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Haas G, Seiler M, Nguyen J, Troxler L, Pennarun S, Lefebvre E, Benamrouche Y, Loizeau L, Reinbolt C, Liang M, Lin X, Li W, Xia Z, Marques JT, Imler JL. Regulation of detoxifying enzymes expression and restriction of picorna-like virus infection by natural polysaccharide extracts in Drosophila cells. Virology 2025; 607:110513. [PMID: 40163969 DOI: 10.1016/j.virol.2025.110513] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/18/2024] [Revised: 03/11/2025] [Accepted: 03/24/2025] [Indexed: 04/02/2025]
Abstract
The world is currently witnessing a rise in viral infections, while the availability of antiviral drugs remains limited. Traditional Chinese medicine (TCM) has historically served as a valuable source of novel compounds for disease treatment. In this study, we assessed the antiviral potential of various TCM compounds using Drosophila melanogaster as a model organism. Our findings reveal that natural polysaccharide extracts, prepared from 10 commonly used medicinal herbs or fungi, exhibit antiviral activity against two picorna-like viruses. Importantly, the antiviral effect is not directly attributable to the compound itself but is instead mediated by cellular responses induced by treatment with the extract. We observed that the polysaccharide extract triggers a broad transcriptional response, which partially overlaps with NF-κB pathway activation in Drosophila. However, the antiviral activity of the extract was independent of classical innate immune pathways, such as RNA interference or NF-κB signaling. Instead, the extract appears to uniquely stimulate detoxification pathways, including upregulation of cytochrome P450 and glutathione S-transferase genes, which correlates with its antiviral effects.
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Affiliation(s)
- Gabrielle Haas
- Université de Strasbourg, CNRS UPR9022, 67084, Strasbourg, France
| | - Mélodie Seiler
- Université de Strasbourg, CNRS UPR9022, 67084, Strasbourg, France
| | - Jenny Nguyen
- Université de Strasbourg, CNRS UPR9022, 67084, Strasbourg, France
| | - Laurent Troxler
- Université de Strasbourg, CNRS UPR9022, 67084, Strasbourg, France
| | - Samuel Pennarun
- Université de Strasbourg, CNRS UPR9022, 67084, Strasbourg, France
| | - Elise Lefebvre
- Université de Strasbourg, CNRS UPR9022, 67084, Strasbourg, France
| | | | - Loriane Loizeau
- Université de Strasbourg, CNRS UPR9022, 67084, Strasbourg, France
| | - Cody Reinbolt
- Université de Strasbourg, CNRS UPR9022, 67084, Strasbourg, France
| | - Ming Liang
- Infinitus (China) Company Ltd., Guangzhou, China
| | | | - Wenzhi Li
- Infinitus (China) Company Ltd., Guangzhou, China
| | - Zumeng Xia
- Infinitus (China) Company Ltd., Guangzhou, China
| | - Joao T Marques
- Université de Strasbourg, INSERM U1257, CNRS UPR9022, 67084, Strasbourg, France; Department of Biochemistry and Immunology, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, 31270-901, Belo Horizonte, Brazil
| | - Jean-Luc Imler
- Université de Strasbourg, CNRS UPR9022, 67084, Strasbourg, France.
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2
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Wei R, Li L, Chen H, Wang X, Chen Y, Liu X. Inhibition of porcine reproductive and respiratory syndrome virus replication by rifampicin in vitro. Front Vet Sci 2024; 11:1439015. [PMID: 39051013 PMCID: PMC11266174 DOI: 10.3389/fvets.2024.1439015] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2024] [Accepted: 06/26/2024] [Indexed: 07/27/2024] Open
Abstract
Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause significant economic losses to the global swine industry, yet effective prevention and control measures remain elusive. The development of novel antivirals is thus urgently needed. Rifampicin (RFP), a semisynthetic derivative of rifamycin, has been previously reported to inhibit the replication of certain mammalian DNA viruses as well as RNA viruses. In this study, we unveil RFP as a potent inhibitor of PRRSV both in Marc-145 cells (half-maximal inhibitory concentration 61.26 μM) and porcine alveolar macrophages (half-maximal inhibitory concentration 53.09 μM). The inhibitory effect of RFP occurred during viral replication rather than binding, internalization and release. We also demonstrated that RFP inhibits PRRSV proteins production in the early stage of infection, without inhibiting host protein synthesis. Moreover, RFP effectively restricted porcine epidemic diarrhea virus (PEDV) and porcine enteric alphacoronavirus (PEAV) infection in Vero cells. In summary, these findings indicate the promising potential of RFP as a therapeutic agent for PRRSV, PEDV and PEAV infection in pig farms.
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Affiliation(s)
| | | | | | | | | | - Xiaohong Liu
- State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong, China
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Wang J, Shahed-Ai-Mahmud M, Chen A, Li K, Tan H, Joyce R. An Overview of Antivirals against Monkeypox Virus and Other Orthopoxviruses. J Med Chem 2023; 66:4468-4490. [PMID: 36961984 DOI: 10.1021/acs.jmedchem.3c00069] [Citation(s) in RCA: 19] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 03/26/2023]
Abstract
The current monkeypox outbreaks during the COVID-19 pandemic have reignited interest in orthopoxvirus antivirals. Monkeypox belongs to the Orthopoxvirus genus of the Poxviridae family, which also includes the variola virus, vaccinia virus, and cowpox virus. Two orally bioavailable drugs, tecovirimat and brincidofovir, have been approved for treating smallpox infections. Given their human safety profiles and in vivo antiviral efficacy in animal models, both drugs have also been recommended to treat monkeypox infection. To facilitate the development of additional orthopoxvirus antivirals, we summarize the antiviral activity, mechanism of action, and mechanism of resistance of orthopoxvirus antivirals. This perspective covers both direct-acting and host-targeting antivirals with an emphasis on drug candidates showing in vivo antiviral efficacy in animal models. We hope to speed the orthopoxvirus antiviral drug discovery by providing medicinal chemists with insights into prioritizing proper drug targets and hits for further development.
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Affiliation(s)
- Jun Wang
- Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, the State University of New Jersey, Piscataway, New Jersey 08854, United States
| | - Md Shahed-Ai-Mahmud
- Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, the State University of New Jersey, Piscataway, New Jersey 08854, United States
| | - Angelo Chen
- Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, the State University of New Jersey, Piscataway, New Jersey 08854, United States
| | - Kan Li
- Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, the State University of New Jersey, Piscataway, New Jersey 08854, United States
| | - Haozhou Tan
- Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, the State University of New Jersey, Piscataway, New Jersey 08854, United States
| | - Ryan Joyce
- Department of Medicinal Chemistry, Ernest Mario School of Pharmacy, Rutgers, the State University of New Jersey, Piscataway, New Jersey 08854, United States
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4
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Guan H, Gul I, Xiao C, Ma S, Liang Y, Yu D, Liu Y, Liu H, Zhang CY, Li J, Qin P. Emergence, phylogeography, and adaptive evolution of mpox virus. New Microbes New Infect 2023; 52:101102. [PMID: 36815201 PMCID: PMC9937731 DOI: 10.1016/j.nmni.2023.101102] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2022] [Revised: 02/14/2023] [Accepted: 02/15/2023] [Indexed: 02/19/2023] Open
Abstract
Mpox (Monkeypox) is a zoonotic disease caused by mpox virus (MPXV). A multi-country MPXV outbreak in non-endemic demographics was identified in May 2022. A systematic evaluation of MPXV evolutionary trajectory and genetic diversity could be a timely addition to the MPXV diagnostics and prophylaxis. Herein, we integrated a systematic evolution analysis including phylogenomic and phylogeographic, followed by an in-depth analysis of the adaptive evolution and amino acid variations in type I interferon binding protein (IFNα/βBP). Mutations in IFNα/βBP protein may impair its binding capacity, affecting the MPXV immune evasion strategy. Based on the equilibrated data, we found an evolutionary rate of 7.75 × 10 - 5 substitutions/site/year, and an earlier original time (2021.25) of the clade IIb. We further discovered significant genetic variations in MPXV genomes from different regions and obtained six plausible spread trajectories from its intricate viral flow network, implying that North America might have acted as a bridge for the spread of MPXV from Africa to other continents. We identified two amino acids under positive selection in the Rifampicin resistance protein and extracellular enveloped virus (EEV) type-I membrane glycoprotein, indicating a role in adaptive evolution. Our research sheds light on the emergence, dispersal, and adaptive evolution of MPXV, providing theoretical support for mitigating and containing its expansion.
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Affiliation(s)
- Haifei Guan
- Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, China
- Tsinghua-Berkeley Shenzhen Institute, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, China
| | - Ijaz Gul
- Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, China
- Tsinghua-Berkeley Shenzhen Institute, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, China
| | - Chufan Xiao
- Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, China
- Tsinghua-Berkeley Shenzhen Institute, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, China
| | - Shuyue Ma
- Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, China
- Tsinghua-Berkeley Shenzhen Institute, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, China
| | - Yingshan Liang
- Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, China
| | - Dongmei Yu
- School of Mechanical, Electrical & Information Engineering, Shandong University, Weihai, Shandong, 264209, China
| | - Ying Liu
- Food Inspection & Quarantine Center, Shenzhen Custom, Shenzhen, Guangdong, 518060, China
| | - Hong Liu
- Food Inspection & Quarantine Center, Shenzhen Custom, Shenzhen, Guangdong, 518060, China
| | - Can Yang Zhang
- Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, China
| | - Juan Li
- Advanced Research Institute for Multidisciplinary Science, Beijing Institute of Technology, Beijing, 100081, China
| | - Peiwu Qin
- Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, China
- Tsinghua-Berkeley Shenzhen Institute, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, China
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5
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Brogna C, Cristoni S, Brogna B, Bisaccia DR, Marino G, Viduto V, Montano L, Piscopo M. Toxin-like Peptides from the Bacterial Cultures Derived from Gut Microbiome Infected by SARS-CoV-2-New Data for a Possible Role in the Long COVID Pattern. Biomedicines 2022; 11:87. [PMID: 36672595 PMCID: PMC9855837 DOI: 10.3390/biomedicines11010087] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2022] [Revised: 12/18/2022] [Accepted: 12/26/2022] [Indexed: 01/01/2023] Open
Abstract
It has been 3 years since the beginning of the SARS-CoV-2 outbreak, however it is as yet little known how to care for the acute COVID-19 and long COVID patients. COVID-19 clinical manifestations are of both pulmonary and extra-pulmonary types. Extra-pulmonary ones include extreme tiredness (fatigue), shortness of breath, muscle aches, hyposmia, dysgeusia, and other neurological manifestations. In other autoimmune diseases, such as Parkinson's disease (PD) or Alzheimer's Disease (AD), it is well known that role of acetylcholine is crucial in olfactory dysfunction. We have already observed the presence of toxin-like peptides in plasma, urine, and faecal samples from COVID-19 patients, which are very similar to molecules known to alter acetylcholine signaling. After observing the production of these peptides in bacterial cultures, we have performed additional proteomics analyses to better understand their behavior and reported the extended data from our latest in vitro experiment. It seems that the gut microbiome continues to produce toxin-like peptides also after the decrease of RNA SARS-CoV-2 viral load at molecular tests. These toxicological interactions between the gut/human microbiome bacteria and the virus suggest a new scenario in the study of the clinical symptoms in long COVID and also in acute COVID-19 patients. It is discussed that in the bacteriophage similar behavior, the presence of toxins produced by bacteria continuously after viral aggression can be blocked using an appropriate combination of certain drugs.
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Affiliation(s)
- Carlo Brogna
- Department of Research, Craniomed Group Facility Srl., 20091 Bresso, Italy
| | | | - Barbara Brogna
- Department of Radiology, Moscati Hospital, Contrada Amoretta, 83100 Avellino, Italy
| | | | - Giuliano Marino
- Marsanconsulting Srl. Public Health Company, Via dei Fiorentini, 80133 Napoli, Italy
| | | | - Luigi Montano
- Andrology Unit and Service of Life Style Medicine in Uro-Andrology, Local Health Authority (ASL), 84124 Salerno, Italy
| | - Marina Piscopo
- Department of Biology, University of Naples Federico II, 80126 Napoli, Italy
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6
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Wang B, Svetlov D, Bartikofsky D, Wobus CE, Artsimovitch I. Going Retro, Going Viral: Experiences and Lessons in Drug Discovery from COVID-19. Molecules 2022; 27:3815. [PMID: 35744940 PMCID: PMC9228142 DOI: 10.3390/molecules27123815] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2022] [Revised: 06/03/2022] [Accepted: 06/07/2022] [Indexed: 12/15/2022] Open
Abstract
The severity of the COVID-19 pandemic and the pace of its global spread have motivated researchers to opt for repurposing existing drugs against SARS-CoV-2 rather than discover or develop novel ones. For reasons of speed, throughput, and cost-effectiveness, virtual screening campaigns, relying heavily on in silico docking, have dominated published reports. A particular focus as a drug target has been the principal active site (i.e., RNA synthesis) of RNA-dependent RNA polymerase (RdRp), despite the existence of a second, and also indispensable, active site in the same enzyme. Here we report the results of our experimental interrogation of several small-molecule inhibitors, including natural products proposed to be effective by in silico studies. Notably, we find that two antibiotics in clinical use, fidaxomicin and rifabutin, inhibit RNA synthesis by SARS-CoV-2 RdRp in vitro and inhibit viral replication in cell culture. However, our mutagenesis studies contradict the binding sites predicted computationally. We discuss the implications of these and other findings for computational studies predicting the binding of ligands to large and flexible protein complexes and therefore for drug discovery or repurposing efforts utilizing such studies. Finally, we suggest several improvements on such efforts ongoing against SARS-CoV-2 and future pathogens as they arise.
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Affiliation(s)
- Bing Wang
- Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA;
| | | | - Dylan Bartikofsky
- Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109, USA; (D.B.); (C.E.W.)
| | - Christiane E. Wobus
- Department of Microbiology and Immunology, University of Michigan, Ann Arbor, MI 48109, USA; (D.B.); (C.E.W.)
| | - Irina Artsimovitch
- Department of Microbiology, The Ohio State University, Columbus, OH 43210, USA;
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7
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Panayiotakopoulos GD, Papadimitriou DT. Rifampicin for COVID-19. World J Virol 2022; 11:90-97. [PMID: 35433334 PMCID: PMC8966591 DOI: 10.5501/wjv.v11.i2.90] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/25/2021] [Revised: 11/29/2021] [Accepted: 02/10/2022] [Indexed: 02/06/2023] Open
Abstract
Vaccinations for coronavirus disease-2019 (COVID-19) have begun more than a year before, yet without specific treatments available. Rifampicin, critically important for human medicine (World Health Organization's list of essential medicines), may prove pharmacologically effective for treatment and chemoprophylaxis of healthcare personnel and those at higher risk. It has been known since 1969 that rifampicin has a direct selective antiviral effect on viruses which have their own RNA polymerase (severe acute respiratory syndrome coronavirus 2), like the main mechanism of action of remdesivir. This involves inhibition of late viral protein synthesis, the virion assembly, and the viral polymerase itself. This antiviral effect is dependent on the administration route, with local application resulting in higher drug concentrations at the site of viral replication. This would suggest also trying lung administration of rifampicin by nebulization to increase the drug's concentration at infection sites while minimizing systemic side effects. Recent in silico studies with a computer-aided approach, found rifampicin among the most promising existing drugs that could be repurposed for the treatment of COVID-19.
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Affiliation(s)
- George D Panayiotakopoulos
- Department of Clinical Pharmacology, University of Patras Medical School, Rion 26504, Greece
- The National Public Health Organization of Greece, Athens 15123, Greece
| | - Dimitrios T Papadimitriou
- Department of Pediatric, Adolescent Endocrinology & Diabetes, Athens Medical Center, Marousi 15125, Greece
- Endocrine Unit, Aretaieion University Hospital, Athens 11528, Greece
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Abdulaziz L, Elhadi E, Abdallah EA, Alnoor FA, Yousef BA. Antiviral Activity of Approved Antibacterial, Antifungal, Antiprotozoal and Anthelmintic Drugs: Chances for Drug Repurposing for Antiviral Drug Discovery. J Exp Pharmacol 2022; 14:97-115. [PMID: 35299994 PMCID: PMC8922315 DOI: 10.2147/jep.s346006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/24/2021] [Accepted: 02/28/2022] [Indexed: 11/29/2022] Open
Abstract
Drug repurposing process aims to identify new uses for the existing drugs to overcome traditional de novo drug discovery and development challenges. At the same time, as viral infections became a serious threat to humans and the viral organism itself has a high ability to mutate genetically, and due to serious adverse effects that result from antiviral drugs, there are crucial needs for the discovery of new antiviral drugs, and to identify new antiviral effects for the exciting approved drugs towards different types of viral infections depending on the observed antiviral activity in preclinical studies or clinical findings is one of the approaches to counter the viral infections problems. This narrative review article summarized mainly the published preclinical studies that evaluated the antiviral activity of drugs that are approved and used mainly as antibacterial, antifungal, antiprotozoal, and anthelmintic drugs, and the preclinical studies included the in silico, in vitro, and in vivo findings, additionally some clinical observations were also included while trying to relate them to the preclinical findings. Finally, the structure used for writing about the antiviral activity of the drugs was according to the families of the viruses used in the studies to form a better image for the target of antiviral activity of different drugs in the different kinds of viruses and to relate between the antiviral activity of the drugs against different strains of viruses within the same viral family.
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Affiliation(s)
- Leena Abdulaziz
- Department of Pharmacology, Faculty of Pharmacy, Omdurman Islamic University, Khartoum, 14415, Sudan
| | - Esraa Elhadi
- Department of Pharmacology, Faculty of Pharmacy, Omdurman Islamic University, Khartoum, 14415, Sudan
- Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing, 210009, People’s Republic of China
| | - Ejlal A Abdallah
- Department of Pharmacology and Pharmacy Practice, Faculty of Pharmacy, Sudan University of Science and Technology, Khartoum, 11111, Sudan
| | - Fadlalbaseer A Alnoor
- Department of Pharmacology, Faculty of Pharmacy, National University, Khartoum, 11111, Sudan
| | - Bashir A Yousef
- Department of Pharmacology, Faculty of Pharmacy, University of Khartoum, Khartoum, 11111, Sudan
- Correspondence: Bashir A Yousef, Department of Pharmacology, Faculty of Pharmacy, University of Khartoum, Al-Qasr Ave, Khartoum, 11111, Sudan, Tel +249 912932418, Fax +249 183780696, Email
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Abstract
Appearance of an eccentric nucleoid within developing poxvirus envelopes is the first ultrastructural evidence of core differentiation. Subsequently, the nucleoid elongates and is flanked by lenticular densities known as lateral bodies. When mature, it collapses into a biconcave form. In ultrathin sections, the eccentric nucleoid usually appears compact; although in the elongate stage central spaces may be apparent. Normally, the stages of poxvirus maturation are asynchronous and difficult to examine in order. Treatment of infected cells with rifampin provides a useful experimental approach for examining the development of nucleoid material.HeLa cells were treated with rifampin (100µg/ml) during an 8 hr. infection with vaccinia virus. Rifampin interrupts vaccinia morphogenesis at a stage prior to the formation of coated envelopes, and membranous precursors of envelopes accumulate. When the drug is washed out, large numbers of virus envelopes form simoultaneously (Fig. 1).
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Moss B. Investigating Viruses During the Transformation of Molecular Biology: Part II. Annu Rev Virol 2020; 7:15-36. [PMID: 32392458 DOI: 10.1146/annurev-virology-021020-100558] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
My scientific career started at an extraordinary time, shortly after the discoveries of the helical structure of DNA, the central dogma of DNA to RNA to protein, and the genetic code. Part I of this series emphasizes my education and early studies highlighted by the isolation and characterization of numerous vaccinia virus enzymes, determination of the cap structure of messenger RNA, and development of poxviruses as gene expression vectors for use as recombinant vaccines. Here I describe a shift in my research focus to combine molecular biology and genetics for a comprehensive understanding of poxvirus biology. The dominant paradigm during the early years was to select a function, isolate the responsible proteins, and locate the corresponding gene, whereas later the common paradigm was to select a gene, make a mutation, and determine the altered function. Motivations, behind-the-scenes insights, importance of new technologies, and the vital roles of trainees and coworkers are emphasized.
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Affiliation(s)
- Bernard Moss
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA;
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11
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Structural basis for the inhibition of poxvirus assembly by the antibiotic rifampicin. Proc Natl Acad Sci U S A 2018; 115:8424-8429. [PMID: 30068608 DOI: 10.1073/pnas.1810398115] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023] Open
Abstract
Poxviruses are large DNA viruses that cause disease in animals and humans. They differ from classical enveloped viruses, because their membrane is acquired from cytoplasmic membrane precursors assembled onto a viral protein scaffold formed by the D13 protein rather than budding through cellular compartments. It was found three decades ago that the antibiotic rifampicin blocks this process and prevents scaffold formation. To elucidate the mechanism of action of rifampicin, we have determined the crystal structures of six D13-rifamycin complexes. These structures reveal that rifamycin compounds bind to a phenylalanine-rich region, or F-ring, at the membrane-proximal opening of the central channel of the D13 trimer. We show by NMR, surface plasmon resonance (SPR), and site-directed mutagenesis that A17, a membrane-associated viral protein, mediates the recruitment of the D13 scaffold by also binding to the F-ring. This interaction is the target of rifampicin, which prevents A17 binding, explaining the inhibition of viral morphogenesis. The F-ring of D13 is both conserved in sequence in mammalian poxviruses and essential for interaction with A17, defining a target for the development of assembly inhibitors. The model of the A17-D13 interaction describes a two-component system for remodeling nascent membranes that may be conserved in other large and giant DNA viruses.
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12
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Weisberg AS, Maruri-Avidal L, Bisht H, Hansen BT, Schwartz CL, Fischer ER, Meng X, Xiang Y, Moss B. Enigmatic origin of the poxvirus membrane from the endoplasmic reticulum shown by 3D imaging of vaccinia virus assembly mutants. Proc Natl Acad Sci U S A 2017; 114:E11001-E11009. [PMID: 29203656 PMCID: PMC5754806 DOI: 10.1073/pnas.1716255114] [Citation(s) in RCA: 26] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/23/2022] Open
Abstract
The long-standing inability to visualize connections between poxvirus membranes and cellular organelles has led to uncertainty regarding the origin of the viral membrane. Indeed, there has been speculation that viral membranes form de novo in cytoplasmic factories. Another possibility, that the connections are too short-lived to be captured by microscopy during a normal infection, motivated us to identify and characterize virus mutants that are arrested in assembly. Five conserved vaccinia virus proteins, referred to as Viral Membrane Assembly Proteins (VMAPs), that are necessary for formation of immature virions were found. Transmission electron microscopy studies of two VMAP deletion mutants had suggested retention of connections between viral membranes and the endoplasmic reticulum (ER). We now analyzed cells infected with each of the five VMAP deletion mutants by electron tomography, which is necessary to validate membrane continuity, in addition to conventional transmission electron microscopy. In all cases, connections between the ER and viral membranes were demonstrated by 3D reconstructions, supporting a role for the VMAPs in creating and/or stabilizing membrane scissions. Furthermore, coexpression of the viral reticulon-like transmembrane protein A17 and the capsid-like scaffold protein D13 was sufficient to form similar ER-associated viral structures in the absence of other major virion proteins. Determination of the mechanism of ER disruption during a normal VACV infection and the likely participation of both viral and cell proteins in this process may provide important insights into membrane dynamics.
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Affiliation(s)
- Andrea S Weisberg
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
| | - Liliana Maruri-Avidal
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
| | - Himani Bisht
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892
| | - Bryan T Hansen
- Research Technologies Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840
| | - Cindi L Schwartz
- Research Technologies Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840
| | - Elizabeth R Fischer
- Research Technologies Branch, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT 59840
| | - Xiangzhi Meng
- Department of Microbiology, Immunology, and Molecular Genetics, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229
| | - Yan Xiang
- Department of Microbiology, Immunology, and Molecular Genetics, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229
| | - Bernard Moss
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892;
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13
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Abstract
This Reflections article describes my early work on viral enzymes and the discovery of mRNA capping, how my training in medicine and biochemistry merged as I evolved into a virologist, the development of viruses as vaccine vectors, and how scientific and technological developments during the 1970s and beyond set the stage for the interrogation of nearly every step in the reproductive cycle of vaccinia virus (VACV), a large DNA virus with about 200 genes. The reader may view this article as a work in progress, because I remain actively engaged in research at the National Institutes of Health (NIH) notwithstanding 50 memorable years there.
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Affiliation(s)
- Bernard Moss
- From the Laboratory of Viral Diseases, NIAID, National Institutes of Health, Bethesda, Maryland 20892
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Poxvirus membrane biogenesis. Virology 2015; 479-480:619-26. [PMID: 25728299 DOI: 10.1016/j.virol.2015.02.003] [Citation(s) in RCA: 44] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2015] [Revised: 02/02/2015] [Accepted: 02/03/2015] [Indexed: 12/18/2022]
Abstract
Poxviruses differ from most DNA viruses by replicating entirely within the cytoplasm. The first discernible viral structures are crescents and spherical immature virions containing a single lipoprotein membrane bilayer with an external honeycomb lattice. Because this viral membrane displays no obvious continuity with a cellular organelle, a de novo origin was suggested. Nevertheless, transient connections between viral and cellular membranes could be difficult to resolve. Despite the absence of direct evidence, the intermediate compartment (ERGIC) between the endoplasmic reticulum (ER) and Golgi apparatus and the ER itself were considered possible sources of crescent membranes. A break-through in understanding poxvirus membrane biogenesis has come from recent studies of the abortive replication of several vaccinia virus null mutants. Novel images showing continuity between viral crescents and the ER and the accumulation of immature virions in the expanded ER lumen provide the first direct evidence for a cellular origin of this poxvirus membrane.
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15
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Duplication of the A17L locus of vaccinia virus provides an alternate route to rifampin resistance. J Virol 2014; 88:11576-85. [PMID: 25078687 DOI: 10.1128/jvi.00618-14] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023] Open
Abstract
UNLABELLED Specific gene duplications can enable double-stranded DNA viruses to adapt rapidly to environmental pressures despite the low mutation rate of their high-fidelity DNA polymerases. We report on the rapid positive selection of a novel vaccinia virus genomic duplication mutant in the presence of the assembly inhibitor rifampin. Until now, all known rifampin-resistant vaccinia virus isolates have contained missense mutations in the D13L gene, which encodes a capsid-like scaffold protein required for stabilizing membrane curvature during the early stage of virion assembly. Here we describe a second pathway to rifampin resistance involving A17, a membrane protein that binds and anchors D13 to the immature virion. After one round of selection, a rifampin-resistant virus that contained a genomic duplication in the A17L-A21L region was recovered. The mutant had both C-terminally truncated and full-length A17L open reading frames. Expression of the truncated A17 protein was retained when the virus was passaged in the presence of rifampin but was lost in the absence of the drug, suggesting that the duplication decreased general fitness. Both forms of A17 were bound to the virion membrane and associated with D13. Moreover, insertion of an additional truncated or inducible full-length A17L open reading frame into the genome of the wild-type virus was sufficient to confer rifampin resistance. In summary, this report contains the first evidence of an alternate mechanism for resistance of poxviruses to rifampin, indicates a direct relationship between A17 levels and the resistance phenotype, and provides further evidence of the ability of double-stranded DNA viruses to acquire drug resistance through gene duplication. IMPORTANCE The present study provides the first evidence of a new mechanism of resistance of a poxvirus to the antiviral drug rifampin. In addition, it affirms the importance of the interaction between the D13 scaffold protein and the A17 membrane protein for assembly of virus particles. Resistance to rifampin was linked to a partial duplication of the gene encoding the A17 protein, similar to the resistance to hydroxyurea enabled by duplication of the gene encoding the small subunit of ribonucleotide reductase and of the K3L gene to allow adaptation to the antiviral action of protein kinase R. Gene duplication may provide a way for poxviruses and other DNA viruses with high-fidelity DNA polymerases to adjust rapidly to changes in the environment.
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Activation of stress response pathways promotes formation of antiviral granules and restricts virus replication. Mol Cell Biol 2014; 34:2003-16. [PMID: 24662051 DOI: 10.1128/mcb.01630-13] [Citation(s) in RCA: 39] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
The formation of protein-RNA granules is a part of both natural cellular function (P-bodies and nuclear HNRNPs) and the response to cellular stress (stress granules and ND10 bodies). To better understand the role of stress-induced granules in viral infection, we have studied the ability of cells to restrict poxvirus replication through the formation of antiviral granules (AVGs). Of cells infected with a wild-type poxvirus, a small number spontaneously formed AVGs. In these AVG-positive cells, viral gene expression was inhibited. The addition of compounds that altered RNA helicase activity, induced oxidative stress, or stimulated translation initiation factor phosphorylation significantly increased the number of AVG-positive cells. When AVGs formed, both viral translation and titers were decreased even when host translation persisted. Treatment with the antiviral compound isatin β-thiosemicarbazone (IBT), a compound that was used to treat smallpox infections, induced AVGs, suggesting a role for these structures in the pharmacological inhibition of poxvirus replication. These findings provide evidence that AVGs are an innate host response that can be exogenously stimulated to combat virus infection. Since small molecules are able to stimulate AVG formation, it is a potential target for new antiviral development.
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Maruri-Avidal L, Weisberg AS, Moss B. Association of the vaccinia virus A11 protein with the endoplasmic reticulum and crescent precursors of immature virions. J Virol 2013; 87:10195-206. [PMID: 23864611 PMCID: PMC3754016 DOI: 10.1128/jvi.01601-13] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2013] [Accepted: 07/03/2013] [Indexed: 12/17/2022] Open
Abstract
The apparent de novo formation of viral membranes within cytoplasmic factories is a mysterious, poorly understood first step in poxvirus morphogenesis. Genetic studies identified several viral proteins essential for membrane formation and the assembly of immature virus particles. Their repression results in abortive replication with the accumulation of dense masses of viroplasm. In the present study, we further characterized one of these proteins, A11, and investigated its association with cellular and viral membranes under normal and abortive replication conditions. We discovered that A11 colocalized in cytoplasmic factories with the endoplasmic reticulum (ER) and L2, another viral protein required for morphogenesis. Confocal microscopy and subcellular fractionation indicated that A11 was not membrane associated in uninfected cells, whereas L2 still colocalized with the ER. Cell-free transcription and translation experiments indicated that both A11 and L2 are tail-anchored proteins that associate posttranslationally with membranes and likely require specific cytoplasmic targeting chaperones. Transmission electron microscopy indicated that A11, like L2, associated with crescent membranes and immature virions during normal infection and with vesicles and tubules near masses of dense viroplasm during abortive infection in the absence of the A17 or A14 protein component of viral membranes. When the synthesis of A11 was repressed, "empty" immature-virion-like structures formed in addition to masses of viroplasm. The immature-virion-like structures were labeled with antibodies to A17 and to the D13 scaffold protein and were closely associated with calnexin-labeled ER. These studies revealed similarities and differences between A11 and L2, both of which may be involved in the recruitment of the ER for virus assembly.
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Affiliation(s)
- Liliana Maruri-Avidal
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA
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Vaccinia virus A19 protein participates in the transformation of spherical immature particles to barrel-shaped infectious virions. J Virol 2013; 87:10700-9. [PMID: 23885081 DOI: 10.1128/jvi.01258-13] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The A19L open reading frame of vaccinia virus encodes a 9-kDa protein that is conserved in all sequenced chordopoxviruses, yet until now it has not been specifically characterized in any species. We appended an epitope tag after the start codon of the A19L open reading frame without compromising infectivity. The protein was synthesized after viral DNA replication and was phosphorylated independently of the vaccinia virus F10 kinase. The A19 protein was present in purified virions and was largely resistant to nonionic detergent extraction, suggesting a location within the core. A conditional lethal mutant virus was constructed by placing the A19 open reading frame under the control of the Escherichia coli lac repressor system. A19 synthesis and infectious virus formation were dependent on inducer. In the absence of inducer, virion morphogenesis was interrupted, and spherical dense particles that had greatly reduced amounts of the D13 scaffold accumulated in place of barrel-shaped mature virions. The infectivity of purified A19-deficient particles was more than 2 log units less than that of A19-containing virions. Nevertheless, the A19-deficient particles contained DNA, and except for the absence of A19 and decreased core protein processing, they appeared to have a similar protein composition as A19-containing virions. Thus, the A19 protein participates in the maturation of immature vaccinia virus virions to infectious particles.
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19
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Maruri-Avidal L, Weisberg AS, Bisht H, Moss B. Analysis of viral membranes formed in cells infected by a vaccinia virus L2-deletion mutant suggests their origin from the endoplasmic reticulum. J Virol 2013; 87:1861-71. [PMID: 23192873 PMCID: PMC3554160 DOI: 10.1128/jvi.02779-12] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2012] [Accepted: 11/20/2012] [Indexed: 12/17/2022] Open
Abstract
Assembly of the poxvirus immature virion (IV) membrane is a poorly understood event that occurs within the cytoplasm. At least eight viral proteins participate in formation of the viral membrane. Of these, A14, A17, and D13 are structural components whereas A6, A11, F10, H7, and L2 participate in membrane biogenesis. L2, the object of this study, is conserved in all chordopoxviruses, expressed early in infection, and associated with the endoplasmic reticulum (ER) throughout the cell and at the edges of crescent-shaped IV precursors. Previous studies with an inducible L2 mutant revealed abortive formation of the crescent membrane. However, possible low-level L2 synthesis under nonpermissive conditions led to ambiguity in interpretation. Here, we constructed a cell line that expresses L2, which allowed the creation of an L2-deletion mutant. In noncomplementing cells, replication was aborted prior to formation of mature virions and two types of aberrant structures were recognized. One consisted of short crescents, at the surface of dense masses of viroplasm, which were labeled with antibodies to the A11, A14, A17, and D13 proteins. The other structure consisted of "empty" IV-like membranes, also labeled with antibodies to the viral proteins, which appeared to be derived from adjacent calnexin-containing ER. A subset of 25 proteins examined, exemplified by components of the entry-fusion complex, were greatly diminished in amount. The primary role of L2 may be to recruit ER and modulate its transformation to viral membranes in juxtaposition with the viroplasm, simultaneously preventing the degradation of viral proteins dependent on viral membranes for stability.
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Affiliation(s)
- Liliana Maruri-Avidal
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland, USA
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21
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Formation of orthopoxvirus cytoplasmic A-type inclusion bodies and embedding of virions are dynamic processes requiring microtubules. J Virol 2012; 86:5905-14. [PMID: 22438543 DOI: 10.1128/jvi.06997-11] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
In cells infected with some orthopoxviruses, numerous mature virions (MVs) become embedded within large, cytoplasmic A-type inclusions (ATIs) that can protect infectivity after cell lysis. ATIs are composed of an abundant viral protein called ATIp, which is truncated in orthopoxviruses such as vaccinia virus (VACV) that do not form ATIs. To study ATI formation and occlusion of MVs within ATIs, we used recombinant VACVs that express the cowpox full-length ATIp or we transfected plasmids encoding ATIp into cells infected with VACV, enabling ATI formation. ATI enlargement and MV embedment required continued protein synthesis and an intact microtubular network. For live imaging of ATIs and MVs, plasmids expressing mCherry fluorescent protein fused to ATIp were transfected into cells infected with VACV expressing the viral core protein A4 fused to yellow fluorescent protein. ATIs appeared as dynamic, mobile bodies that enlarged by multiple coalescence events, which could be prevented by disrupting microtubules. Coalescence of ATIs was confirmed in cells infected with cowpox virus. MVs were predominantly at the periphery of ATIs early in infection. We determined that coalescence contributed to the distribution of MVs within ATIs and that microtubule-disrupting drugs abrogated coalescence-mediated MV embedment. In addition, MVs were shown to move from viral factories at speeds consistent with microtubular transport to the peripheries of ATIs, whereas disruption of microtubules prevented such trafficking. The data indicate an important role for microtubules in the coalescence of ATIs into larger structures, transport of MVs to ATIs, and embedment of MVs within the ATI matrix.
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22
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Maruri-Avidal L, Weisberg AS, Moss B. Vaccinia virus L2 protein associates with the endoplasmic reticulum near the growing edge of crescent precursors of immature virions and stabilizes a subset of viral membrane proteins. J Virol 2011; 85:12431-41. [PMID: 21917978 PMCID: PMC3209352 DOI: 10.1128/jvi.05573-11] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2011] [Accepted: 09/04/2011] [Indexed: 12/11/2022] Open
Abstract
The initial step in poxvirus morphogenesis, the formation of crescent membranes, occurs within cytoplasmic factories. L2 is one of several vaccinia virus proteins known to be necessary for formation of crescents and the only one synthesized early in infection. Virus replication was unaffected when the L2R open reading frame was replaced by L2R containing an N-terminal epitope tag while retaining the original promoter. L2 colocalized with the endoplasmic reticulum (ER) protein calnexin throughout the cytoplasm of infected and transfected cells. Topological studies indicated that the N terminus of L2 is exposed to the cytoplasm with the hydrophobic C terminus anchored in the ER. Using immunogold labeling and electron microscopy, L2 was detected in tubular membranes outside factories and inside factories near crescents and close to the edge or rim of crescents; a similar labeling pattern was found for the ER luminal protein disulfide isomerase (PDI). The phenotype of L2 conditional lethal mutants and the localization of L2 suggest that it participates in elongation of crescents by the addition of ER membrane to the growing edge. Small amounts of L2 and PDI were detected within immature and mature virions, perhaps trapped during assembly. The repression of L2, as well as A11 and A17, two other proteins that are required for viral crescent formation, profoundly decreased the stability of a subset of viral membrane proteins including those comprising the entry-fusion complex. To avoid degradation, these unstable membrane proteins may need to directly insert into the viral membrane or be rapidly shunted there from the ER.
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Affiliation(s)
- Liliana Maruri-Avidal
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-3210
| | - Andrea S. Weisberg
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-3210
| | - Bernard Moss
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-3210
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Membrane remodeling by the double-barrel scaffolding protein of poxvirus. PLoS Pathog 2011; 7:e1002239. [PMID: 21931553 PMCID: PMC3169552 DOI: 10.1371/journal.ppat.1002239] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2011] [Accepted: 07/11/2011] [Indexed: 11/24/2022] Open
Abstract
In contrast to most enveloped viruses, poxviruses produce infectious particles that do not acquire their internal lipid membrane by budding through cellular compartments. Instead, poxvirus immature particles are generated from atypical crescent-shaped precursors whose architecture and composition remain contentious. Here we describe the 2.6 Å crystal structure of vaccinia virus D13, a key structural component of the outer scaffold of viral crescents. D13 folds into two jellyrolls decorated by a head domain of novel fold. It assembles into trimers that are homologous to the double-barrel capsid proteins of adenovirus and lipid-containing icosahedral viruses. We show that, when tethered onto artificial membranes, D13 forms a honeycomb lattice and assembly products structurally similar to the viral crescents and immature particles. The architecture of the D13 honeycomb lattice and the lipid-remodeling abilities of D13 support a model of assembly that exhibits similarities with the giant mimivirus. Overall, these findings establish that the first committed step of poxvirus morphogenesis utilizes an ancestral lipid-remodeling strategy common to icosahedral DNA viruses infecting all kingdoms of life. Furthermore, D13 is the target of rifampicin and its structure will aid the development of poxvirus assembly inhibitors. Poxviruses are arguably the largest viruses infecting humans. The unique brick-shape architecture of their infectious virus particles sets them apart from any other viral family in the virosphere. The infectious particles are produced through a series of assembly steps where intermediates of distinct composition and architecture can be identified. In particular, atypical crescent-shaped precursors of immature particles have generated much controversy regarding their structure and the origin of their lipidic membrane. Here, we used a combination of X-ray crystallography and electron microscopy to investigate the role of a crucial structural component of viral crescents called D13. Our atomic structure of D13 firmly establishes an evolutionary link between poxviruses and a group of large DNA viruses. In addition, we show that, when tethered to artificial membranes, this protein assembles into a scaffold analogous to that in immature particles. The resulting pseudo-atomic model of the honeycomb lattice reveals similarities to the mimivirus, which suggests that giant viral shells use common assembly principles. Overall, our findings reveal that poxviruses utilize an ancestral lipid-remodeling strategy common to DNA viruses infecting all kingdoms of life. They also provide a basis for structure-based design of assembly inhibitors against poxvirus pathogens.
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24
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Bahar M, Graham S, Stuart D, Grimes J. Insights into the evolution of a complex virus from the crystal structure of vaccinia virus D13. Structure 2011; 19:1011-20. [PMID: 21742267 PMCID: PMC3136756 DOI: 10.1016/j.str.2011.03.023] [Citation(s) in RCA: 73] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2011] [Revised: 03/29/2011] [Accepted: 03/31/2011] [Indexed: 11/27/2022]
Abstract
The morphogenesis of poxviruses such as vaccinia virus (VACV) sees the virion shape mature from spherical to brick-shaped. Trimeric capsomers of the VACV D13 protein form a transitory, stabilizing lattice on the surface of the initial spherical immature virus particle. The crystal structure of D13 reveals that this major scaffolding protein comprises a double β barrel "jelly-roll" subunit arranged as pseudo-hexagonal trimers. These structural features are characteristic of the major capsid proteins of a lineage of large icosahedral double-stranded DNA viruses including human adenovirus and the bacteriophages PRD1 and PM2. Structure-based phylogenetic analysis confirms that VACV belongs to this lineage, suggesting that (analogously to higher organism embryogenesis) early poxvirus morphogenesis reflects their evolution from a lineage of viruses sharing a common icosahedral ancestor.
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Affiliation(s)
- Mohammad W. Bahar
- The Division of Structural Biology and the Oxford Protein Production Facility, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK
| | - Stephen C. Graham
- The Division of Structural Biology and the Oxford Protein Production Facility, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK
| | - David I. Stuart
- The Division of Structural Biology and the Oxford Protein Production Facility, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK
- Science Division, Diamond Light Source Ltd., Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, UK
| | - Jonathan M. Grimes
- The Division of Structural Biology and the Oxford Protein Production Facility, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, OX3 7BN, UK
- Science Division, Diamond Light Source Ltd., Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire OX11 0DE, UK
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25
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Maruri-Avidal L, Domi A, Weisberg AS, Moss B. Participation of vaccinia virus l2 protein in the formation of crescent membranes and immature virions. J Virol 2011; 85:2504-11. [PMID: 21228235 PMCID: PMC3067936 DOI: 10.1128/jvi.02505-10] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2010] [Accepted: 12/30/2010] [Indexed: 11/20/2022] Open
Abstract
Morphogenesis of vaccinia virus begins with the appearance of crescent-shaped membrane precursors of immature virions in cytoplasmic factories. During the initial characterization of the product of the L2R reading frame, we discovered that it plays an important role in crescent formation. The L2 protein was expressed early in infection and was associated with the detergent-soluble membrane fraction of mature virions, consistent with two potential membrane-spanning domains. All chordopoxviruses have L2 homologs, suggesting an important function. Indeed, we were unable to isolate an infectious L2R deletion mutant. Consequently, we constructed an inducible mutant with a conditional lethal phenotype. When L2 expression was repressed, proteolytic processing of the major core proteins and the A17 protein, which is an essential component of the immature virion membrane, failed to occur, suggesting an early block in viral morphogenesis. At 8 h after infection in the presence of inducer, immature and mature virions were abundantly seen by electron microscopy. In contrast, those structures were rare in the absence of inducer and were replaced by large, dense aggregates of viroplasm. A minority of these aggregates had short spicule-coated membranes, which resembled the beginnings of crescent formation, at their periphery. These short membrane segments at the edge of the dense viroplasm increased in number at later times, and some immature virions were seen. Although the L2 protein was not detected under nonpermissive conditions, minute amounts could account for stunted and delayed viral membrane formation. These findings suggested that L2 is required for the formation or elongation of crescent membranes.
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Affiliation(s)
- Liliana Maruri-Avidal
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-3210
| | - Arban Domi
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-3210
| | - Andrea S. Weisberg
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-3210
| | - Bernard Moss
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-3210
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26
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Suárez C, Gutiérrez-Berzal J, Andrés G, Salas ML, Rodríguez JM. African swine fever virus protein p17 is essential for the progression of viral membrane precursors toward icosahedral intermediates. J Virol 2010; 84:7484-99. [PMID: 20504920 PMCID: PMC2897610 DOI: 10.1128/jvi.00600-10] [Citation(s) in RCA: 45] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/18/2010] [Accepted: 05/17/2010] [Indexed: 11/20/2022] Open
Abstract
The first morphological evidence of African swine fever virus (ASFV) assembly is the appearance of precursor viral membranes, thought to derive from the endoplasmic reticulum, within the assembly sites. We have shown previously that protein p54, a viral structural integral membrane protein, is essential for the generation of the viral precursor membranes. In this report, we study the role of protein p17, an abundant transmembrane protein localized at the viral internal envelope, in these processes. Using an inducible virus for this protein, we show that p17 is essential for virus viability and that its repression blocks the proteolytic processing of polyproteins pp220 and pp62. Electron microscopy analyses demonstrate that when the infection occurs under restrictive conditions, viral morphogenesis is blocked at an early stage, immediately posterior to the formation of the viral precursor membranes, indicating that protein p17 is required to allow their progression toward icosahedral particles. Thus, the absence of this protein leads to an accumulation of these precursors and to the delocalization of the major components of the capsid and core shell domains. The study of ultrathin serial sections from cells infected with BA71V or the inducible virus under permissive conditions revealed the presence of large helicoidal structures from which immature particles are produced, suggesting that these helicoidal structures represent a previously undetected viral intermediate.
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Affiliation(s)
- Cristina Suárez
- Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
| | - Javier Gutiérrez-Berzal
- Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
| | - Germán Andrés
- Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
| | - María L. Salas
- Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
| | - Javier M. Rodríguez
- Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
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27
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Li Z, Ling L, Liu X, Laus R, Delcayre A. A flow cytometry-based immuno-titration assay for rapid and accurate titer determination of modified vaccinia Ankara virus vectors. J Virol Methods 2010; 169:87-94. [PMID: 20637240 DOI: 10.1016/j.jviromet.2010.07.003] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2009] [Revised: 07/01/2010] [Accepted: 07/06/2010] [Indexed: 11/17/2022]
Abstract
A flow cytometry-based immuno-titration titer assay was established to determine infectious unit (IU) and transducing unit (TU) of modified vaccinia Ankara (MVA) virus vectors. This titration method enumerates infected cells by measuring the expression of viral protein for IU and transgene protein for TU in individual cells after staining with fluorophore-conjugated antibodies. It presents many advantages over standard virus titration approaches, such as TCID(50) or plaque assay, for its convenience, rapidity and accuracy as illustrated by excellent assay linearity and reproducibility. Importantly, the IU and the TCID(50) assays generated similar batch-specific titer values when testing varied MVA-derived virus preparations. Assay development revealed that the post-infection time at which viral protein expression is evaluated, host cell type, and blocking the formation and release of progeny virion with nocodazole, an anti-microtubule agent or rifampin, a specific vaccinia virus assembly inhibitor, are critical parameters for the precision, robustness, and accuracy of IU titer determination. An added advantage of this assay is that it enables the concurrent determination of IU and transducing units (TU) by measuring the expression of a transgene product when testing recombinant viruses. The latter was demonstrated using a MVA vector carrying a human HER-2 gene fragment as model. Hence, this assay is very versatile in that it can be used to determine IU as well as multiple TU titers simultaneously. Furthermore, it can readily be adapted to other poxvirus vectors.
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Affiliation(s)
- Zengji Li
- BN ImmunoTherapeutics, 2425 Garcia Avenue, Mountain View, CA 94043, United States.
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McNulty S, Bornmann W, Schriewer J, Werner C, Smith SK, Olson VA, Damon IK, Buller RM, Heuser J, Kalman D. Multiple phosphatidylinositol 3-kinases regulate vaccinia virus morphogenesis. PLoS One 2010; 5:e10884. [PMID: 20526370 PMCID: PMC2878334 DOI: 10.1371/journal.pone.0010884] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2009] [Accepted: 05/07/2010] [Indexed: 12/22/2022] Open
Abstract
Poxvirus morphogenesis is a complex process that involves the successive wrapping of the virus in host cell membranes. We screened by plaque assay a focused library of kinase inhibitors for those that caused a reduction in viral growth and identified several compounds that selectively inhibit phosphatidylinositol 3-kinase (PI3K). Previous studies demonstrated that PI3Ks mediate poxviral entry. Using growth curves and electron microscopy in conjunction with inhibitors, we show that that PI3Ks additionally regulate morphogenesis at two distinct steps: immature to mature virion (IMV) transition, and IMV envelopment to form intracellular enveloped virions (IEV). Cells derived from animals lacking the p85 regulatory subunit of Type I PI3Ks (p85α−/−β−/−) presented phenotypes similar to those observed with PI3K inhibitors. In addition, VV appear to redundantly use PI3Ks, as PI3K inhibitors further reduce plaque size and number in p85α−/−β−/− cells. Together, these data provide evidence for a novel regulatory mechanism for virion morphogenesis involving phosphatidylinositol dynamics and may represent a new therapeutic target to contain poxviruses.
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Affiliation(s)
- Shannon McNulty
- Microbiology and Molecular Genetics Graduate Program, Emory University School of Medicine, Atlanta, Georgia, United States of America
| | - William Bornmann
- MD Anderson Cancer Center, University of Texas, Houston, Texas, United States of America
| | - Jill Schriewer
- Department of Molecular Microbiology and Immunology, Saint Louis University Health Sciences Center, St. Louis, Missouri, United States of America
| | - Chas Werner
- Department of Molecular Microbiology and Immunology, Saint Louis University Health Sciences Center, St. Louis, Missouri, United States of America
| | - Scott K. Smith
- Poxvirus Team, Poxvirus and Rabies Branch, Division of Viral and Rickettsial Diseases, National Center for Zoonotic, Viral and Enteric Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America
| | - Victoria A. Olson
- Poxvirus Team, Poxvirus and Rabies Branch, Division of Viral and Rickettsial Diseases, National Center for Zoonotic, Viral and Enteric Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America
| | - Inger K. Damon
- Poxvirus Team, Poxvirus and Rabies Branch, Division of Viral and Rickettsial Diseases, National Center for Zoonotic, Viral and Enteric Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America
| | - R. Mark Buller
- Department of Molecular Microbiology and Immunology, Saint Louis University Health Sciences Center, St. Louis, Missouri, United States of America
| | - John Heuser
- Department of Cell Biology, Washington University School of Medicine, St. Louis, Missouri, United States of America
| | - Daniel Kalman
- Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia, United States of America
- * E-mail:
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Congregation of orthopoxvirus virions in cytoplasmic A-type inclusions is mediated by interactions of a bridging protein (A26p) with a matrix protein (ATIp) and a virion membrane-associated protein (A27p). J Virol 2010; 84:7592-602. [PMID: 20484506 DOI: 10.1128/jvi.00704-10] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Some orthopoxviruses, e.g., the cowpox, ectromelia, and raccoonpox viruses, form large, discrete cytoplasmic inclusions within which mature virions (MVs) are embedded by a process called occlusion. These inclusions, which may protect occluded MVs in the environment, are composed of aggregates of the A-type inclusion protein (ATIp), which is truncated in orthopoxviruses such as vaccinia virus (VACV) and variola virus that fail to form inclusions. In addition to an intact ATIp, occlusion requires the A26 protein (A26p). Although VACV contains a functional A26p, determined by complementation of a cowpox virus occlusion-defective mutant, its role in occlusion was unknown. We found that restoration of the full-length ATI gene was sufficient for VACV inclusion formation and the ensuing occlusion of MVs. A26p was present in inclusions even when virion assembly was inhibited, suggesting a direct interaction of A26p with ATIp. Analysis of a panel of ATIp mutants indicated that the C-terminal repeat region was required for inclusion formation and the N-terminal domain for interaction with A26p and occlusion. A26p is tethered to MVs via interaction with the A27 protein (A27p); A27p was not required for association of A26p with ATIp but was necessary for occlusion. In addition, the C-terminal domain of A26p, which mediates A26p-A27p interactions, was necessary but insufficient for occlusion. Taken together, the data suggest a model for occlusion in which A26p has a bridging role between ATIp and A27p, and A27p provides a link to the MV membrane.
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30
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Abstract
The increasing emergence of antimicrobial-resistant organisms, especially methicillin-resistant Staphylococcus aureus (MRSA), has resulted in the increased use of rifampin combination therapy. The data supporting rifampin combination therapy in nonmycobacterial infections are limited by a lack of significantly controlled clinical studies. Therefore, its current use is based upon in vitro or in vivo data or retrospective case series, all with major limitations. A prominent observation from this review is that rifampin combination therapy appears to have improved treatment outcomes in cases in which there is a low organism burden, such as biofilm infections, but is less effective when effective surgery to obtain source control is not performed. The clinical data support rifampin combination therapy for the treatment of prosthetic joint infections due to methicillin-sensitive S. aureus (MSSA) after extensive debridement and for the treatment of prosthetic heart valve infections due to coagulase-negative staphylococci. Importantly, rifampin-vancomycin combination therapy has not shown any benefit over vancomycin monotherapy against MRSA infections either clinically or experimentally. Rifampin combination therapy with daptomycin, fusidic acid, and linezolid needs further exploration for these severe MRSA infections. Lastly, an assessment of the risk-benefits is needed before the addition of rifampin to other antimicrobials is considered to avoid drug interactions or other drug toxicities.
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31
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Satheshkumar PS, Weisberg A, Moss B. Vaccinia virus H7 protein contributes to the formation of crescent membrane precursors of immature virions. J Virol 2009; 83:8439-50. [PMID: 19553304 PMCID: PMC2738178 DOI: 10.1128/jvi.00877-09] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2009] [Accepted: 06/17/2009] [Indexed: 11/20/2022] Open
Abstract
Crescent membranes are the first viral structures that can be discerned during poxvirus morphogenesis. The crescents consist of a lipoprotein membrane and an outer lattice scaffold, which provides uniform curvature. Relatively little is known regarding the composition of the crescent membrane or its mode of formation. Here, we show that the H7 protein, which is conserved in all vertebrate poxviruses but has no discernible functional motifs or nonpoxvirus homologs, contributes to the formation of crescents and immature virions. Synthesis of the 17-kDa H7 protein was dependent on DNA replication and occurred late during vaccinia virus infection. Unlike many late proteins, however, H7 was not incorporated into mature virions or localized in cellular organelles. To gain insight into the role of H7, an inducible mutant was constructed and shown to have a conditional lethal phenotype: H7 expression and infectious virus formation were dependent on isopropyl-beta-D-thiogalactopyranoside. In the absence of inducer, viral late proteins were made, but membrane and core proteins were not processed by the I7 protease. A block in morphogenesis was demonstrated by transmission electron microscopy: neither typical crescents nor immature virions were detected in the absence of inducer. Instead, factory areas of the cytoplasm contained large, electron-dense inclusions, some of which had partially coated membrane segments at their surfaces. Separate, lower-density inclusions containing the D13 scaffold protein and endoplasmic reticulum membranes were also present. These features are most similar to those previously seen when expression of A11, another conserved nonvirion protein, is repressed.
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Affiliation(s)
- P S Satheshkumar
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 33 North Drive, Bethesda, MD 20892-3210, USA
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32
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Assembly and disassembly of the capsid-like external scaffold of immature virions during vaccinia virus morphogenesis. J Virol 2009; 83:9140-50. [PMID: 19570860 DOI: 10.1128/jvi.00875-09] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023] Open
Abstract
Infectious poxvirus particles are unusual in that they are brick shaped and lack symmetry. Nevertheless, an external honeycomb lattice comprised of a capsid-like protein dictates the spherical shape and size of immature poxvirus particles. In the case of vaccinia virus, trimers of 63-kDa D13 polypeptides form the building blocks of the lattice. In the present study, we addressed two questions: how D13, which has no transmembrane domain, associates with the immature virion (IV) membrane to form the lattice structure and how this scaffold is removed during the subsequent stage of morphogenesis. Interaction of D13 with the A17 membrane protein was demonstrated by immunoaffinity purification and Western blot analysis. In addition, the results of immunogold electron microscopy indicated a close association of A17 and D13 in crescents, as well as in vesicular structures when crescent formation was prevented. Further studies indicated that binding of A17 to D13 was abrogated by truncation of the N-terminal segment of A17. The N-terminal region of A17 was also required for the formation of crescent and IV structures. Disassembly of the D13 scaffold correlated with the processing of A17 by the I7 protease. When I7 expression was repressed, D13 was retained on aberrant virus particles. Furthermore, the morphogenesis of IVs to mature virions was blocked by mutation of the N-terminal but not the C-terminal cleavage site on A17. Taken together, these data indicate that A17 and D13 interactions regulate the assembly and disassembly of the IV scaffold.
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33
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Resch W, Weisberg AS, Moss B. Expression of the highly conserved vaccinia virus E6 protein is required for virion morphogenesis. Virology 2009; 386:478-85. [PMID: 19217136 PMCID: PMC2714823 DOI: 10.1016/j.virol.2009.01.009] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2008] [Revised: 01/05/2009] [Accepted: 01/09/2009] [Indexed: 11/29/2022]
Abstract
The vaccinia virus E6R gene (VACVWR062) is conserved in all members of the poxvirus family and encodes a protein associated with the mature virion. We confirmed this association and provided evidence for an internal location. An inducible mutant that conditionally expresses E6 was constructed. In the absence of inducer, plaque formation and virus production were severely inhibited in several cell lines, whereas some replication occurred in others. This difference could be due to variation in the stringency of repression, since we could not isolate a stable deletion mutant even in the more "permissive" cells. Under non-permissive conditions, viral late proteins were synthesized but processing of core proteins was inefficient, indicative of an assembly block. Transmission electron microscopy of sections of cells infected with the mutant in the absence of inducer revealed morphogenetic defects with crescents and empty immature virions adjacent to dense inclusions of viroplasm. Mature virions were infrequent and cores appeared to have lucent centers.
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Affiliation(s)
| | - Andrea S. Weisberg
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20814
| | - Bernard Moss
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20814
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34
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The vaccinia virus gene I2L encodes a membrane protein with an essential role in virion entry. J Virol 2008; 82:10247-61. [PMID: 18701587 DOI: 10.1128/jvi.01035-08] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023] Open
Abstract
The previously unstudied vaccinia virus gene I2L is conserved in all orthopoxviruses. We show here that the 8-kDa I2 protein is expressed at late times of infection, is tightly associated with membranes, and is encapsidated in mature virions. We have generated a recombinant virus in which I2 expression is dependent upon the inclusion of tetracycline in the culture medium. In the absence of I2, the biochemical events of the viral life cycle progress normally, and virion morphogenesis culminates in the production of mature virions. However, these virions show an approximately 400-fold reduction in specific infectivity due to an inability to enter target cells. Several proteins that have been previously identified as components of an essential entry/fusion complex are present at reduced levels in I2-deficient virions, although other membrane proteins, core proteins, and DNA are encapsidated at normal levels. A preliminary structure/function analysis of I2 has been performed using a transient complementation assay: the C-terminal hydrophobic domain is essential for protein stability, and several regions within the N-terminal hydrophilic domain are essential for biological competency. I2 is thus yet another component of the poxvirus virion that is essential for the complex process of entry into target cells.
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35
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McAuslan BR. Enzymes specified by DNA-containing animal viruses. In: strategy of the viral genome. CIBA FOUNDATION SYMPOSIUM 2008:25-44. [PMID: 4337203 DOI: 10.1002/9780470719824.ch3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/10/2023]
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36
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Netherton C, Moffat K, Brooks E, Wileman T. A guide to viral inclusions, membrane rearrangements, factories, and viroplasm produced during virus replication. Adv Virus Res 2007; 70:101-82. [PMID: 17765705 PMCID: PMC7112299 DOI: 10.1016/s0065-3527(07)70004-0] [Citation(s) in RCA: 171] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
Virus replication can cause extensive rearrangement of host cell cytoskeletal and membrane compartments leading to the “cytopathic effect” that has been the hallmark of virus infection in tissue culture for many years. Recent studies are beginning to redefine these signs of viral infection in terms of specific effects of viruses on cellular processes. In this chapter, these concepts have been illustrated by describing the replication sites produced by many different viruses. In many cases, the cellular rearrangements caused during virus infection lead to the construction of sophisticated platforms in the cell that concentrate replicase proteins, virus genomes, and host proteins required for replication, and thereby increase the efficiency of replication. Interestingly, these same structures, called virus factories, virus inclusions, or virosomes, can recruit host components that are associated with cellular defences against infection and cell stress. It is possible that cellular defence pathways can be subverted by viruses to generate sites of replication. The recruitment of cellular membranes and cytoskeleton to generate virus replication sites can also benefit viruses in other ways. Disruption of cellular membranes can, for example, slow the transport of immunomodulatory proteins to the surface of infected cells and protect against innate and acquired immune responses, and rearrangements to cytoskeleton can facilitate virus release.
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Affiliation(s)
- Christopher Netherton
- Vaccinology Group, Pirbright Laboratories, Institute for Animal Health, Surrey, United Kingdom
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37
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Hyun JK, Coulibaly F, Turner AP, Baker EN, Mercer AA, Mitra AK. The structure of a putative scaffolding protein of immature poxvirus particles as determined by electron microscopy suggests similarity with capsid proteins of large icosahedral DNA viruses. J Virol 2007; 81:11075-83. [PMID: 17670837 PMCID: PMC2045580 DOI: 10.1128/jvi.00594-07] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Orf virus, the prototype parapoxvirus, is responsible for contagious ecthyma in sheep and goats. The central region of the viral genome codes for proteins highly conserved among vertebrate poxviruses and which are frequently essential for viral proliferation. Analysis of the recently published genome sequence of orf virus revealed that among such essential proteins, the protein orfv075 is an orthologue of D13, the rifampin resistance gene product critical for vaccinia virus morphogenesis. Previous studies showed that D13, arranged as "spicules," is necessary for the formation of vaccinia virus immature virions, a mandatory intermediate in viral maturation. We have determined the three-dimensional structure of recombinant orfv075 at approximately 25-A resolution by electron microscopy of two-dimensional crystals. orfv075 organizes as trimers with a tripod-like main body and a propeller-like smaller domain. The molecular envelope of orfv075 shows unexpectedly good agreement to that of a distant homologue, VP54, the major capsid protein of Paramecium bursaria Chlorella virus type 1. Our structural analysis suggests that orfv075 belongs in the double-barreled capsid protein family found in many double-stranded DNA icosahedral viruses and supports the hypothesis that the nonicosahedral poxviruses and the large icosahedral DNA viruses are evolutionarily related.
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Affiliation(s)
- Jae-Kyung Hyun
- School of Biological Sciences, University of Auckland, Thomas Building, 3A Symonds Street, Auckland, New Zealand
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38
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Charity JC, Katz E, Moss B. Amino acid substitutions at multiple sites within the vaccinia virus D13 scaffold protein confer resistance to rifampicin. Virology 2006; 359:227-32. [PMID: 17055024 PMCID: PMC1817899 DOI: 10.1016/j.virol.2006.09.031] [Citation(s) in RCA: 27] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2006] [Revised: 09/19/2006] [Accepted: 09/20/2006] [Indexed: 11/20/2022]
Abstract
D13 protein trimers, which form an external lattice providing curvature to the membrane of vaccinia virus immature virions, are the target of the drug rifampicin. We obtained 63 rifampicin-resistant mutants following random PCR mutagenesis of the D13L gene and 5 that arose spontaneously. Sequencing indicated that 26 mutants contained a single, unique, amino acid substitution whereas others contained 2 or more. The single mutations, including 6 previously identified, mapped to 24 different amino acids that were distributed over the length of the protein with the majority clustered between amino acids 17 to 33, 222 to 243 and 480 to 488. Two or three different substitutions occurred in six of the 24 amino acids. Representative mutant viruses of each cluster replicated to wild-type levels in the absence of rifampicin and nearly two logs higher than wild-type in the presence of drug. The large number and fitness of the mutations are remarkable in view of the extreme sequence conservation (99-100% amino acid identity amongst all orthopoxviruses). Clustering of mutations could suggest the presence of a rifampicin-binding pocket comprised of discontinuous regions of D13.
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39
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Abstract
Poxviruses comprise a large family of viruses characterized by a large, linear dsDNA genome, a cytoplasmic site of replication and a complex virion morphology. The most notorious member of the poxvirus family is variola, the causative agent of smallpox. The laboratory prototype virus used for the study of poxviruses is vaccinia, the virus that was used as a live, naturally attenuated vaccine for the eradication of smallpox. Both the morphogenesis and structure of poxvirus virions are unique among viruses. Poxvirus virions apparently lack any of the symmetry features common to other viruses such as helical or icosahedral capsids or nucleocapsids. Instead poxvirus virions appear as "brick shaped" or "ovoid" membrane-bound particles with a complex internal structure featuring a walled, biconcave core flanked by "lateral bodies." The virion assembly pathway involves a remarkable fabrication of membrane-containing crescents and immature virions, which evolve into mature virions in a process that is unparalleled in virology. As a result of significant advances in poxvirus genetics and molecular biology during the past 15 years, we can now positively identify over 70 specific gene products contained in poxvirus virions, and we can describe the effects of mutations in over 50 specific genes on poxvirus assembly. This review summarizes these advances and attempts to assemble them into a comprehensible and thoughtful picture of poxvirus structure and assembly.
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Affiliation(s)
- Richard C Condit
- Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, 32610, USA
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40
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Hsiao JC, Chao CC, Young MJ, Chang YT, Cho EC, Chang W. A poxvirus host range protein, CP77, binds to a cellular protein, HMG20A, and regulates its dissociation from the vaccinia virus genome in CHO-K1 cells. J Virol 2006; 80:7714-28. [PMID: 16840350 PMCID: PMC1563734 DOI: 10.1128/jvi.00207-06] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Vaccinia virus does not grow in Chinese hamster ovary (CHO-K1) cells in the absence of a viral host range factor, cowpox protein CP77. In this study, CP77 was fused to the C terminus of green fluorescence protein (GFP-CP77) and a series of nested deletion mutants of GFP-CP77 was constructed for insertion into a vaccinia virus host range mutant, VV-hr, and expressed from a viral early promoter. Deletion mapping analyses demonstrated that the N-terminal 352 amino acids of CP77 were sufficient to support vaccinia virus growth in CHO-K1 cells, whereas the C-terminal residues 353 to 668 were dispensable. In yeast two-hybrid analyses, CP77 bound to a cellular protein, HMG20A, and GST pulldown analyses showed that residues 1 to 234 of CP77 were sufficient for this interaction. After VV-hr virus infection of CHO-K1 cells, HMG20A was translocated from the nucleus to viral factories and bound to the viral genome via the HMG box region. In control VV-hr-infected CHO-K1 cells, binding of HMG20A to the viral genome persisted from 2 to 8 h postinfection (h p.i.); in contrast, when CP77 was expressed, the association of HMG20A with viral genome was transient, with little HMG20A remaining bound at 8 h p.i. This indicates that dissociation of HMG20A from viral factories correlates well with CP77 host range activity in CHO-K1 cells. Finally, in cells expressing a CP77 deletion protein (amino acids 277 to 668) or a DeltaANK5 mutant that did not support vaccinia virus growth and did not contain the HMG20A binding site, HMG20A remained bound to viral DNA, demonstrating that the binding of CP77 to HMG20A is essential for its host range function. In summary, our data revealed that a novel cellular protein, HMG20A, the dissociation of which from viral DNA is regulated by CP77, providing the first cellular target regulated by viral host range CP77 protein.
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Affiliation(s)
- Jye-Chian Hsiao
- Graduate Institute of Life Sciences, National Defense Medical Center, Nankang, Taipei, Taiwan
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41
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Lin G, Murphy SL, Gaulton GN, Hoxie JA. Modification of a viral envelope glycoprotein cell-cell fusion assay by utilizing plasmid encoded bacteriophage RNA polymerase. J Virol Methods 2005; 128:135-42. [PMID: 15941597 DOI: 10.1016/j.jviromet.2005.04.011] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2005] [Accepted: 04/28/2005] [Indexed: 11/23/2022]
Abstract
Many viruses enter cells via an interaction of the viral envelope glycoprotein (Env) with receptor inducing fusion of viral and cellular membranes. These interactions are often evaluated in cell-cell fusion, gene-reporting systems with effector cells expressing Env and target cells expressing receptors. A common system utilizes vaccinia virus encoding T7 RNA polymerase (RNAP) in effector cells and a T7 promoted reporter plasmid in target cells. Fusion is quantified with expression of the reporter plasmid. However, direct activation of reporter plasmid from vaccinia virus can occur increasing background activity. We report here a modification of this assay in which T7 RNAP is expressed from a plasmid rather than vaccinia. This modification increased sensitivity with a ten-fold reduction in background. A novel dual T7/SP6 RNAP fusion assay was also developed to allow rapid screening for functional Env clones. Using these assays, we show that Envs from two CD4-independent HIV-2 isolates (VCP and ROD/B), which are able to fuse with chemokine receptor CXCR4 in a CD4-independent manner, are also able to fuse with alternative coreceptors GPR1 and GPR15 in the absence of CD4. The assay could also detect fusion of murine leukemia virus on target cells expressing the ecotropic MCAT-1 receptor showing its broad utility in other viral systems.
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Affiliation(s)
- George Lin
- Department of Medicine, Hematology-Oncology Division, University of Pennsylvania, Philadelphia, PA 19104, USA.
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42
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Szajner P, Weisberg AS, Lebowitz J, Heuser J, Moss B. External scaffold of spherical immature poxvirus particles is made of protein trimers, forming a honeycomb lattice. ACTA ACUST UNITED AC 2005; 170:971-81. [PMID: 16144903 PMCID: PMC2171439 DOI: 10.1083/jcb.200504026] [Citation(s) in RCA: 82] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022]
Abstract
During morphogenesis, poxviruses undergo a remarkable transition from spherical immature forms to brick-shaped infectious particles lacking helical or icosahedral symmetry. In this study, we show that the transitory honeycomb lattice coating the lipoprotein membrane of immature vaccinia virus particles is formed from trimers of a 62-kD protein encoded by the viral D13L gene. Deep-etch electron microscopy demonstrated that anti-D13 antibodies bound to the external protein coat and that lattice fragments were in affinity-purified D13 preparations. Soluble D13 appeared mostly trimeric by gel electrophoresis and ultracentrifugation, which is consistent with structural requirements for a honeycomb. In the presence or absence of other virion proteins, a mutated D13 with one amino acid substitution formed stacks of membrane-unassociated flat sheets that closely resembled the curved honeycombs of immature virions except for the absence of pentagonal facets. A homologous domain that is present in D13 and capsid proteins of certain other lipid-containing viruses support the idea that the developmental stages of poxviruses reflect their evolution from an icosahedral ancestor.
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Affiliation(s)
- Patricia Szajner
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health, Bethesda, MD 20892, USA
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43
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Mercer J, Traktman P. Genetic and cell biological characterization of the vaccinia virus A30 and G7 phosphoproteins. J Virol 2005; 79:7146-61. [PMID: 15890954 PMCID: PMC1112092 DOI: 10.1128/jvi.79.11.7146-7161.2005] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
The vaccinia virus proteins A30 and G7 are known to play essential roles in early morphogenesis, acting prior to the formation of immature virions. Their repression or inactivation results in the accumulation of large virosomes, detached membrane crescents, and empty immature virions. We have undertaken further study of these proteins to place them within the context of the F10 kinase, the A14 membrane protein, and the H5 phosphoprotein, which have been the focus of previous studies within our laboratory. Here we confirm that both A30 and G7 undergo F10 kinase-dependent phosphorylation in vivo and recapitulate that modification of A30 in vitro. Although the detached crescents observed upon loss of A30 or G7 echo those seen upon repression of A14, no interaction between A30/G7 and A14 could be detected. We did, however, determine that the A30 and G7 proteins are unstable during nonpermissive tsH5 infections, suggesting that the loss of A30/G7 is the underlying cause for the formation of lacy or curdled virosomes. We also determined that the temperature-sensitive phenotype of the Cts11 virus is due to mutations in two codons of the G7L gene. Phenotypic analysis of nonpermissive Cts11 infections indicated that these amino acid substitutions compromise G7 function without impairing the stability of either G7 or A30. Utilizing Cts11 in conjunction with a rifampin release assay, we determined that G7 acts at multiple stages of virion morphogenesis that can be distinguished both by ultrastructural analysis and by monitoring the phosphorylation status of several viral proteins that undergo F10-mediated phosphorylation.
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Affiliation(s)
- Jason Mercer
- Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
| | - Paula Traktman
- Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
- Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Rd., BSB-273, Milwaukee, WI 53226. Phone: (414) 456-8253. Fax: (414) 456-6535. E-mail:
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44
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Resch W, Weisberg AS, Moss B. Vaccinia virus nonstructural protein encoded by the A11R gene is required for formation of the virion membrane. J Virol 2005; 79:6598-609. [PMID: 15890898 PMCID: PMC1112135 DOI: 10.1128/jvi.79.11.6598-6609.2005] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
The vaccinia virus A11R gene has orthologs in all known poxvirus genomes, and the A11 protein has been previously reported to interact with the putative DNA packaging protein A32 in a yeast two-hybrid screen. Using antisera raised against A11 peptides, we show that the A11 protein was (i) expressed at late times with an apparent mass of 40 kDa, (ii) not incorporated into virus particles, (iii) phosphorylated independently of the viral F10 kinase, (iv) coimmunoprecipitated with A32, and (v) localized to the viral factory. To determine the role of the A11 protein and test whether it is indeed involved in DNA packaging, we constructed a recombinant vaccinia virus with an inducible A11R gene. This recombinant was dependent on inducer for single-cycle growth and plaque formation. In the absence of inducer, viral late proteins were produced at normal levels, but proteolytic processing and other posttranslational modifications of some proteins were inhibited, suggesting a block in virus particle assembly. Consistent with this observation, electron microscopy of cells infected in the absence of inducer showed virus factories with abnormal electron-dense viroplasms and intermediate density regions associated with membranes and containing the D13 protein. However, no viral membrane crescents, immature virions, or mature virions were produced. The requirement for nonvirion protein A11 in order to make normal viral membranes was an unexpected and exciting finding, since neither the origin of these membranes nor their mechanism of formation in the cytoplasm of infected cells is understood.
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Affiliation(s)
- Wolfgang Resch
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-0445, USA
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45
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Heuser J. Deep-etch EM reveals that the early poxvirus envelope is a single membrane bilayer stabilized by a geodetic "honeycomb" surface coat. J Cell Biol 2005; 169:269-83. [PMID: 15851517 PMCID: PMC2171873 DOI: 10.1083/jcb.200412169] [Citation(s) in RCA: 93] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2004] [Accepted: 03/15/2005] [Indexed: 02/07/2023] Open
Abstract
Three-dimensional "deep-etch" electron microscopy (DEEM) resolves a longstanding controversy concerning poxvirus morphogenesis. By avoiding fixative-induced membrane distortions that confounded earlier studies, DEEM shows that the primary poxvirus envelope is a single membrane bilayer coated on its external surface by a continuous honeycomb lattice. Freeze fracture of quick-frozen poxvirus-infected cells further shows that there is only one fracture plane through this primary envelope, confirming that it consists of a single lipid bilayer. DEEM also illustrates that the honeycomb coating on this envelope is completely replaced by a different paracrystalline coat as the poxvirus matures. Correlative thin section images of infected cells freeze substituted after quick-freezing, plus DEEM imaging of Tokuyasu-type cryo-thin sections of infected cells (a new application introduced here) all indicate that the honeycomb network on immature poxvirus virions is sufficiently continuous and organized, and tightly associated with the envelope throughout development, to explain how its single lipid bilayer could remain stable in the cytoplasm even before it closes into a complete sphere.
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Affiliation(s)
- John Heuser
- Department of Cell Biology, Washington University School of Medicine, St. Louis, MO 63110, USA.
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Weli SC, Nilssen O, Traavik T. Avipoxvirus multiplication in a mammalian cell line. Virus Res 2005; 109:39-49. [PMID: 15826911 DOI: 10.1016/j.virusres.2004.10.009] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2004] [Revised: 10/07/2004] [Accepted: 10/07/2004] [Indexed: 11/16/2022]
Abstract
Avipoxviruses have many advantages and are being increasingly employed as recombinant vaccine vectors. One attractive feature is that while inserted transgenes are expressed in immunologically favourable ways, avipoxvirus infections of mammalian cells are believed to be abortive. The experimental evidence supporting this belief is, however, based on a limited number of mammalian cell-types and a few avipoxvirus species. We evaluated two avian and eight mammalian cell lines for permissivity to three avipoxvirus strains, one reference fowlpoxvirus and two newly isolated strains from sparrow and pigeon, respectively. Both avian cell lines were, as expected, permissive for all three avipoxvirus strains. However, by multiplication assays, we found to our surprise that Syrian baby hamster kidney (BHK-21) cells were equally permissive to all virus strains. Results from electron microscopy of infected BHK-21 cells revealed viral morphogenesis proceeding to various forms of infectious viruses. These results were supported by the demonstration of avipoxvirus specific late gene expression and avipoxvirus specific DNA restriction pattern in BHK-21 infected cells.
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Affiliation(s)
- Simon Chioma Weli
- Department of Microbiology and Virology, University of Tromsø, Brevika, N-9037 Tromsø, Norway.
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Szajner P, Jaffe H, Weisberg AS, Moss B. A complex of seven vaccinia virus proteins conserved in all chordopoxviruses is required for the association of membranes and viroplasm to form immature virions. Virology 2005; 330:447-59. [PMID: 15567438 DOI: 10.1016/j.virol.2004.10.008] [Citation(s) in RCA: 33] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2004] [Revised: 09/28/2004] [Accepted: 10/05/2004] [Indexed: 10/26/2022]
Abstract
Early events in vaccinia virus (VAC) morphogenesis, particularly the formation of viral membranes and their association with viroplasm, are poorly understood. Recently, we showed that repression of A30 or G7 expression results in the accumulation of normal viral membranes that form empty-looking immature virions (IV), which are separated from large masses of electron-dense viroplasm. In addition, A30 and G7 physically and functionally interact with each other and with the F10 protein kinase. To identify other proteins involved in early morphogenesis, proteins from cells that had been infected with vaccinia virus expressing an epitope-tagged copy of F10 were purified by immunoaffinity chromatography and analyzed by gel electrophoresis. In addition to F10, A30, and G7, viral proteins A15, D2, D3, and J1 were identified by mass spectrometry of tryptic peptides. Further evidence for the complex was obtained by immunopurification of proteins associated with epitope-tagged A15, D2, and D3. The previously unstudied A15, like other proteins in the complex, was expressed late in infection, associated with virus cores, and required for the stability and kinase activity of F10. Biochemical and electron microscopic analyses indicated that mutants in which A15 or D2 expression was regulated by the Escherichia coli lac operator system exhibited phenotypes characterized by the presence of large numbers of empty immature virions, similar to the results obtained with inducible A30 and G7 mutants. Empty immature virions were also seen by electron microscopy of cells infected with temperature-sensitive mutants of D2 or D3, though the numbers of membrane forms were reduced perhaps due to additional effects of high temperature.
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Affiliation(s)
- Patricia Szajner
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, USA
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Byrd CM, Bolken TC, Mjalli AM, Arimilli MN, Andrews RC, Rothlein R, Andrea T, Rao M, Owens KL, Hruby DE. New class of orthopoxvirus antiviral drugs that block viral maturation. J Virol 2004; 78:12147-56. [PMID: 15507601 PMCID: PMC525040 DOI: 10.1128/jvi.78.22.12147-12156.2004] [Citation(s) in RCA: 38] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2004] [Accepted: 07/09/2004] [Indexed: 11/20/2022] Open
Abstract
By using a homology-based bioinformatics approach, a structural model of the vaccinia virus (VV) I7L proteinase was developed. A unique chemical library of approximately 51,000 compounds was computationally queried to identify potential active site inhibitors. The resulting biased subset of compounds was assayed for both toxicity and the ability to inhibit the growth of VV in tissue culture cells. A family of chemotypically related compounds was found which exhibits selective activity against orthopoxviruses, inhibiting VV with 50% inhibitory concentrations of 3 to 12 microM. These compounds exhibited no significant cytotoxicity in the four cell lines tested and did not inhibit the growth of other organisms such as Saccharomyces cerevisiae, Pseudomonas aeruginosa, adenovirus, or encephalomyocarditis virus. Phenotypic analyses of virus-infected cells were conducted in the presence of active compounds to verify that the correct biochemical step (I7L-mediated core protein processing) was being inhibited. Electron microscopy of compound-treated VV-infected cells indicated a block in morphogenesis. Compound-resistant viruses were generated and resistance was mapped to the I7L open reading frame. Transient expression with the mutant I7L gene rescued the ability of wild-type virus to replicate in the presence of compound, indicating that this is the only gene necessary for resistance. This novel class of inhibitors has potential for development as an efficient antiviral drug against pathogenic orthopoxviruses, including smallpox.
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Affiliation(s)
- Chelsea M Byrd
- Department of Microbiology, 220 Nash Hall, Oregon State University, Corvallis, OR 97331, USA
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Unger B, Traktman P. Vaccinia virus morphogenesis: a13 phosphoprotein is required for assembly of mature virions. J Virol 2004; 78:8885-901. [PMID: 15280497 PMCID: PMC479082 DOI: 10.1128/jvi.78.16.8885-8901.2004] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
The 70-amino-acid A13L protein is a component of the vaccinia virus membrane. We demonstrate here that the protein is expressed at late times of infection, undergoes phosphorylation at a serine residue(s), and becomes encapsidated in a monomeric form. Phosphorylation is dependent on Ser40, which lies within the proline-rich motif SPPP. Because phosphorylation of the A13 protein is only minimally affected by disruption of the viral F10 kinase or H1 phosphatase, a cellular kinase is likely to be involved. We generated an inducible recombinant in which A13 protein expression is dependent upon the inclusion of tetracycline in the culture medium. Repression of the A13L protein spares the biochemical progression of the viral life cycle but arrests virion morphogenesis. Virion assembly progresses through the formation of immature virions (IVs); however, these virions do not acquire nucleoids, and DNA crystalloids accumulate in the cytoplasm. Further development into intracellular mature virions is blocked, causing a 1,000-fold decrease in the infectious virus yield relative to that obtained in the presence of the inducer. We also determined that the temperature-sensitive phenotype of the viral mutant Cts40 is due to a nucleotide transition within the A13L gene that causes a Thr(48)-->Ile substitution. This substitution disrupts the function of the A13 protein but does not cause thermolability of the protein; at the nonpermissive temperature, virion morphogenesis arrests at the stage of IV formation. The A13L protein, therefore, is part of a newly recognized group of membrane proteins that are dispensable for the early biogenesis of the virion membrane but are essential for virion maturation.
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Affiliation(s)
- Bethany Unger
- Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, 8701 Watertown Plank Rd., BSB-273, Milwaukee, WI 53226, USA
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Ansarah-Sobrinho C, Moss B. Role of the I7 protein in proteolytic processing of vaccinia virus membrane and core components. J Virol 2004; 78:6335-43. [PMID: 15163727 PMCID: PMC416539 DOI: 10.1128/jvi.78.12.6335-6343.2004] [Citation(s) in RCA: 65] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Certain core and membrane proteins of vaccinia virus undergo proteolytic cleavage at consensus AG/X sites. The processing of core proteins is coupled to morphogenesis and is inhibited by the drug rifampin, whereas processing of the A17 membrane protein occurs at an earlier stage of assembly and is unaffected by the drug. A temperature-sensitive mutant with a lesion in the I7L gene exhibits blocks in morphogenesis and in cleavage of core proteins. We found that the mutant also failed to cleave the A17 membrane protein. To further investigate the role of the putative I7 protease, we constructed a conditional lethal mutant in which the I7L gene was regulated by the Escherichia coli lac repressor. In the absence of an inducer, the synthesis of I7 was repressed, proteolytic processing of the A17 membrane protein and the L4 core protein was inhibited, and virus morphogenesis was blocked. Under these conditions, expression of the wild-type I7 protein in trans restored protein processing. In contrast, rescue did not occur when the putative protease active site residue histidine 241 or cysteine 328 of I7 was converted to alanine. The mutation of an authentic AG/A and an alternative AG/S motif of L4 prevented substrate cleavage. Similarly, when AG/X sites of A17 were mutated, I7-induced cleavages at the N and C termini failed to occur. In conclusion, we provide evidence that I7 is a viral protease that is required for AG/X-specific cleavages of viral membrane and core proteins, which occur at early and late stages of virus assembly, respectively.
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Affiliation(s)
- Camilo Ansarah-Sobrinho
- Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 4 Center Drive, MSC 0445, Bethesda, MD 20892-0445, USA
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