
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Affiliation(s)
- Justyna M Meissner
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Jay M Bhatt
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Eunjoo Lee
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
| | - Melanie L Styers
- Department of Biology, Birmingham-Southern College, Birmingham, AL 35254, USA
| | - Anna A Ivanova
- Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Richard A Kahn
- Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Elizabeth Sztul
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA
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33
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Farhat R, Ankavay M, Lebsir N, Gouttenoire J, Jackson CL, Wychowski C, Moradpour D, Dubuisson J, Rouillé Y, Cocquerel L. Identification of GBF1 as a cellular factor required for hepatitis E virus RNA replication. Cell Microbiol 2017; 20. [PMID: 29112323 PMCID: PMC7162332 DOI: 10.1111/cmi.12804] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/14/2017] [Revised: 10/30/2017] [Accepted: 10/31/2017] [Indexed: 12/23/2022]
Abstract
The hepatitis E virus (HEV) genome is a single‐stranded, positive‐sense RNA that encodes three proteins including the ORF1 replicase. Mechanisms of HEV replication in host cells are unclear, and only a few cellular factors involved in this step have been identified so far. Here, we used brefeldin A (BFA) that blocks the activity of the cellular Arf guanine nucleotide exchange factors GBF1, BIG1, and BIG2, which play a major role in reshuffling of cellular membranes. We showed that BFA inhibits HEV replication in a dose‐dependent manner. The use of siRNA and Golgicide A identified GBF1 as a host factor critically involved in HEV replication. Experiments using cells expressing a mutation in the catalytic domain of GBF1 and overexpression of wild type GBF1 or a BFA‐resistant GBF1 mutant rescuing HEV replication in BFA‐treated cells, confirmed that GBF1 is the only BFA‐sensitive factor required for HEV replication. We demonstrated that GBF1 is likely required for the activity of HEV replication complexes. However, GBF1 does not colocalise with the ORF1 protein, and its subcellular distribution is unmodified upon infection or overexpression of viral proteins, indicating that GBF1 is likely not recruited to replication sites. Together, our results suggest that HEV replication involves GBF1‐regulated mechanisms.
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Affiliation(s)
- Rayan Farhat
- Pasteur Institute of Lille, U1019-UMR 8204-CIIL- Center for Infection and Immunity of Lille, University of Lille, CNRS, INSERM, CHU Lille, Lille, France
| | - Maliki Ankavay
- Pasteur Institute of Lille, U1019-UMR 8204-CIIL- Center for Infection and Immunity of Lille, University of Lille, CNRS, INSERM, CHU Lille, Lille, France
| | - Nadjet Lebsir
- Pasteur Institute of Lille, U1019-UMR 8204-CIIL- Center for Infection and Immunity of Lille, University of Lille, CNRS, INSERM, CHU Lille, Lille, France
| | - Jérôme Gouttenoire
- Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland
| | - Catherine L Jackson
- Institut Jacques Monod, CNRS UMR 7592, Université Paris Diderot, Sorbonne Paris Cité, Paris, France
| | - Czeslaw Wychowski
- Pasteur Institute of Lille, U1019-UMR 8204-CIIL- Center for Infection and Immunity of Lille, University of Lille, CNRS, INSERM, CHU Lille, Lille, France
| | - Darius Moradpour
- Division of Gastroenterology and Hepatology, Centre Hospitalier Universitaire Vaudois, University of Lausanne, Lausanne, Switzerland
| | - Jean Dubuisson
- Pasteur Institute of Lille, U1019-UMR 8204-CIIL- Center for Infection and Immunity of Lille, University of Lille, CNRS, INSERM, CHU Lille, Lille, France
| | - Yves Rouillé
- Pasteur Institute of Lille, U1019-UMR 8204-CIIL- Center for Infection and Immunity of Lille, University of Lille, CNRS, INSERM, CHU Lille, Lille, France
| | - Laurence Cocquerel
- Pasteur Institute of Lille, U1019-UMR 8204-CIIL- Center for Infection and Immunity of Lille, University of Lille, CNRS, INSERM, CHU Lille, Lille, France
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Enterovirus 3A Facilitates Viral Replication by Promoting Phosphatidylinositol 4-Kinase IIIβ-ACBD3 Interaction. J Virol 2017; 91:JVI.00791-17. [PMID: 28701404 DOI: 10.1128/jvi.00791-17] [Citation(s) in RCA: 51] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2017] [Accepted: 07/05/2017] [Indexed: 01/27/2023] Open
Abstract
Like other enteroviruses, enterovirus 71 (EV71) relies on phosphatidylinositol 4-kinase IIIβ (PI4KB) for genome RNA replication. However, how PI4KB is recruited to the genome replication sites of EV71 remains elusive. Recently, we reported that a host factor, ACBD3, is needed for EV71 replication by interacting with viral 3A protein. Here, we show that ACBD3 is required for the recruitment of PI4KB to RNA replication sites. Overexpression of viral 3A or EV71 infection stimulates the interaction of PI4KB and ACBD3. Consistently, EV71 infection induces the production of phosphatidylinositol-4-phosphate (PI4P). Furthermore, PI4KB, ACBD3, and 3A are all localized to the viral-RNA replication sites. Accordingly, PI4KB or ACBD3 depletion by small interfering RNA (siRNA) leads to a reduction in PI4P production after EV71 infection. I44A or H54Y substitution in 3A interrupts the stimulation of PI4KB and ACBD3. Further analysis suggests that stimulation of ACBD3-PI4KB interaction is also important for the replication of enterovirus 68 but disadvantageous to human rhinovirus 16. These results reveal a mechanism of enterovirus replication that involves a selective strategy for recruitment of PI4KB to the RNA replication sites.IMPORTANCE Enterovirus 71, like other human enteroviruses, replicates its genome within host cells, where viral proteins efficiently utilize cellular machineries. While multiple factors are involved, it is largely unclear how viral replication is controlled. We show that the 3A protein of enterovirus 71 recruits an enzyme, phosphatidylinositol 4-kinase IIIβ, by interacting with ACBD3, which alters cellular membranes through the production of a lipid, PI4P. Consequently, the viral and host proteins form a large complex that is necessary for RNA synthesis at replication sites. Notably, PI4KB-ACBD3 interaction also differentially mediates the replication of enterovirus 68 and rhinovirus 16. These results provide new insight into the molecular network of enterovirus replication.
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35
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Ignashkova TI, Gendarme M, Peschk K, Eggenweiler HM, Lindemann RK, Reiling JH. Cell survival and protein secretion associated with Golgi integrity in response to Golgi stress-inducing agents. Traffic 2017; 18:530-544. [PMID: 28485883 DOI: 10.1111/tra.12493] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2017] [Revised: 05/05/2017] [Accepted: 05/05/2017] [Indexed: 12/29/2022]
Abstract
The Golgi apparatus is part of the secretory pathway and of central importance for modification, transport and sorting of proteins and lipids. ADP-ribosylation factors, whose activation can be blocked by brefeldin A (BFA), play a major role in functioning of the Golgi network and regulation of membrane traffic and are also involved in proliferation and migration of cancer cells. Due to high cytotoxicity and poor bioavailability, BFA has not passed the preclinical stage of drug development. Recently, AMF-26 and golgicide A have been described as novel inhibitors of the Golgi system with antitumor or bactericidal properties. We provide here further evidence that AMF-26 closely mirrors the mode of action of BFA but is less potent. Using several human cancer cell lines, we studied the effects of AMF-26, BFA and golgicide A on cell homeostasis including Golgi structure, endoplasmic reticulum (ER) stress markers, secretion and viability, and found overall a significant correlation between these parameters. Furthermore, modulation of ADP-ribosylation factor expression has a profound impact on Golgi organization and survival in response to Golgi stress inducers.
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Affiliation(s)
- Tatiana I Ignashkova
- Metabolism and Signaling in Cancer, BioMed X Innovation Center, Heidelberg, Germany
| | - Mathieu Gendarme
- Metabolism and Signaling in Cancer, BioMed X Innovation Center, Heidelberg, Germany
| | - Katrin Peschk
- Medicinal Chemistry, Merck Biopharma, Merck KGaA, Darmstadt, Germany
| | | | - Ralph K Lindemann
- Translational Innovation Platform Oncology, Merck Biopharma, Merck KGaA, Darmstadt, Germany
| | - Jan H Reiling
- Metabolism and Signaling in Cancer, BioMed X Innovation Center, Heidelberg, Germany
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36
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Tyrphostin AG1478 Inhibits Encephalomyocarditis Virus and Hepatitis C Virus by Targeting Phosphatidylinositol 4-Kinase IIIα. Antimicrob Agents Chemother 2016; 60:6402-6. [PMID: 27480860 DOI: 10.1128/aac.01331-16] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2016] [Accepted: 07/27/2016] [Indexed: 02/07/2023] Open
Abstract
Encephalomyocarditis virus (EMCV), like hepatitis C virus (HCV), requires phosphatidylinositol 4-kinase IIIα (PI4KA) for genome replication. Here, we demonstrate that tyrphostin AG1478, a known epidermal growth factor receptor (EGFR) inhibitor, also inhibits PI4KA activity, both in vitro and in cells. AG1478 impaired replication of EMCV and HCV but not that of an EMCV mutant previously shown to escape PI4KA inhibition. This work uncovers novel cellular and antiviral properties of AG1478, a compound previously regarded only as a cancer chemotherapy agent.
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Yadav V, Panganiban AT, Honer Zu Bentrup K, Voss TG. Influenza infection modulates vesicular trafficking and induces Golgi complex disruption. Virusdisease 2016; 27:357-368. [PMID: 28004015 DOI: 10.1007/s13337-016-0347-3] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2016] [Accepted: 09/07/2016] [Indexed: 12/18/2022] Open
Abstract
Influenza A virus (IFV) replicates its genome in the nucleus of infected cells and uses the cellular protein transport system for genome trafficking from the nucleus to the plasma membrane. However, many details of the mechanism of this process, and its relationship to subsequent cytoplasmic virus trafficking, have not been elucidated. We examined the effect of nuclear transport inhibitors Leptomycin B (LB), 5,6 dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB), the vesicular transport inhibitor Brefeldin A (BFA), the caspase inhibitor ZWEHD, and microtubule inhibitor Nocodazole (NOC) on virus replication and intracellular trafficking of viral nucleoprotein (NP) from the nucleus to the ER and Golgi. Also, we carried out complementary studies to determine the effect of IFV on intracellular membranes. Inhibition of the CRM1 and TAP-P15 nuclear transport pathways by DRB and LB blocked completely the export of virus. Inhibition of vesicular trafficking by BFA, NOC, and ZWEHD also affected influenza infection. Interestingly, IFV infection induced fragmentation of the Golgi complex resulting in diffuse distribution of large and small vesicles throughout the cytoplasm. Live-cell microscopy revealed expansion of Golgi localization signals indicating progressive dispersion of Golgi positive structures, resulting in the disassembly of the Golgi ribbon structure. Other vesicular components (Rab1b, ARF1 and GBF1) were also found to be required for IFV infection. Furthermore, the exact step at which IFV infection disrupts vesicle trafficking was identified as the ER-Golgi intermediate compartment. These findings suggest that IFV NP is trafficked from the nucleus via the CRM1 and TAP pathways. IFV modulates vesicular trafficking inducing disruption of the Golgi complex. These studies provide insight on the ways in which IFV affects intracellular trafficking of different host proteins and will facilitate identification of useful pharmaceutical targets to abrogate virus replication.
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Affiliation(s)
- Vibha Yadav
- Division of Microbiology, Tulane National Primate Research Center, Covington, LA USA
| | - Antonito T Panganiban
- Division of Microbiology, Tulane National Primate Research Center, Covington, LA USA
| | - Kerstin Honer Zu Bentrup
- Department of Microbiology and Immunology, Tulane School of Medicine, Tulane University, New Orleans, LA USA
| | - Thomas G Voss
- Department of Microbiology and Immunology, Tulane School of Medicine, Tulane University, New Orleans, LA USA
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Illuminating the Sites of Enterovirus Replication in Living Cells by Using a Split-GFP-Tagged Viral Protein. mSphere 2016; 1:mSphere00104-16. [PMID: 27390781 PMCID: PMC4935779 DOI: 10.1128/msphere.00104-16] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/13/2016] [Accepted: 06/02/2016] [Indexed: 12/13/2022] Open
Abstract
Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for genome replication. Electron microscopy has revealed the morphology of enterovirus ROs, and immunofluorescence studies have been conducted to investigate their origin and formation. Yet, immunofluorescence analysis of fixed cells results in a rather static view of RO formation, and the results may be compromised by immunolabeling artifacts. While live-cell imaging of ROs would be preferred, enteroviruses encoding a membrane-anchored viral protein fused to a large fluorescent reporter have thus far not been described. Here, we tackled this constraint by introducing a small tag from a split-GFP system into an RO-resident enterovirus protein. This new tool bridges a methodological gap by circumventing the need for immunolabeling fixed cells and allows the study of the dynamics and formation of enterovirus ROs in living cells. Like all other positive-strand RNA viruses, enteroviruses generate new organelles (replication organelles [ROs]) with a unique protein and lipid composition on which they multiply their viral genome. Suitable tools for live-cell imaging of enterovirus ROs are currently unavailable, as recombinant enteroviruses that carry genes that encode RO-anchored viral proteins tagged with fluorescent reporters have not been reported thus far. To overcome this limitation, we used a split green fluorescent protein (split-GFP) system, comprising a large fragment [strands 1 to 10; GFP(S1-10)] and a small fragment [strand 11; GFP(S11)] of only 16 residues. The GFP(S11) (GFP with S11 fragment) fragment was inserted into the 3A protein of the enterovirus coxsackievirus B3 (CVB3), while the large fragment was supplied by transient or stable expression in cells. The introduction of GFP(S11) did not affect the known functions of 3A when expressed in isolation. Using correlative light electron microscopy (CLEM), we showed that GFP fluorescence was detected at ROs, whose morphologies are essentially identical to those previously observed for wild-type CVB3, indicating that GFP(S11)-tagged 3A proteins assemble with GFP(S1-10) to form GFP for illumination of bona fide ROs. It is well established that enterovirus infection leads to Golgi disintegration. Through live-cell imaging of infected cells expressing an mCherry-tagged Golgi marker, we monitored RO development and revealed the dynamics of Golgi disassembly in real time. Having demonstrated the suitability of this virus for imaging ROs, we constructed a CVB3 encoding GFP(S1-10) and GFP(S11)-tagged 3A to bypass the need to express GFP(S1-10) prior to infection. These tools will have multiple applications in future studies on the origin, location, and function of enterovirus ROs. IMPORTANCE Enteroviruses induce the formation of membranous structures (replication organelles [ROs]) with a unique protein and lipid composition specialized for genome replication. Electron microscopy has revealed the morphology of enterovirus ROs, and immunofluorescence studies have been conducted to investigate their origin and formation. Yet, immunofluorescence analysis of fixed cells results in a rather static view of RO formation, and the results may be compromised by immunolabeling artifacts. While live-cell imaging of ROs would be preferred, enteroviruses encoding a membrane-anchored viral protein fused to a large fluorescent reporter have thus far not been described. Here, we tackled this constraint by introducing a small tag from a split-GFP system into an RO-resident enterovirus protein. This new tool bridges a methodological gap by circumventing the need for immunolabeling fixed cells and allows the study of the dynamics and formation of enterovirus ROs in living cells.
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Endoplasmic Reticulum: The Favorite Intracellular Niche for Viral Replication and Assembly. Viruses 2016; 8:v8060160. [PMID: 27338443 PMCID: PMC4926180 DOI: 10.3390/v8060160] [Citation(s) in RCA: 132] [Impact Index Per Article: 14.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/16/2016] [Revised: 05/23/2016] [Accepted: 05/26/2016] [Indexed: 02/07/2023] Open
Abstract
The endoplasmic reticulum (ER) is the largest intracellular organelle. It forms a complex network of continuous sheets and tubules, extending from the nuclear envelope (NE) to the plasma membrane. This network is frequently perturbed by positive-strand RNA viruses utilizing the ER to create membranous replication factories (RFs), where amplification of their genomes occurs. In addition, many enveloped viruses assemble progeny virions in association with ER membranes, and viruses replicating in the nucleus need to overcome the NE barrier, requiring transient changes of the NE morphology. This review first summarizes some key aspects of ER morphology and then focuses on the exploitation of the ER by viruses for the sake of promoting the different steps of their replication cycles.
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Vázquez-Calvo Á, Caridi F, González-Magaldi M, Saiz JC, Sobrino F, Martín-Acebes MA. The Amino Acid Substitution Q65H in the 2C Protein of Swine Vesicular Disease Virus Confers Resistance to Golgi Disrupting Drugs. Front Microbiol 2016; 7:612. [PMID: 27199941 PMCID: PMC4846857 DOI: 10.3389/fmicb.2016.00612] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2016] [Accepted: 04/13/2016] [Indexed: 11/13/2022] Open
Abstract
Swine vesicular disease virus (SVDV) is a porcine pathogen and a member of the species Enterovirus B within the Picornaviridae family. Brefeldin A (BFA) is an inhibitor of guanine nucleotide exchange factors of Arf proteins that induces Golgi complex disassembly and alters the cellular secretory pathway. Since BFA has been shown to inhibit the RNA replication of different enteroviruses, including SVDV, we have analyzed the effect of BFA and of golgicide A (GCA), another Golgi disrupting drug, on SVDV multiplication. BFA and GCA similarly inhibited SVDV production. To investigate the molecular basis of the antiviral effect of BFA, SVDV mutants with increased resistance to BFA were isolated. A single amino acid substitution, Q65H, in the non-structural protein 2C was found to be responsible for increased resistance to BFA. These results provide new insight into the relationship of enteroviruses with the components of the secretory pathway and on the role of SVDV 2C protein in this process.
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Affiliation(s)
- Ángela Vázquez-Calvo
- Centro de Biología Molecular Severo Ochoa (CSIC-UAM)Madrid, Spain; Departamento de Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)Madrid, Spain
| | - Flavia Caridi
- Centro de Biología Molecular Severo Ochoa (CSIC-UAM) Madrid, Spain
| | | | - Juan-Carlos Saiz
- Departamento de Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) Madrid, Spain
| | | | - Miguel A Martín-Acebes
- Centro de Biología Molecular Severo Ochoa (CSIC-UAM)Madrid, Spain; Departamento de Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA)Madrid, Spain
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Berryman S, Moffat K, Harak C, Lohmann V, Jackson T. Foot-and-mouth disease virus replicates independently of phosphatidylinositol 4-phosphate and type III phosphatidylinositol 4-kinases. J Gen Virol 2016; 97:1841-1852. [PMID: 27093462 PMCID: PMC5156328 DOI: 10.1099/jgv.0.000485] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
Picornaviruses form replication complexes in association with membranes in structures called replication organelles. Common themes to emerge from studies of picornavirus replication are the need for cholesterol and phosphatidylinositol 4-phosphate (PI4P). In infected cells, type III phosphatidylinositol 4-kinases (PI4KIIIs) generate elevated levels of PI4P, which is then exchanged for cholesterol at replication organelles. For the enteroviruses, replication organelles form at Golgi membranes in a process that utilizes PI4KIIIβ. Other picornaviruses, for example the cardioviruses, are believed to initiate replication at the endoplasmic reticulum and subvert PI4KIIIα to generate PI4P. Here we investigated the role of PI4KIII in foot-and-mouth disease virus (FMDV) replication. Our results showed that, in contrast to the enteroviruses and the cardioviruses, FMDV replication does not require PI4KIII (PI4KIIIα and PI4KIIIβ), and PI4P levels do not increase in FMDV-infected cells and PI4P is not seen at replication organelles. These results point to a unique requirement towards lipids at the FMDV replication membranes.
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Affiliation(s)
- Stephen Berryman
- The Pirbright Institute, Ash Rd, Pirbright, Surrey, GU24 0NF, UK
| | - Katy Moffat
- The Pirbright Institute, Ash Rd, Pirbright, Surrey, GU24 0NF, UK
| | - Christian Harak
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany
| | - Volker Lohmann
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany
| | - Terry Jackson
- The Pirbright Institute, Ash Rd, Pirbright, Surrey, GU24 0NF, UK
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Fat(al) attraction: Picornaviruses Usurp Lipid Transfer at Membrane Contact Sites to Create Replication Organelles. Trends Microbiol 2016; 24:535-546. [PMID: 27020598 PMCID: PMC7126954 DOI: 10.1016/j.tim.2016.02.017] [Citation(s) in RCA: 90] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2016] [Revised: 02/23/2016] [Accepted: 02/25/2016] [Indexed: 12/23/2022]
Abstract
All viruses that carry a positive-sense RNA genome (+RNA), such as picornaviruses, hepatitis C virus, dengue virus, and SARS- and MERS-coronavirus, confiscate intracellular membranes of the host cell to generate new compartments (i.e., replication organelles) for amplification of their genome. Replication organelles (ROs) are membranous structures that not only harbor viral proteins but also contain a specific array of hijacked host factors that create a unique lipid microenvironment optimal for genome replication. While some lipids may be locally synthesized de novo, other lipids are shuttled towards ROs. In picornavirus-infected cells, lipids are exchanged at membrane contact sites between ROs and other organelles. In this paper, we review recent advances in our understanding of how picornaviruses exploit host membrane contact site machinery to generate ROs, a mechanism that is used by some other +RNA viruses as well. Picornaviruses create replication organelles with a unique protein and lipid composition to amplify their genome. Picornaviruses hijack membrane contact site machinery to shuttle lipids to their replication organelles. Picornaviruses from different genera employ a cholesterol/PI4P counterflux mechanism to accumulate cholesterol at replication organelles.
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Farhat R, Séron K, Ferlin J, Fénéant L, Belouzard S, Goueslain L, Jackson CL, Dubuisson J, Rouillé Y. Identification of class II ADP-ribosylation factors as cellular factors required for hepatitis C virus replication. Cell Microbiol 2016; 18:1121-33. [PMID: 26814617 DOI: 10.1111/cmi.12572] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2015] [Revised: 01/12/2016] [Accepted: 01/21/2016] [Indexed: 12/21/2022]
Abstract
GBF1 is a host factor required for hepatitis C virus (HCV) replication. GBF1 functions as a guanine nucleotide exchange factor for G-proteins of the Arf family, which regulate membrane dynamics in the early secretory pathway and the metabolism of cytoplasmic lipid droplets. Here we established that the Arf-guanine nucleotide exchange factor activity of GBF1 is critical for its function in HCV replication, indicating that it promotes viral replication by activating one or more Arf family members. Arf involvement was confirmed with the use of two dominant negative Arf1 mutants. However, siRNA-mediated depletion of Arf1, Arf3 (class I Arfs), Arf4 or Arf5 (class II Arfs), which potentially interact with GBF1, did not significantly inhibit HCV infection. In contrast, the simultaneous depletion of both Arf4 and Arf5, but not of any other Arf pair, imposed a significant inhibition of HCV infection. Interestingly, the simultaneous depletion of both Arf4 and Arf5 had no impact on the activity of the secretory pathway and induced a compaction of the Golgi and an accumulation of lipid droplets. A similar phenotype of lipid droplet accumulation was also observed when GBF1 was inhibited by brefeldin A. In contrast, the simultaneous depletion of both Arf1 and Arf4 resulted in secretion inhibition and Golgi scattering, two actions reminiscent of GBF1 inhibition. We conclude that GBF1 could regulate different metabolic pathways through the activation of different pairs of Arf proteins.
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Affiliation(s)
- Rayan Farhat
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204, CIIL - Center for Infection and Immunity of Lille, Lille, France
| | - Karin Séron
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204, CIIL - Center for Infection and Immunity of Lille, Lille, France
| | - Juliette Ferlin
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204, CIIL - Center for Infection and Immunity of Lille, Lille, France
| | - Lucie Fénéant
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204, CIIL - Center for Infection and Immunity of Lille, Lille, France
| | - Sandrine Belouzard
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204, CIIL - Center for Infection and Immunity of Lille, Lille, France
| | - Lucie Goueslain
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204, CIIL - Center for Infection and Immunity of Lille, Lille, France.,Institut Jacques Monod, CNRS UMR 7592, Université Paris Diderot, Sorbonne Paris Cité, Paris, France
| | - Catherine L Jackson
- Institut Jacques Monod, CNRS UMR 7592, Université Paris Diderot, Sorbonne Paris Cité, Paris, France
| | - Jean Dubuisson
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204, CIIL - Center for Infection and Immunity of Lille, Lille, France
| | - Yves Rouillé
- Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 8204, CIIL - Center for Infection and Immunity of Lille, Lille, France
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Arita M. Mechanism of Poliovirus Resistance to Host Phosphatidylinositol-4 Kinase III β Inhibitor. ACS Infect Dis 2016; 2:140-8. [PMID: 27624965 DOI: 10.1021/acsinfecdis.5b00122] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
Phosphatidylinositol-4 kinase III β (PI4KB) and oxysterol-binding protein (OSBP) family I have been identified as the major targets of anti-enterovirus drug candidates. Resistance mutations in poliovirus (PV) to these inhibitors have been identified in viral 3A protein, represented by a G5318A (3A-Ala70Thr) mutation, but the mechanism of viral resistance to host PI4KB/OSBP inhibitors remained unknown. In this study, we found that a G5318A mutation enhances the basal levels of phosphatidylinositol 4-phosphate (PI4P) and of the 3A protein and decreases the levels of the 3AB protein during PV replication. The 3A protein acted as a major effector responsible for the resistance to PI4KB inhibitor, but did not enhance the PI4KB activity in vitro in contrast to the 2C, 2BC, 3AB, and 3D proteins. The 3AB protein acted as the primary target of a G5318A mutation and also as an effector. We identified novel resistance mutations to a PI4KB inhibitor [C5151U (3A-T14M) and C5366U (3A-H86Y) mutations] and found that there is a positive correlation between the extent of the resistance phenotype and the levels of the 3A proteins. These results suggested that the 3A protein overproduced by enhanced processing of the 3AB protein with the resistance mutations overcomes the inhibitory effect of PI4KB inhibitor on PV replication independently of the hyperactivation of the PI4KB/OSBP pathway.
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Affiliation(s)
- Minetaro Arita
- Department
of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama-shi, Tokyo 208-0011, Japan
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Bhatt JM, Viktorova EG, Busby T, Wyrozumska P, Newman LE, Lin H, Lee E, Wright J, Belov GA, Kahn RA, Sztul E. Oligomerization of the Sec7 domain Arf guanine nucleotide exchange factor GBF1 is dispensable for Golgi localization and function but regulates degradation. Am J Physiol Cell Physiol 2015; 310:C456-69. [PMID: 26718629 DOI: 10.1152/ajpcell.00185.2015] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2015] [Accepted: 12/14/2015] [Indexed: 12/15/2022]
Abstract
Members of the large Sec7 domain-containing Arf guanine nucleotide exchange factor (GEF) family have been shown to dimerize through their NH2-terminal dimerization and cyclophilin binding (DCB) and homology upstream of Sec7 (HUS) domains. However, the importance of dimerization in GEF localization and function has not been assessed. We generated a GBF1 mutant (91/130) in which two residues required for oligomerization (K91 and E130 within the DCB domain) were replaced with A and assessed the effects of these mutations on GBF1 localization and cellular functions. We show that 91/130 is compromised in oligomerization but that it targets to the Golgi in a manner indistinguishable from wild-type GBF1 and that it rapidly exchanges between the cytosolic and membrane-bound pools. The 91/130 mutant appears active as it integrates within the functional network at the Golgi, supports Arf activation and COPI recruitment, and sustains Golgi homeostasis and cargo secretion when provided as a sole copy of functional GBF1 in cells. In addition, like wild-type GBF1, the 91/130 mutant supports poliovirus RNA replication, a process requiring GBF1 but believed to be independent of GBF1 catalytic activity. However, oligomerization appears to stabilize GBF1 in cells, and the 91/130 mutant is degraded faster than the wild-type GBF1. Our data support a model in which oligomerization is not a key regulator of GBF1 activity but impacts its function by regulating the cellular levels of GBF1.
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Affiliation(s)
- Jay M Bhatt
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama
| | - Ekaterina G Viktorova
- Department of Veterinary Medicine, Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland; and
| | - Theodore Busby
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama
| | - Paulina Wyrozumska
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama
| | - Laura E Newman
- Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia
| | - Helen Lin
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama
| | - Eunjoo Lee
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama
| | - John Wright
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama
| | - George A Belov
- Department of Veterinary Medicine, Virginia-Maryland Regional College of Veterinary Medicine, University of Maryland, College Park, Maryland; and
| | - Richard A Kahn
- Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia
| | - Elizabeth Sztul
- Department of Cell, Developmental and Integrative Biology, University of Alabama at Birmingham, Birmingham, Alabama;
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van der Linden L, Wolthers KC, van Kuppeveld FJM. Replication and Inhibitors of Enteroviruses and Parechoviruses. Viruses 2015; 7:4529-62. [PMID: 26266417 PMCID: PMC4576193 DOI: 10.3390/v7082832] [Citation(s) in RCA: 104] [Impact Index Per Article: 10.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2015] [Accepted: 08/03/2015] [Indexed: 01/11/2023] Open
Abstract
The Enterovirus (EV) and Parechovirus genera of the picornavirus family include many important human pathogens, including poliovirus, rhinovirus, EV-A71, EV-D68, and human parechoviruses (HPeV). They cause a wide variety of diseases, ranging from a simple common cold to life-threatening diseases such as encephalitis and myocarditis. At the moment, no antiviral therapy is available against these viruses and it is not feasible to develop vaccines against all EVs and HPeVs due to the great number of serotypes. Therefore, a lot of effort is being invested in the development of antiviral drugs. Both viral proteins and host proteins essential for virus replication can be used as targets for virus inhibitors. As such, a good understanding of the complex process of virus replication is pivotal in the design of antiviral strategies goes hand in hand with a good understanding of the complex process of virus replication. In this review, we will give an overview of the current state of knowledge of EV and HPeV replication and how this can be inhibited by small-molecule inhibitors.
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Affiliation(s)
- Lonneke van der Linden
- Laboratory of Clinical Virology, Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, Amsterdam 1105 AZ, The Netherlands.
| | - Katja C Wolthers
- Laboratory of Clinical Virology, Department of Medical Microbiology, Academic Medical Center, University of Amsterdam, Meibergdreef 15, Amsterdam 1105 AZ, The Netherlands.
| | - Frank J M van Kuppeveld
- Virology Division, Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, Utrecht 3584 CL, The Netherlands.
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Reid CR, Airo AM, Hobman TC. The Virus-Host Interplay: Biogenesis of +RNA Replication Complexes. Viruses 2015; 7:4385-413. [PMID: 26287230 PMCID: PMC4576186 DOI: 10.3390/v7082825] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2015] [Revised: 07/21/2015] [Accepted: 07/24/2015] [Indexed: 12/22/2022] Open
Abstract
Positive-strand RNA (+RNA) viruses are an important group of human and animal pathogens that have significant global health and economic impacts. Notable members include West Nile virus, Dengue virus, Chikungunya, Severe acute respiratory syndrome (SARS) Coronavirus and enteroviruses of the Picornaviridae family.Unfortunately, prophylactic and therapeutic treatments against these pathogens are limited. +RNA viruses have limited coding capacity and thus rely extensively on host factors for successful infection and propagation. A common feature among these viruses is their ability to dramatically modify cellular membranes to serve as platforms for genome replication and assembly of new virions. These viral replication complexes (VRCs) serve two main functions: To increase replication efficiency by concentrating critical factors and to protect the viral genome from host anti-viral systems. This review summarizes current knowledge of critical host factors recruited to or demonstrated to be involved in the biogenesis and stabilization of +RNA virus VRCs.
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Affiliation(s)
- Colleen R Reid
- Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB T6G 2E1, Canada.
| | - Adriana M Airo
- Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB T6G 2E1, Canada.
| | - Tom C Hobman
- Department of Medical Microbiology and Immunology, University of Alberta, Edmonton, AB T6G 2E1, Canada.
- Department of Cell Biology, University of Alberta, Edmonton, AB T6G 2H7, Canada.
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Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells. J Virol 2015; 89:10359-70. [PMID: 26246569 DOI: 10.1128/jvi.01225-15] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2015] [Accepted: 07/28/2015] [Indexed: 01/25/2023] Open
Abstract
UNLABELLED Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in association with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellular membranes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles were found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this membrane population in infected and mock-infected cells were identified by tandem mass spectrometry. The results indicated that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein interaction network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly enriched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the role that some of the identified proteins might have in the replication cycle of the virus. Silencing of the expression of genes involved in cholesterol (DHCR7, CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIIIβ) and inositol phosphate (ITPR3) metabolism, and RNA helicase activity (DDX23) significantly decreased the amounts of Yuc8 genomic and antigenomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA replication and particle assembly take place in association with modified membranes potentially derived from multiple cell organelles. IMPORTANCE Astroviruses are common etiological agents of acute gastroenteritis in children and immunocompromised patients. More recently, they have been associated with neurological diseases in mammals, including humans, and are also responsible for different pathologies in birds. In this work, we provide evidence that astrovirus RNA replication and virus assembly occur in contact with cell membranes potentially derived from multiple cell organelles and show that membrane-associated cellular proteins involved in lipid metabolism are required for efficient viral replication. Our findings provide information to enhance our knowledge of astrovirus biology and provide information that might be useful for the development of therapeutic interventions to prevent virus replication.
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Abstract
The Picornaviridae represent a large family of small plus-strand RNA viruses that cause a bewildering array of important human and animal diseases. Morphogenesis is the least-understood step in the life cycle of these viruses, and this process is difficult to study because encapsidation is tightly coupled to genome translation and RNA replication. Although the basic steps of assembly have been known for some time, very few details are available about the mechanism and factors that regulate this process. Most of the information available has been derived from studies of enteroviruses, in particular poliovirus, where recent evidence has shown that, surprisingly, the specificity of encapsidation is governed by a viral protein-protein interaction that does not involve an RNA packaging signal. In this review, we make an attempt to summarize what is currently known about the following topics: (i) encapsidation intermediates, (ii) the specificity of encapsidation (iii), viral and cellular factors that are required for encapsidation, (iv) inhibitors of encapsidation, and (v) a model of enterovirus encapsidation. Finally, we compare some features of picornavirus morphogenesis with those of other plus-strand RNA viruses.
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Harak C, Lohmann V. Ultrastructure of the replication sites of positive-strand RNA viruses. Virology 2015; 479-480:418-33. [PMID: 25746936 PMCID: PMC7111692 DOI: 10.1016/j.virol.2015.02.029] [Citation(s) in RCA: 118] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2014] [Revised: 01/06/2015] [Accepted: 02/16/2015] [Indexed: 12/13/2022]
Abstract
Positive strand RNA viruses replicate in the cytoplasm of infected cells and induce intracellular membranous compartments harboring the sites of viral RNA synthesis. These replication factories are supposed to concentrate the components of the replicase and to shield replication intermediates from the host cell innate immune defense. Virus induced membrane alterations are often generated in coordination with host factors and can be grouped into different morphotypes. Recent advances in conventional and electron microscopy have contributed greatly to our understanding of their biogenesis, but still many questions remain how viral proteins capture membranes and subvert host factors for their need. In this review, we will discuss different representatives of positive strand RNA viruses and their ways of hijacking cellular membranes to establish replication complexes. We will further focus on host cell factors that are critically involved in formation of these membranes and how they contribute to viral replication.
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Affiliation(s)
- Christian Harak
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany
| | - Volker Lohmann
- Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 345, D-69120 Heidelberg, Germany.
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