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Luczo JM, Spackman E. Molecular Evolution of the H5 and H7 Highly Pathogenic Avian Influenza Virus Haemagglutinin Cleavage Site Motif. Rev Med Virol 2025; 35:e70012. [PMID: 39730318 DOI: 10.1002/rmv.70012] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2024] [Revised: 11/19/2024] [Accepted: 11/21/2024] [Indexed: 12/29/2024]
Abstract
Avian influenza viruses are ubiquitous in the Anatinae subfamily of aquatic birds and occasionally spill over to poultry. Infection with low pathogenicity avian influenza viruses generally leads to subclinical or mild clinical disease. In contrast, highly pathogenic avian influenza viruses emerge from low pathogenic forms and can cause severe disease associated with extraordinarily high mortality rates. Here, we describe the natural history of avian influenza virus, with a focus on H5Nx and H7Nx subtypes, and the emergence of highly pathogenic forms; we review the biology of AIV; we examine cleavage of haemagglutinin by host cell enzymes with a particular emphasis on the biochemical properties of the proprotein convertases, and trypsin and trypsin-like proteases; we describe mechanisms implicated in the functional evolution of the haemagglutinin cleavage site motif that leads to emergence of HPAIVs; and finally, we discuss the diversity of H5 and H7 haemagglutinin cleavage site sequence motifs. It is crucial to understand the molecular attributes that drive the emergence and evolution of HPAIVs with pandemic potential to inform risk assessments and mitigate the threat of HPAIVs to poultry and human populations.
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Affiliation(s)
- Jasmina M Luczo
- Australian Animal Health Laboratory, Australian Centre for Disease Preparedness, Commonwealth Scientific and Industrial Research Organisation, East Geelong, Australia
- United States Department of Agriculture, Exotic & Emerging Avian Viral Diseases Research, Southeast Poultry Research Laboratory, United States National Poultry Research Center, Agricultural Research Service, Athens, Georgia, USA
| | - Erica Spackman
- United States Department of Agriculture, Exotic & Emerging Avian Viral Diseases Research, Southeast Poultry Research Laboratory, United States National Poultry Research Center, Agricultural Research Service, Athens, Georgia, USA
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2
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Gamblin SJ, Vachieri SG, Xiong X, Zhang J, Martin SR, Skehel JJ. Hemagglutinin Structure and Activities. Cold Spring Harb Perspect Med 2021; 11:a038638. [PMID: 32513673 PMCID: PMC8485738 DOI: 10.1101/cshperspect.a038638] [Citation(s) in RCA: 37] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
Hemagglutinins (HAs) are the receptor-binding and membrane fusion glycoproteins of influenza viruses. They recognize sialic acid-containing, cell-surface glycoconjugates as receptors but have limited affinity for them, and, as a consequence, virus attachment to cells requires their interaction with several virus HAs. Receptor-bound virus is transferred into endosomes where membrane fusion by HAs is activated at pH between 5 and 6.5, depending on the strain of virus. Fusion activity requires extensive rearrangements in HA conformation that include extrusion of a buried "fusion peptide" to connect with the endosomal membrane, form a bridge to the virus membrane, and eventually bring both membranes close together. In this review, we give an overview of the structures of the 16 genetically and antigenically distinct subtypes of influenza A HA in relation to these two functions in virus replication and in relation to recognition of HA by antibodies that neutralize infection.
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Affiliation(s)
- Steven J Gamblin
- Structural Biology of Disease Processes Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom
| | - Sébastien G Vachieri
- Structural Biology of Disease Processes Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom
| | - Xiaoli Xiong
- Structural Biology of Disease Processes Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom
| | - Jie Zhang
- Structural Biology of Disease Processes Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom
| | - Stephen R Martin
- Structural Biology of Disease Processes Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom
| | - John J Skehel
- Structural Biology of Disease Processes Laboratory, The Francis Crick Institute, London NW1 1AT, United Kingdom
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Abstract
In 1918, a strain of influenza A virus caused a human pandemic resulting in the deaths of 50 million people. A century later, with the advent of sequencing technology and corresponding phylogenetic methods, we know much more about the origins, evolution and epidemiology of influenza epidemics. Here we review the history of avian influenza viruses through the lens of their genetic makeup: from their relationship to human pandemic viruses, starting with the 1918 H1N1 strain, through to the highly pathogenic epidemics in birds and zoonoses up to 2018. We describe the genesis of novel influenza A virus strains by reassortment and evolution in wild and domestic bird populations, as well as the role of wild bird migration in their long-range spread. The emergence of highly pathogenic avian influenza viruses, and the zoonotic incursions of avian H5 and H7 viruses into humans over the last couple of decades are also described. The threat of a new avian influenza virus causing a human pandemic is still present today, although control in domestic avian populations can minimize the risk to human health. This article is part of the theme issue ‘Modelling infectious disease outbreaks in humans, animals and plants: approaches and important themes’. This issue is linked with the subsequent theme issue ‘Modelling infectious disease outbreaks in humans, animals and plants: epidemic forecasting and control’.
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Affiliation(s)
| | | | - Paul Digard
- The Roslin Institute, University of Edinburgh , Edinburgh , UK
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4
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Braun E, Sauter D. Furin-mediated protein processing in infectious diseases and cancer. Clin Transl Immunology 2019; 8:e1073. [PMID: 31406574 PMCID: PMC6682551 DOI: 10.1002/cti2.1073] [Citation(s) in RCA: 217] [Impact Index Per Article: 36.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2019] [Revised: 07/10/2019] [Accepted: 07/13/2019] [Indexed: 12/17/2022] Open
Abstract
Proteolytic cleavage regulates numerous processes in health and disease. One key player is the ubiquitously expressed serine protease furin, which cleaves a plethora of proteins at polybasic recognition motifs. Mammalian substrates of furin include cytokines, hormones, growth factors and receptors. Thus, it is not surprising that aberrant furin activity is associated with a variety of disorders including cancer. Furthermore, the enzymatic activity of furin is exploited by numerous viral and bacterial pathogens, thereby enhancing their virulence and spread. In this review, we describe the physiological and pathophysiological substrates of furin and discuss how dysregulation of a simple proteolytic cleavage event may promote infectious diseases and cancer. One major focus is the role of furin in viral glycoprotein maturation and pathogenicity. We also outline cellular mechanisms regulating the expression and activation of furin and summarise current approaches that target this protease for therapeutic intervention.
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Affiliation(s)
- Elisabeth Braun
- Institute of Molecular VirologyUlm University Medical CenterUlmGermany
| | - Daniel Sauter
- Institute of Molecular VirologyUlm University Medical CenterUlmGermany
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Böttcher-Friebertshäuser E, Garten W, Klenk HD. Characterization of Proprotein Convertases and Their Involvement in Virus Propagation. ACTIVATION OF VIRUSES BY HOST PROTEASES 2018. [PMCID: PMC7122180 DOI: 10.1007/978-3-319-75474-1_9] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Affiliation(s)
| | - Wolfgang Garten
- Institut für Virologie, Philipps Universität, Marburg, Germany
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6
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Luczo JM, Stambas J, Durr PA, Michalski WP, Bingham J. Molecular pathogenesis of H5 highly pathogenic avian influenza: the role of the haemagglutinin cleavage site motif. Rev Med Virol 2015; 25:406-30. [PMID: 26467906 PMCID: PMC5057330 DOI: 10.1002/rmv.1846] [Citation(s) in RCA: 50] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/10/2014] [Revised: 06/09/2015] [Accepted: 06/11/2015] [Indexed: 11/22/2022]
Abstract
The emergence of H5N1 highly pathogenic avian influenza has caused a heavy socio‐economic burden through culling of poultry to minimise human and livestock infection. Although human infections with H5N1 have to date been limited, concerns for the pandemic potential of this zoonotic virus have been greatly intensified following experimental evidence of aerosol transmission of H5N1 viruses in a mammalian infection model. In this review, we discuss the dominance of the haemagglutinin cleavage site motif as a pathogenicity determinant, the host‐pathogen molecular interactions driving cleavage activation, reverse genetics manipulations and identification of residues key to haemagglutinin cleavage site functionality and the mechanisms of cell and tissue damage during H5N1 infection. We specifically focus on the disease in chickens, as it is in this species that high pathogenicity frequently evolves and from which transmission to the human population occurs. With >75% of emerging infectious diseases being of zoonotic origin, it is necessary to understand pathogenesis in the primary host to explain spillover events into the human population. © 2015 The Authors. Reviews in Medical Virology published by John Wiley & Sons Ltd.
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Affiliation(s)
- Jasmina M Luczo
- Australian Animal Health Laboratory, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Geelong, Victoria, Australia.,School of Medicine, Deakin University, Geelong, Victoria, Australia
| | - John Stambas
- School of Medicine, Deakin University, Geelong, Victoria, Australia
| | - Peter A Durr
- Australian Animal Health Laboratory, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Geelong, Victoria, Australia
| | - Wojtek P Michalski
- Australian Animal Health Laboratory, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Geelong, Victoria, Australia
| | - John Bingham
- Australian Animal Health Laboratory, Commonwealth Scientific and Industrial Research Organisation (CSIRO), Geelong, Victoria, Australia
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7
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Barbosa EGV, Aburjaile FF, Ramos RTJ, Carneiro AR, Le Loir Y, Baumbach J, Miyoshi A, Silva A, Azevedo V. Value of a newly sequenced bacterial genome. World J Biol Chem 2014; 5:161-168. [PMID: 24921006 PMCID: PMC4050110 DOI: 10.4331/wjbc.v5.i2.161] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/11/2013] [Accepted: 04/03/2014] [Indexed: 02/05/2023] Open
Abstract
Next-generation sequencing (NGS) technologies have made high-throughput sequencing available to medium- and small-size laboratories, culminating in a tidal wave of genomic information. The quantity of sequenced bacterial genomes has not only brought excitement to the field of genomics but also heightened expectations that NGS would boost antibacterial discovery and vaccine development. Although many possible drug and vaccine targets have been discovered, the success rate of genome-based analysis has remained below expectations. Furthermore, NGS has had consequences for genome quality, resulting in an exponential increase in draft (partial data) genome deposits in public databases. If no further interests are expressed for a particular bacterial genome, it is more likely that the sequencing of its genome will be limited to a draft stage, and the painstaking tasks of completing the sequencing of its genome and annotation will not be undertaken. It is important to know what is lost when we settle for a draft genome and to determine the “scientific value” of a newly sequenced genome. This review addresses the expected impact of newly sequenced genomes on antibacterial discovery and vaccinology. Also, it discusses the factors that could be leading to the increase in the number of draft deposits and the consequent loss of relevant biological information.
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Ohnishi SI. Chapter 9 Fusion of Viral Envelopes with Cellular Membranes. CURRENT TOPICS IN MEMBRANES AND TRANSPORT 2008; 32:257-296. [PMID: 32287479 PMCID: PMC7146812 DOI: 10.1016/s0070-2161(08)60137-9] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 02/12/2023]
Abstract
This chapter reviews some characteristic features of membrane fusion activity for each virus and discusses the mechanisms of membrane fusion, especially low pH-induced membrane fusion. It concentrates on the interaction of the hydrophobic segment with the target cell membrane lipid bilayer and suggests the entrance of the segment into the lipid bilayer hydrophobic core as a key step in fusion. The envelope is a lipid bilayer membrane with the virus specific glycoproteins spanning it. The bilayer originates from the host cell membrane and has a lipid composition and transbilayer distribution quite similar to the host's. The viral glycoproteins have the functions of binding to the target cell surface and fusion with the cell membranes. The two functions are carried by a single glycoprotein in influenza virus (HA), vesicular stomatitis virus (VSV) G glycoprotein, and Semliki Forest virus SFV E glycoprotein. In Sendai virus (HVJ), the functions are carried by separate glycoproteins, hemagglutinin-neuraminidase (HN) for binding and fusion glycoprotein (F) for fusion. When viruses encounter target cells, they first bind to the cell surface through an interaction of the viral glycoprotein with receptors.
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Affiliation(s)
- Shun-Ichi Ohnishi
- Department of Biophysics Facurlty of Science Kyoto University Sakyo-ku. Kyoto 606, Japan
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9
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Rodriguez-Boulan E, Misek DE, Salas DVD, Salas PJI, Bard E. Chapter 6 Protein Sorting in the Secretory Pathway. CURRENT TOPICS IN MEMBRANES AND TRANSPORT 2008; 24:251-294. [PMID: 32287478 PMCID: PMC7146842 DOI: 10.1016/s0070-2161(08)60328-7] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
Abstract
This chapter focuses on protein sorting in the secretory pathway. From primary and secondary biosynthetic sites in the cytosol and mitochondrial matrix, respectively, proteins and lipids are distributed to more than 30 final destinations in membranes or membrane-bound spaces, where they carry out their programmed function. Molecular sorting is defined, in its most general sense, as the sum of the mechanisms that determine the distribution of a given molecule from its site of synthesis to its site of function in the cell. The final site of residence of a protein in a eukaryotic cell is determined by a combination of various factors, acting in concert: (1) site of synthesis, (2) sorting signals or zip codes, (3) signal recognition or decoding mechanisms, (4) cotranslational or posttranslational mechanisms for translocation across membranes, (5) specific fusion-fission interactions between intracellular vesicular compartments, and (6) restrictions to the lateral mobility in the plane of the bilayer. Improvements in cell fractionation, protein separation, and immune precipitation procedures in the past decade have made them possible. Very little is known about the mechanisms that mediate the localization and concentration of specific proteins and lipids within organelles. Various experimental model systems have become available for their study. The advent of recombinant DNA technology has shortened the time needed for obtaining the primary structure of proteins to a few months.
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Affiliation(s)
| | - David E Misek
- Department of Pathology, State University of New York, Downstate Medical Center, Brooklyn, New York
| | - Dora Vega De Salas
- Department of Cell Biology and Anatomy, Cornell University Medical College, New York, New York
| | - Pedro J I Salas
- Department of Cell Biology and Anatomy, Cornell University Medical College, New York, New York
| | - Enzo Bard
- Department of Pathology, State University of New York, Downstate Medical Center, Brooklyn, New York
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Chapter 3 Antiviral drugs: general considerations. PERSPECTIVES IN MEDICAL VIROLOGY 2008; 1:93-126. [PMID: 32287578 PMCID: PMC7133937 DOI: 10.1016/s0168-7069(08)70011-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 03/29/2024]
Abstract
The development of an antiviral drug as well as of other drugs is a long process. In most programmes the screening and evaluation start using inhibition of virus multiplication in cell cultures, but in some instances the screening starts in animal models of different viral diseases. In these cases, the mechanism of action has to be analyzed after the in vivo effect has been found. It is not possible to specify precisely the time and resources required in a newly started project to find a compound active against a virus infection but 5-10 years is a reasonable estimation. For some viruses such as herpesviruses, where a number of active inhibitors are already known, the task is simpler than it is to find inhibitors of a virus such as influenza against which only a few active inhibitors have been reported. Evaluation of clinical efficacy in humans is a large and difficult part of the development of an antiviral drug. The number of uncontrolled clinical studies claiming efficacy of different drugs against viral diseases is depressingly large. It is essential to perform double-blind, placebo-controlled and statistically well evaluated studies to be able to judge the clinical efficacy of an antiviral drug. As the knowledge of the detailed natural history and molecular biology of viral diseases and viruses themselves increases, one will obviously have better opportunities to find new drugs. Methods such as X-ray diffraction measurement and NMR determinations will probably lead to a detailed understanding of the structures and interactions taking place at the active site of viral enzymes and their cellular counterparts.
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11
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Chapter 7 Orthomyxovirus infections. PERSPECTIVES IN MEDICAL VIROLOGY 2008; 1:255-343. [PMID: 32287580 PMCID: PMC7134264 DOI: 10.1016/s0168-7069(08)70015-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 11/23/2022]
Abstract
The earth is a unity for influenza A virus in a manner not yet found for probably any other parasite and epidemics occur in all inhabited parts of the globe regardless of latitude, longitude, altitude, climate, rainfall, temperature, humidity, race and sex. Influenza A is the classic pandemic virus infection of man and influenza B virus also can cause sharp outbreaks, resulting in significant mortality. An overwhelming amount of data has accumulated on the biochemistry, cell biology, and epidemiology of influenza, but prospects of control of epidemics in the near future are dim. Meanwhile, a holding operation can be achieved using inactivated vaccine and rimantadine (100 mg/daily) in special risk groups in the population until new more effective vaccines and broad spectrum antivirals (active against influenza A and B virus) are developed. Research work is centered on biotechnology to produce immunogenic peptides and proteins and more logical searches for antivirals using amino acid sequence data and also virus specific enzymes such as the virion transcriptase as targets.
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Reina J. [Factors affecting the virulence and pathogenicity of avian and human viral strains (influenza virus type A)]. Enferm Infecc Microbiol Clin 2002; 20:346-53. [PMID: 12237002 DOI: 10.1016/s0213-005x(02)72814-0] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022]
Abstract
Most studies performed in avian viral strains seem to indicate that virulence is a polygenic phenomenon. However, hemagglutinin and neuraminidase and the genes codifying these substances (genes 4 and 6) play an essential role in viral pathogenesis. Avian strains can be classified as avirulent or virulent according to the ability of hemagglutinin to be activated by endoproteases of the respiratory tract only or by proteases from other tissues. This ability is based on the progressive development of mutations that lead to the substitution of the normal amino acids at the point of hemagglutinin hydrolysis by the other basic amino acids that determine the amplification of the spectrum of hydrolysis and activation. Neuraminidase participates in the acquisition of virulence through its capacity to bind to plasminogen and by increasing the concentration of activating proteases. Adaptation to the host, through recognition of the cell receptor, is another factor determining the virulence and interspecies transmission of avian strains. From an epidemiological point of view, viral strains should be subtyped and the activating capacity of hemagglutinin should be determined to identify their degree of virulence.
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Affiliation(s)
- Jordi Reina
- Unidad de Virología, Servicio de Microbiología Clínica, Hospital Universitario Son Dureta, Palma de Mallorca, España.
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Abstract
Although human epidemics of influenza occur on nearly an annual basis and result in a significant number of "excess deaths," the viruses responsible are not generally considered highly pathogenic. On occasion, however, an outbreak occurs that demonstrates the potential lethality of influenza viruses. The human pandemic of 1918 spread worldwide and killed millions, and the limited human outbreak of highly pathogenic avian viruses in Hong Kong at the end of 1997 is a warning that this could happen again. In avian species such as chickens and turkeys, several outbreaks of highly pathogenic influenza viruses have been documented. Although the reason for the lethality of the human 1918 viruses remains unclear, the pathogenicity of the avian viruses, including those that caused the human 1997 outbreak, relates primarily to properties of the hemagglutinin glycoprotein (HA). Cleavage of the HA precursor molecule HA0 is required to activate virus infectivity, and the distribution of activating proteases in the host is one of the determinants of tropism and, as such, pathogenicity. The HAs of mammalian and nonpathogenic avian viruses are cleaved extracellularly, which limits their spread in hosts to tissues where the appropriate proteases are encountered. On the other hand, the HAs of pathogenic viruses are cleaved intracellularly by ubiquitously occurring proteases and therefore have the capacity to infect various cell types and cause systemic infections. The x-ray crystal structure of HA0 has been solved recently and shows that the cleavage site forms a loop that extends from the surface of the molecule, and it is the composition and structure of the cleavage loop region that dictate the range of proteases that can potentially activate infectivity. Here influenza virus pathogenicity is discussed, with an emphasis on the role of HA0 cleavage as a determining factor.
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Affiliation(s)
- D A Steinhauer
- National Institute for Medical Research, The Ridgeway, London, Mill Hill, NW7 1AA, United Kingdom.
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Wood GW, Banks J, McCauley JW, Alexander DJ. Deduced amino acid sequences of the haemagglutinin of H5N1 avian influenza virus isolates from an outbreak in turkeys in Norfolk, England. Arch Virol 1994; 134:185-94. [PMID: 7506519 DOI: 10.1007/bf01379117] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023]
Abstract
The deduced amino acid sequences of the haemagglutinins of avian influenza viruses, isolated from an outbreak in turkeys in Norfolk, England in 1991/92, were determined by PCR amplification and cycle sequencing. Both the highly pathogenic and avirulent isolates had the same cleavage site sequence with multiple-basic amino acids, which normally would be expected only for the former. Clones derived by plaque picking from the highly pathogenic isolate ranged from low to very high pathogenicity in vivo and these, and the original isolates, showed nucleotide and amino acid variation at one or more of five possible sites, none of which were at the cleavage site. None of these site variations correlated with pathogenicity, suggesting that the factor responsible for the suppression of the expected effects of the multiple-basic amino acid haemagglutinin cleavage site in the avirulent isolate may not have been part of the haemagglutinin amino acid sequence.
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Affiliation(s)
- G W Wood
- Central Veterinary Laboratory, Addlestone, Surrey, U.K
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Abstract
The majority of viral glycoproteins that undergo post-translational proteolysis are cleaved by ubiquitous intracellular proteases; however, a minority are cleaved by secreted proteases available only in a few host systems. The interplay of viral glycoproteins and cellular proteases may have a pivotal role in the spread of infection, host range and pathogenicity.
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Affiliation(s)
- H D Klenk
- Institut für Virologie, Philipps-Universität Marburg, Germany
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17
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Ponnuswamy PK, Gromiha MM. Prediction of transmembrane helices from hydrophobic characteristics of proteins. INTERNATIONAL JOURNAL OF PEPTIDE AND PROTEIN RESEARCH 1993; 42:326-41. [PMID: 8244628 DOI: 10.1111/j.1399-3011.1993.tb00502.x] [Citation(s) in RCA: 49] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/29/2023]
Abstract
Membrane proteins, requiring to be embedded into the lipid bilayers, have evolved to have amino acid sequences that will fold with a hydrophobic surface in contact with the alkane chains of the lipids and polar surface in contact with the aqueous phases on both sides of the membrane and the polar head groups of the lipids. It is generally assumed that the characteristics of the aqueous parts of the membrane proteins are similar to those of normal globular proteins, and the embedded parts are highly hydrophobic. In our earlier works, we introduced the concept of 'surrounding hydrophobicity' and developed a hydrophobicity scale for the 20 amino acid residues, and applied it successfully to the study of the family of globular proteins. In this work we use the concept of surrounding hydrophobicity to indicate quantitatively how the aqueous parts of membrane proteins compare with the normal globular proteins, and how rich the embedded parts are in their hydrophobic activity. We then develop a surrounding hydrophobicity scale applicable to membrane proteins, by mixing judicially the surrounding hydrophobicities observed in the crystals of the membrane protein, photosynthetic reaction center from the bacterium Rhodopseudomonas viridis, porin from Rhodobacter capsulatus and a set of 64 globular proteins. A predictive scheme based on this scale predicts from amino acid sequence, transmembrane segments in PRC and randomly selected 26 membrane proteins to 80% level of accuracy. This is a much higher predictive power when compared to the existing popular methods. A new procedure to measure the amphipathicity of sequence segments is proposed, and it is used to characterize the transmembrane parts of the sample membrane proteins.
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Affiliation(s)
- P K Ponnuswamy
- Department of Physics, Bharathidasan University, Tiruchirapalli, Tamil Nadu, India
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18
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Roberts PC, Garten W, Klenk HD. Role of conserved glycosylation sites in maturation and transport of influenza A virus hemagglutinin. J Virol 1993; 67:3048-60. [PMID: 8497042 PMCID: PMC237641 DOI: 10.1128/jvi.67.6.3048-3060.1993] [Citation(s) in RCA: 114] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023] Open
Abstract
The role of three N-linked glycans which are conserved among various hemagglutinin (HA) subtypes of influenza A viruses was investigated by eliminating the conserved glycosylation (cg) sites at asparagine residues 12 (cg1), 28 (cg2), and 478 (cg3) by site-directed mutagenesis. An additional mutant was constructed by eliminating the cg3 site and introducing a novel site 4 amino acids away, at position 482. Expression of the altered HA proteins in eukaryotic cells by a panel of recombinant vaccinia viruses revealed that rates and efficiency of intracellular transport of HA are dependent upon both the number of conserved N-linked oligosaccharides and their respective positions on the polypeptide backbone. Glycosylation at two of the three sites was sufficient for maintenance of transport of the HA protein. Conserved glycosylation at either the cg1 or cg2 site alone also promoted efficient transport of HA. However, the rates of transport of these mutants were significantly reduced compared with the wild-type protein or single-site mutants of HA. The transport of HA proteins lacking all three conserved sites or both amino-terminally located sites was temperature sensitive, implying that a polypeptide folding step had been affected. Analysis of trimer assembly by these mutants indicated that the presence of a single oligosaccharide in the stem domain of the HA molecule plays an important role in preventing aggregation of molecules in the endoplasmic reticulum, possibly by maintaining the hydrophilic properties of this domain. The conformational change observed after loss of all three conserved oligosaccharides also resulted in exposure of a normally mannose-rich oligosaccharide at the tip of the large stem helix that allowed its conversion to a complex type of structure. Evidence was also obtained suggesting that carbohydrate-carbohydrate interactions between neighboring oligosaccharides at positions 12 and 28 influence the accessibility of the cg2 oligosaccharide for processing enzymes. We also showed that terminal glycosylation of the cg3 oligosaccharide is site specific, since shifting of this site 4 amino acids away, to position 482, yielded an oligosaccharide that was arrested in the mannose-rich form. In conclusion, carbohydrates at conserved positions not only act synergistically by promoting and stabilizing a conformation compatible with transport, they also enhance trimerization and/or folding rates of the HA protein.
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Affiliation(s)
- P C Roberts
- Institut für Virologie, Philipps-Universität Marburg, Germany
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19
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Wood GW, McCauley JW, Bashiruddin JB, Alexander DJ. Deduced amino acid sequences at the haemagglutinin cleavage site of avian influenza A viruses of H5 and H7 subtypes. Arch Virol 1993; 130:209-17. [PMID: 8503786 DOI: 10.1007/bf01319010] [Citation(s) in RCA: 105] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The amino acid sequences at the haemagglutinin cleavage sites of 9 avian influenza A viruses of H5 subtype (5 high and 4 low pathogenicity for chickens) and 21 of H7 subtype (13 high and 8 low pathogenicity for chickens) were determined by direct RNA sequencing, PCR amplification sequencing or both. None of the viruses of low pathogenicity had multiple basic amino acids at the cleavage site. All highly pathogenic viruses had an insert of basic amino acids at the cleavage site, except A/chicken/Scotland/59 (H5N1) for which the multiple basic amino acids appeared as substitutions and not insertions. All highly pathogenic viruses examined conformed to the amino acid motif of R-X-R/K-R at the cleavage site which is considered to be essential for high pathogenicity in chickens, with the notable exception of highly pathogenic virus A/turkey/England/50-92/91 (H5N1) which had the sequence R-K-R-K-T-R adjacent to the cleavage site.
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Affiliation(s)
- G W Wood
- Central Veterinary Laboratory, Weybridge, Surrey, United Kingdom
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Bashiruddin JB, Gould AR, Westbury HA. Molecular pathotyping of two avian influenza viruses isolated during the Victoria 1976 outbreak. Aust Vet J 1992; 69:140-2. [PMID: 1642597 DOI: 10.1111/j.1751-0813.1992.tb07485.x] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022]
Affiliation(s)
- J B Bashiruddin
- CSIRO Division of Animal Health, Australian Animal Health Laboratory, Geelong, Victoria
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21
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Vey M, Orlich M, Adler S, Klenk HD, Rott R, Garten W. Hemagglutinin activation of pathogenic avian influenza viruses of serotype H7 requires the protease recognition motif R-X-K/R-R. Virology 1992; 188:408-13. [PMID: 1566583 PMCID: PMC7172898 DOI: 10.1016/0042-6822(92)90775-k] [Citation(s) in RCA: 108] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
The hemagglutinin of influenza virus A/FPV/Rostock/34 (H7) was altered at its multibasic cleavage site by site-directed mutagenesis and assayed for proteolytic activation after expression in CV-1 cells. The results indicated that the cellular protease responsible for activation recognizes the tetrapeptide motif R-X-K/R-R that must be presented in the correct sequence position. Studies on plaque variants of influenza virus A/fowl/Victoria/75 (H7N7) showed that alteration of the consensus sequence resulted in a loss of pathogenicity for chickens.
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Affiliation(s)
- M Vey
- Institut für Virologie, Philipps-Universität Marburg, Germany
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22
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Garten W, Will C, Buckard K, Kuroda K, Ortmann D, Munk K, Scholtissek C, Schnittler H, Drenckhahn D, Klenk HD. Structure and assembly of hemagglutinin mutants of fowl plague virus with impaired surface transport. J Virol 1992; 66:1495-505. [PMID: 1738202 PMCID: PMC240875 DOI: 10.1128/jvi.66.3.1495-1505.1992] [Citation(s) in RCA: 21] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
Five temperature-sensitive mutants of influenza virus A/FPV/Rostock/34 (H7N1), ts206, ts293, ts478, ts482, and ts651, displaying correct hemagglutinin (HA) insertion into the apical plasma membrane of MDCK cells at the permissive temperature but defective transport to the cell surface at the restrictive temperature, have been investigated. Nucleotide sequence analysis of the HA gene of the mutants and their revertants demonstrated that with each mutant a single amino acid change is responsible for the transport block. The amino acid substitutions were compared with those of mutants ts1 and ts227, which have been analyzed previously (W. Schuy, C. Will, K. Kuroda, C. Scholtissek, W. Garten, and H.-D. Klenk, EMBO J. 5:2831-2836, 1986). With the exception of ts206, the changed amino acids of all mutants and revertants accumulate in three distinct areas of the three-dimensional HA model: (i) at the tip of the 80-A (8-nm)-long alpha helix, (ii) at the connection between the globular region and stem, and (iii) in the basal domain of the stem. The concept that these areas are critical for HA assembly and hence for transport is supported by the finding that the mutants that are unable to leave the endoplasmic reticulum at the nonpermissive temperature do not correctly trimerize. Upon analysis by density gradient centrifugation, cross-linking, and digestion with trypsin and endoglucosaminidase H, two groups can be discriminated among these mutants: with ts1, ts227, and ts478, the HA forms large irreversible aggregates, whereas with ts206 and ts293, it is retained in the monomeric form in the endoplasmic reticulum. With a third group, comprising mutants ts482 and ts651 that enter the Golgi apparatus, trimerization was not impaired.
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Affiliation(s)
- W Garten
- Institut für Virologie, Philipps-Universität, Marburg, Germany
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23
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Klimov A, Prösch S, Schäfer J, Bucher D. Subtype H7 influenza viruses: comparative antigenic and molecular analysis of the HA-, M-, and NS-genes. Arch Virol 1992; 122:143-61. [PMID: 1530908 DOI: 10.1007/bf01321124] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
Antigenic analysis of the haemagglutinin and matrix protein with corresponding sets of monoclonal antibodies as well as sequence analysis of HA-, M-, and NS-genes were carried out to establish antigenic and genetic relationships between four fowl plague virus (FPV) strains of H7 subtype. The data obtained revealed close genetic relatedness between the oldest known influenza A virus, A/chicken/Brescia/1902 (H7N7), and two FPV strains, A/FPV/Dobson (H7N7) and A/FPV/Weybridge (H7N7). These three strains apparently differ in all genes investigated from the A/FPV/Rostock isolate.
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Affiliation(s)
- A Klimov
- Research Institute for Viral Preparations, Academy of Medical Sciences of the U.S.S.R., Moscow
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24
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Gibson CA, Daniels RS, Oxford JS, McCauley JW. Sequence analysis of the equine H7 influenza virus haemagglutinin gene. Virus Res 1992; 22:93-106. [PMID: 1566601 DOI: 10.1016/0168-1702(92)90037-a] [Citation(s) in RCA: 22] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
The nucleotide sequences of ten haemagglutinin genes of representative H7N7 equine influenza viruses isolated between 1956 and 1977 have been determined by primer extension sequencing. Their nucleotide and deduced amino acid sequences demonstrate a high degree of homology. These equine viruses can be divided into two distinct subgroups, the prototype-like, and a group comprising the early American isolates and the remaining equine viruses. The equine H7 haemagglutinins form a quite distinct group compared to H7 haemagglutinins isolated from other species. Each of these equine H7 haemagglutinins possess a tetrabasic amino acid cleavage site separating the HA1 and HA2 domains but, in addition, all ten contain a nine amino acid insertion prior to the tetrabasic sequence. The haemagglutinin glycoproteins of all ten viruses are capable of cleavage activation in virus infected primary chicken embryo fibroblast cells.
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Affiliation(s)
- C A Gibson
- National Institute for Biological Standards and Control, Potters Bar, U.K
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25
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Nobusawa E, Aoyama T, Kato H, Suzuki Y, Tateno Y, Nakajima K. Comparison of complete amino acid sequences and receptor-binding properties among 13 serotypes of hemagglutinins of influenza A viruses. Virology 1991; 182:475-85. [PMID: 2024485 DOI: 10.1016/0042-6822(91)90588-3] [Citation(s) in RCA: 385] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022]
Abstract
We determined the sequences of 7 serotypes (H4, H6, H8, H9, H11, H12, and H13) of hemagglutinin (HA) genes, which have not been reported so far. The coding regions consisted of 1692 nucleotides in H4, 1698 in H6, 1695 in H8, 1680 in H9, 1695 in H11, 1692 in H12, and 1698 in H13, and specified 564, 566, 565, 560, 565, 564, and 566 amino acids, respectively. By comparison of amino acid sequences, 13 HA serotypes could be divided into two families, i.e., an H1 group (H1, H2, H5, H6, H8, H9, H11, H12, and H13) and an H3 group (H3, H4, H7, and H10). The relationship was essentially similar to that reported by Air from the comparison of 80 amino-terminal amino acid sequence of 12 HA serotypes (G.M. Air, 1981, Proc. Natl. Acad. Sci. USA 78, 7639-7643). Though a considerable amino acid sequence difference exists between certain HA serotypes, several amino acid residues in fusion peptides (HA2(1-11)) and receptor-binding sites (HA1(98), -134, -138, -153, -183, and -195) were shown to be conserved among the 13 HA serotypes. Human H1 and avian H3, H4, H8, and H10 viruses preferentially bound NeuAc alpha 2,3Gal sequences, whereas human H2 and H3 and avian H6 and H9 viruses bound NeuAc alpha 2,6Gal sequences, although the amino acid residues at position 226 of human H2 and avian H6 and H9 serotype HAs are glutamine. These results show that the amino acid residue at position 226 is not necessarily a determinant of receptor specificity for all serotypes.
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Affiliation(s)
- E Nobusawa
- Department of Microbiology, Institute of Public Health, Tokyo, Japan
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26
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Veit M, Kretzschmar E, Kuroda K, Garten W, Schmidt MF, Klenk HD, Rott R. Site-specific mutagenesis identifies three cysteine residues in the cytoplasmic tail as acylation sites of influenza virus hemagglutinin. J Virol 1991; 65:2491-500. [PMID: 1901916 PMCID: PMC240604 DOI: 10.1128/jvi.65.5.2491-2500.1991] [Citation(s) in RCA: 95] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/29/2022] Open
Abstract
The hemagglutinin (HA) of influenza virus is a type I transmembrane glycoprotein which is acylated with long-chain fatty acids. In this study we have used oligonucleotide-directed mutagenesis of cloned cDNA and a simian virus 40 expression system to determine the fatty acid binding site in HA and to examine possible functions of covalently linked fatty acids. The results show that the HA is acylated through thioester linkages at three highly conserved cysteine residues located in the cytoplasmic domain and at the carboxy-terminal end of the transmembrane region, whereas a cysteine located in the middle of the membrane-spanning domain is not acylated. Mutants lacking fatty acids at individual or all three attachment sites acquire endoglycosidase H-resistant oligosaccharide side chains, are cleaved into HA1 and HA2 subunits, and are transported to the plasma membrane at rates similar to that of wild-type HA. All mutants are membrane bound and not secreted into the medium. These results exclude transport signal and membrane-anchoring functions of covalently linked fatty acids for this integral membrane glycoprotein. Furthermore, lack of acylation has no obvious influence on the biological activities of HA: cells expressing fatty acid-free HA bind to and, after brief exposure to mildly acidic pH, fuse with erythrocytes; the HA-induced polykaryon formation is not impaired, either. Other possible functions of covalently linked fatty acids in integral membrane glycoproteins which cannot be examined in conventional cDNA expression systems are discussed.
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Affiliation(s)
- M Veit
- Institut für Virologie, Philipps-Universität Marburg, Germany
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27
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Kawaoka Y, Yamnikova S, Chambers TM, Lvov DK, Webster RG. Molecular characterization of a new hemagglutinin, subtype H14, of influenza A virus. Virology 1990; 179:759-67. [PMID: 2238469 DOI: 10.1016/0042-6822(90)90143-f] [Citation(s) in RCA: 91] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022]
Abstract
Two influenza A viruses whose hemagglutinin (HA) did not react with any of the reference antisera for the 13 recognized HA subtypes were isolated from mallard ducks in the USSR. Antigenic analysis by hemagglutination inhibition and double immunodiffusion tests showed that the HAs of these viruses are similar to each other but distinct from the HAs of other influenza A viruses. Nucleotide sequence analysis showed that these HA genes differ from each other by only 21 nucleotides. However, they differ from all other HA subtypes at the amino acid level by at least 31% in HAI. Thus, we propose that the HAs of these viruses [A/Mallard/Gurjev/263/82 (H14N5) and A/Mallard/Gurjev/244/82 (H14N6) belong to a previously unrecognized subtype, and are designated H14. Unlike any other HAs of influenza viruses, the H14 HAs contained lysine at the cleavage site between HA1 and HA2 instead of arginine. Experimental infection of domestic poultry and ferrets with A/Mallard/Gurjev/263/82 (H14N5) showed that the virus is avirulent for these animals. Based on comparative sequence analysis of different HA genes, it is suggested that differences of 30% or more at the amino acid level in HA1 constitute separate subtypes. Phylogenetic analysis of representatives of each HA subtype showed that H14 is one of the most recently diverged lineages while H8 and H12 branched off early during the evolution of the HA subtypes. These latter two subtypes (H8 and H12) have been isolated very infrequently in recent years, suggesting that these old subtypes may be disappearing from the influenza reservoirs in nature.
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Affiliation(s)
- Y Kawaoka
- St. Jude Children's Research Hospital, Department of Virology and Molecular Biology, Memphis, Tennessee 38101
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28
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Characterization of a cDNA encoding a cysteine-rich cell surface protein located in the flagellar pocket of the protozoan Trypanosoma brucei. Mol Cell Biol 1990. [PMID: 1697030 DOI: 10.1128/mcb.10.9.4506] [Citation(s) in RCA: 48] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
We have characterized a cDNA encoding a cysteine-rich, acidic integral membrane protein (CRAM) of the parasitic protozoa Trypanosoma brucei and Trypanosoma equiperdum. Unlike other membrane proteins of T. brucei, which are distributed throughout the cell surface, CRAM is concentrated in the flagellar pocket, an invagination of the cell surface of the trypanosome where endocytosis has been documented. Accordingly, CRAM also locates to vesicles located underneath the pocket, providing evidence of its internalization. CRAM has a predicted molecular mass of 130 kilodaltons and has a signal peptide, a transmembrane domain, and a 41-amino-acid cytoplasmic extension. A characteristic feature of CRAM is a large extracellular domain with a roughly 66-fold acidic, cysteine-rich 12-amino-acid repeat. CRAM is conserved among different protozoan species, including Trypanosoma cruzi, and CRAM has structural similarities with eucaryotic cell surface receptors. The most striking homology of CRAM is to the human low-density-lipoprotein receptor. We propose that CRAM functions as a cell surface receptor of different trypanosome species.
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29
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Lee MG, Bihain BE, Russell DG, Deckelbaum RJ, Van der Ploeg LH. Characterization of a cDNA encoding a cysteine-rich cell surface protein located in the flagellar pocket of the protozoan Trypanosoma brucei. Mol Cell Biol 1990; 10:4506-17. [PMID: 1697030 PMCID: PMC361037 DOI: 10.1128/mcb.10.9.4506-4517.1990] [Citation(s) in RCA: 25] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/28/2022] Open
Abstract
We have characterized a cDNA encoding a cysteine-rich, acidic integral membrane protein (CRAM) of the parasitic protozoa Trypanosoma brucei and Trypanosoma equiperdum. Unlike other membrane proteins of T. brucei, which are distributed throughout the cell surface, CRAM is concentrated in the flagellar pocket, an invagination of the cell surface of the trypanosome where endocytosis has been documented. Accordingly, CRAM also locates to vesicles located underneath the pocket, providing evidence of its internalization. CRAM has a predicted molecular mass of 130 kilodaltons and has a signal peptide, a transmembrane domain, and a 41-amino-acid cytoplasmic extension. A characteristic feature of CRAM is a large extracellular domain with a roughly 66-fold acidic, cysteine-rich 12-amino-acid repeat. CRAM is conserved among different protozoan species, including Trypanosoma cruzi, and CRAM has structural similarities with eucaryotic cell surface receptors. The most striking homology of CRAM is to the human low-density-lipoprotein receptor. We propose that CRAM functions as a cell surface receptor of different trypanosome species.
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Affiliation(s)
- M G Lee
- Department of Genetics and Development, Columbia University, New York, New York 10032
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30
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Orlich M, Khátchikian D, Teigler A, Rott R. Structural variation occurring in the hemagglutinin of influenza virus A/turkey/Oregon/71 during adaptation to different cell types. Virology 1990; 176:531-8. [PMID: 2345964 DOI: 10.1016/0042-6822(90)90023-k] [Citation(s) in RCA: 32] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The influenza virus A/turkey/Oregon/71 (H7N3) has been adapted to grow in MDCK or chicken embryo cells (CEC) in the absence of trypsin. Changes occurred in the biological properties of the virus variants selected, depending on the cell type used for adaptation. They coincided with enhanced hemagglutinin (HA) activation by intracellular proteolytic cleavage. In the case of MDCK cell selected variants growth, plaque formation, and HA cleavability were restricted to this cell type, whereas the CEC-derived variants displayed altered activities in a broad range of host cells. Unlike the wild-type virus and its MDCK cell-derived variants, CEC variants had acquired pathogenic properties for chickens. By nucleotide sequence analysis of the HA genes of the MDCK cell variants several point mutations were found, which were localized predominantly at the distal, globular part of the HA molecule. The mechanism by which these point mutations increased HA cleavability has not been defined. In the CEC-derived variants besides point mutations, an insertion of 54 nucleotides adjacent to the cleavage site was observed, which corresponds in its sequence to a region in the 28 S ribosomal RNA. This insertion is probably responsible for the altered cleavability of the CEC variants' HA, leading to increased growth potential and pathogenicity.
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Affiliation(s)
- M Orlich
- Institut für Virologie, Justus-Liebig-Universität Giessen, Federal Republic of Germany
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31
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Kuroda K, Geyer H, Geyer R, Doerfler W, Klenk HD. The oligosaccharides of influenza virus hemagglutinin expressed in insect cells by a baculovirus vector. Virology 1990; 174:418-29. [PMID: 2407026 DOI: 10.1016/0042-6822(90)90095-9] [Citation(s) in RCA: 163] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
The hemagglutinin of fowl plague virus has been expressed in Spodoptera frugiperda (SF) cell cultures using a baculovirus vector. To elucidate the structure of the carbohydrate side chains, radioactively labeled oligosaccharides were liberated by treatment with endoglucosaminidase H and glycopeptidase F. Sequential degradation with exoglycosidases and chromatographic analyses revealed the presence of oligomannosidic side chains, predominantly of the structures Man5-9GlcNAc2, and the truncated oligosaccharide cores Man3GlcNAc2 and Man3[Fuc]GlcNAc2. Polyacrylamide gel electrophoresis of endoglycosidase-treated hemagglutinin showed that most side chains of the HA1 subunit are truncated, whereas the HA2 subunit has one oligomannosidic and one truncated oligosaccharide. Comparison of these results with the glycosylation pattern of hemagglutinin obtained from vertebrate cells allowed a tentative allocation of the oligosaccharides to individual glycosylation sites. The results indicate that SF cells have the capacity to trim N-glycans to trimannosyl cores and to further process these by the addition of fucose. Thus, the complex oligosaccharides found on hemagglutinin from vertebrate hosts are replaced on hemagglutinin derived from insect cells by small truncated side chains.
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Affiliation(s)
- K Kuroda
- Institut für Virologie, Philipps-Universität Marburg, Federal Republic of Germany
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32
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Deber CM, Glibowicka M, Woolley GA. Conformations of proline residues in membrane environments. Biopolymers 1990; 29:149-57. [PMID: 2328283 DOI: 10.1002/bip.360290120] [Citation(s) in RCA: 51] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Although noted as hydrophilic residues with helix-breaking potential, proline residues are observed in putatively alpha-helical transmembrane (TM) segments of many channel-forming integral membrane proteins. In addition to the recognized property of X-Pro peptide bonds (where X = any amino acid) to occur in cis as well as trans isomeric states, the tertiary amide character of the X-Pro bond confers increased propensity for involvement of its carbonyl group in specific H-bonded structures (e.g., beta- and gamma-turns) and/or liganding interactions with positively charged species. To examine this latter situation in further detail, we identified Leu-Pro-Phe as a consensus sequence triad based on actual occurrences of intramembranous Pro residues in transport protein TM segments. Accordingly, we have undertaken the synthesis of hydrophobic peptides with potential membrane affinity, of which t-butyloxycarbonyl-L-Ala-L-Ala-L-Ala-L-Leu-L-Pro-L-Phe-OH (t-Boc-AAALPF-OH) is an initial compound. Partitioning of this peptide into model membrane environments composed of lipid micelles induces specific conformation(s) for the membrane-bound hexapeptide, as monitored by 75-MHz 13C-nmr spectral behavior of 13C-enriched Leu and Pro carbonyl carbons, and by 300-MHz 1H-nmr spectra of peptide alpha, beta, and aromatic protons. Data are interpreted in terms of an intramolecularly H-bonded inverse gamma-turn conformation in the membrane environment involving the Leu-Pro-Phe triad. The inherent structural instability of a Pro-containing segment in a TM helix due to the multiplicity of possible local conformations is discussed as a functional aspect of membrane-buried prolines in transport proteins.
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Affiliation(s)
- C M Deber
- Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada
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33
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Affiliation(s)
- E Hunter
- Department of Microbiology, University of Alabama, Birmingham 35294
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34
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Abstract
Knowledge of the structure and function of the genes and proteins of the rotaviruses has expanded rapidly. Information obtained in the last 5 years has revealed unexpected and unique molecular properties of rotavirus proteins of general interest to virologists, biochemists, and cell biologists. Rotaviruses share some features of replication with reoviruses, yet antigenic and molecular properties of the outer capsid proteins, VP4 (a protein whose cleavage is required for infectivity, possibly by mediating fusion with the cell membrane) and VP7 (a glycoprotein), show more similarities with those of other viruses such as the orthomyxoviruses, paramyxoviruses, and alphaviruses. Rotavirus morphogenesis is a unique process, during which immature subviral particles bud through the membrane of the endoplasmic reticulum (ER). During this process, transiently enveloped particles form, the outer capsid proteins are assembled onto particles, and mature particles accumulate in the lumen of the ER. Two ER-specific viral glycoproteins are involved in virus maturation, and these glycoproteins have been shown to be useful models for studying protein targeting and retention in the ER and for studying mechanisms of virus budding. New ideas and approaches to understanding how each gene functions to replicate and assemble the segmented viral genome have emerged from knowledge of the primary structure of rotavirus genes and their proteins and from knowledge of the properties of domains on individual proteins. Localization of type-specific and cross-reactive neutralizing epitopes on the outer capsid proteins is becoming increasingly useful in dissecting the protective immune response, including evaluation of vaccine trials, with the practical possibility of enhancing the production of new, more effective vaccines. Finally, future analyses with recently characterized immunologic and gene probes and new animal models can be expected to provide a basic understanding of what regulates the primary interactions of these viruses with the gastrointestinal tract and the subsequent responses of infected hosts.
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35
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Bray M, Zhao BT, Markoff L, Eckels KH, Chanock RM, Lai CJ. Mice immunized with recombinant vaccinia virus expressing dengue 4 virus structural proteins with or without nonstructural protein NS1 are protected against fatal dengue virus encephalitis. J Virol 1989; 63:2853-6. [PMID: 2724416 PMCID: PMC250798 DOI: 10.1128/jvi.63.6.2853-2856.1989] [Citation(s) in RCA: 73] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
We have constructed vaccinia virus recombinants expressing dengue virus proteins from cloned DNA for use in experimental immunoprophylaxis. A recombinant virus containing a 4.0-kilobase DNA sequence that codes for three structural proteins, capsid (C), premembrane (pre-M), and envelope (E), and for nonstructural proteins NS1 and NS2a produced authentic pre-M, E, and NS1 in infected CV-1 cells. Mice immunized with this recombinant were protected against an intracerebral injection of 100 50% lethal doses of dengue 4 virus. A recombinant containing only genes C, pre-M, and E also induced solid resistance to challenge. Deletion of the putative C-terminal hydrophobic anchor of the E glycoprotein did not result in secretion of E from recombinant-virus-infected cells. Recombinants expressing only the E protein preceded by its own predicted N-terminal hydrophobic signal or by the signal of influenza A virus hemagglutinin or by the N-terminal 71 amino acids of the G glycoprotein of respiratory syncytial virus produced glycosylated E protein products of expected molecular sizes. These vaccinia virus recombinants also protected mice.
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Affiliation(s)
- M Bray
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892
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36
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Hoekstra D, Kok JW. Entry mechanisms of enveloped viruses. Implications for fusion of intracellular membranes. Biosci Rep 1989; 9:273-305. [PMID: 2673423 DOI: 10.1007/bf01114682] [Citation(s) in RCA: 77] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
Enveloped viruses infect cells by a mechanism involving membrane fusion. This process is mediated and triggered by specific viral membrane glycoproteins. Evidence is accumulating that fusion of intracellular membranes, as occurs during endocytosis and transport between intracellular organelles, also requires the presence of specific proteins. The relevance of elucidating the mechanisms of virus fusion for a better understanding of fusion of intracellular membranes is discussed.
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Affiliation(s)
- D Hoekstra
- Laboratory of Physiological Chemistry, University of Groningen, The Netherlands
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37
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Paterson RG, Shaughnessy MA, Lamb RA. Analysis of the relationship between cleavability of a paramyxovirus fusion protein and length of the connecting peptide. J Virol 1989; 63:1293-301. [PMID: 2644448 PMCID: PMC247826 DOI: 10.1128/jvi.63.3.1293-1301.1989] [Citation(s) in RCA: 65] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
The relationship between the length of the connecting peptide in a paramyxovirus F0 protein and cleavage of F0 into the F1 and F2 subunits has been examined by constructing a series of mutant F proteins via site-directed mutagenesis of a cDNA clone encoding the simian virus 5 F protein. The mutant F proteins had one to five arginine residues deleted from the connecting peptide. The minimum number of arginine residues required for cleavage-activation of the simian virus 5 F0 protein by host cell proteases was found to be four. F proteins with two or three arginine residues in the connecting peptide were not cleaved by host cell proteases but could be cleaved by exogenously added trypsin. The mutant F protein possessing a connecting peptide consisting of one arginine residue was not cleaved by trypsin. The altered F proteins were all transported to the infected-cell plasma membrane as shown by cell surface immunofluorescence or cell surface trypsinization. However, the only mutant F protein found to be biologically active as detected by syncytium formation was the F protein which has four arginine residues at the cleavage site. The results presented here suggest that in the paramyxovirus F protein the number of basic amino acid residues in the connecting peptide is important for cleavage of the precursor protein by host cell proteases but is not the only structural feature involved. In addition, the data indicate that cleavage of F0 into F1 and F2 does not necessarily result in biological activity and that the connecting peptide may affect the local conformation of the F polypeptide.
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Affiliation(s)
- R G Paterson
- Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500
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38
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de Curtis I, Simons K. Dissection of Semliki Forest virus glycoprotein delivery from the trans-Golgi network to the cell surface in permeabilized BHK cells. Proc Natl Acad Sci U S A 1988; 85:8052-6. [PMID: 3186706 PMCID: PMC282352 DOI: 10.1073/pnas.85.21.8052] [Citation(s) in RCA: 137] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
In this paper mechanically permeabilized cells have been used to dissect the transport of Semliki Forest virus glycoproteins from the trans-Golgi network to the plasma membrane. Transport from the Golgi complex was monitored by measuring the proteolytic cleavage of the Semliki Forest virus p62 glycoprotein into the E2 and E3 polypeptide chains. Cell surface appearance was measured by the exposure of the exoplasmic domain to antibodies directed against the viral glycoprotein. Both the cleavage of the p62 protein and the transport of the glycoprotein to the cell surface were reconstituted in permeabilized BHK cells when calcium and glucose were present in the medium. Detailed analysis showed that the cleavage of the p62 protein occurred before arrival to the plasma membrane.
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Affiliation(s)
- I de Curtis
- European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany
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39
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Abstract
Proteins with molecular weights of up to 500K can be enclosed in erythrocyte ghosts by exposing the ghosts to hypotonic solution containing these proteins. The proteins can then be introduced into recipient cells by fusing the ghosts with the cells using HVJ, PEG, or influenza virus. Some applications of this method are described. By an improved method, 15 kbp DNA and IgM (900 kDa) can be entrapped in erythrocyte membranes and these are then treated with liposomes containing gangliosides and HVJ. These treated membranes containing large macromolecules fuse with almost 100% of the recipient cells used. Naked liposomes infrequently fuse with cultured cells, so introduction of their contents into cells is very inefficient. However, liposomes constituted from lipid and glycoproteins (HN and F) of HVJ (Sendai virus), by removing a nonionic detergent, fuse with cells about 200 times more efficiently than naked liposomes. Naked liposomes can fuse with specific cells, such as cells infected with subacute sclerosing panencephalitis virus or with human immunodeficiency virus. Plasmid DNA and mRNA of up to about 40 kbp can be entrapped efficiently in liposomes associated with gangliosides formed by reverse-phase evaporation, and then reacted with HVJ. The contents of the resulting liposomes with HVJ can be introduced efficiently into cultured cells in a suspended or plated state, and nearly all the cells then express the gene transiently. This procedure is also effective for obtaining stable transformants of many kinds of cultured cells.
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Affiliation(s)
- T Uchida
- Institute for Molecular and Cellular Biology, Osaka University, Japan
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40
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Tsai WP, Oroszlan S. Novel glycosylation pathways of retroviral envelope proteins identified with avian reticuloendotheliosis virus. J Virol 1988; 62:3167-74. [PMID: 2841469 PMCID: PMC253434 DOI: 10.1128/jvi.62.9.3167-3174.1988] [Citation(s) in RCA: 20] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
Previously, we identified two mature glycoproteins, gp90, the surface glycoprotein, and gp20, the transmembrane protein, from avian reticuloendotheliosis virus and an avian reticuloendotheliosis virus env gene-encoded intracellular polyprotein gPr77env, but the precise relationship of gPr77env to the mature envelope proteins was not determined (W.-P. Tsai, T.D. Copeland, and S. Oroszlan, Virology 155:567-583, 1986). In the present study, using metabolic labeling of viral proteins with [35S]cysteine, radioimmunoprecipitation, and carbohydrate structure analysis, we have identified a higher-molecular-weight endo-H-resistant env gene-encoded polyprotein designated gPr115env in addition to the endo-H-sensitive gPr77env. It appears that gPr77env is the primary polyprotein precursor, modified with mannosyloligosaccharides that are processed into sialic-acid-rich extraordinarily large complex-type carbohydrates (up to 17 kilodaltons for each N-linked site) on the gp90 domain but not on the gPr22 domain. In this process, gPr77env is converted into the apparently endo-H-resistant secondary polyprotein, gPr115env, which is rapidly processed into gp90 and gPr22. The proteolytic processing which occurs only after the appearance of an endo-H resistant precursor is now clearly demonstrated for a retrovirus. Some important aspects of carbohydrate structure, including the site-specific glycosylation, as well as the intracellular location and nature of the potential enzyme involved in the proteolytic cleavage of gPr115env are discussed.
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Affiliation(s)
- W P Tsai
- Laboratory of Molecular Virology and Carcinogenesis, NCI-Frederick Cancer Research Facility, Maryland 21701
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41
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Ahne A, Müller-Derlich J, Merlos-Lange AM, Kanbay F, Wolf K, Lang BF. Two distinct mechanisms for deletion in mitochondrial DNA of Schizosaccharomyces pombe mutator strains. Slipped mispairing mediated by direct repeats and erroneous intron splicing. J Mol Biol 1988; 202:725-34. [PMID: 3172236 DOI: 10.1016/0022-2836(88)90553-0] [Citation(s) in RCA: 31] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
Mutator strains of the fission yeast Schizosaccharomyces pombe produce mitochondrial respiratory deficient mutants at a high rate, and roughly 20% of these mutants carry deletions in the range of 50 to 1500 base-pairs. To elucidate the mechanism of deletion we have sequenced ten deletion mutants in the mosaic gene encoding apocytochrome b (cob) and three in the split gene coding for the first subunit of cytochrome c oxidase (cox1). Of 13 deletions, ten are correlated with the presence of direct repeats, which could promote deletions by slipped mispairing during DNA replication. In some of these mutants, the termini are located in possible DNA secondary structures. In three independently isolated mutants with identical deletions in the cob gene, the 5' deletion endpoint coincides with the 3' splice point of the intron, whereas the 3' endpoint of the deletion exhibits pronounced homology with the 5' splice point of the intron. This result suggests that these deletions might be initiated by erroneous RNA splicing.
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Affiliation(s)
- A Ahne
- Institut für Genetik und Mikrobiologie, Universität München, FRG
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42
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Sutou S, Sato S, Okabe T, Nakai M, Sasaki N. Cloning and sequencing of genes encoding structural proteins of avian infectious bronchitis virus. Virology 1988; 165:589-95. [PMID: 2841803 DOI: 10.1016/0042-6822(88)90603-4] [Citation(s) in RCA: 53] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
Affiliation(s)
- S Sutou
- NRI Life Science, Kanagawa, Japan
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43
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Feldmann H, Kretzschmar E, Klingeborn B, Rott R, Klenk HD, Garten W. The structure of serotype H10 hemagglutinin of influenza A virus: comparison of an apathogenic avian and a mammalian strain pathogenic for mink. Virology 1988; 165:428-37. [PMID: 3407149 DOI: 10.1016/0042-6822(88)90586-7] [Citation(s) in RCA: 35] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
The primary structure of the hemagglutinin of the apathogenic avian influenza virus A/chick/Germany/N/49 (H10N7) and of the serologically related strain A/mink/Sweden/84 (H10N4) pathogenic for mink has been elucidated by nucleotide sequence analysis, and the carbohydrates attached to the polypeptide have been determined. The H10 hemagglutinin has 65, 52, 46, 45, and 44% amino acid sequence homology with serotypes H7, H3, H1, H2, and H5, respectively. H10 and H7 hemagglutinins are also most closely related in their glycosylation patterns. There is a high sequence homology between both H10 strains supporting the concept that the mink virus has obtained its hemagglutinin from an avian strain. The sequence homology includes the cleavage site which consists of a single arginine as is the case with most other hemagglutinins exhibiting low susceptibility to proteolytic activation. The similarity in hemagglutinin structure between both H10 strains is discussed in light of the distinct differences in the pathogenicity of both viruses.
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Affiliation(s)
- H Feldmann
- Institut für Virologie, Philipps-Universität, Marburg, Germany
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44
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Brandl CJ, Deber RB, Hsu LC, Woolley GA, Young XK, Deber CM. Evidence for similar function of transmembrane segments in receptor and membrane-anchored proteins. Biopolymers 1988; 27:1171-82. [PMID: 2850033 DOI: 10.1002/bip.360270710] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023]
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45
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Abstract
Integral membrane proteins are characterized by long apolar segments that cross the lipid bilayer. Polar domains flanking these apolar segments have a more balanced amino acid composition, typical for soluble proteins. We show that the apolar segments from three different kinds of membrane-assembly signals do not differ significantly in amino acid content, but that the inside/outside location of the polar domains correlates strongly with their content of arginyl and lysyl residues, not only for bacterial inner-membrane proteins, but also for eukaryotic.proteins from the endoplasmic reticulum, the plasma membrane, the inner mitochondrial membrane, and the chloroplast thylakoid membrane. A positive-inside rule thus seems to apply universally to all integral membrane proteins, with apolar regions targeting for membrane integration and charged residues providing the topological information.
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Affiliation(s)
- G von Heijne
- Department of Molecular Biology, Karolinska Institutet, Huddinge, Sweden
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46
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Hull JD, Gilmore R, Lamb RA. Integration of a small integral membrane protein, M2, of influenza virus into the endoplasmic reticulum: analysis of the internal signal-anchor domain of a protein with an ectoplasmic NH2 terminus. J Cell Biol 1988; 106:1489-98. [PMID: 2836432 PMCID: PMC2115032 DOI: 10.1083/jcb.106.5.1489] [Citation(s) in RCA: 59] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
The M2 protein of influenza A virus is a small integral membrane protein of 97 residues that is expressed on the surface of virus-infected cells. M2 has an unusual structure as it lacks a cleavable signal sequence yet contains an ectoplasmic amino-terminal domain of 23 residues, a 19 residue hydrophobic transmembrane spanning segment, and a cytoplasmic carboxyl-terminal domain of 55 residues. Oligonucleotide-mediated deletion mutagenesis was used to construct a series of M2 mutants lacking portions of the hydrophobic segment. Membrane integration of the M2 protein was examined by in vitro translation of synthetic mRNA transcripts prepared using bacteriophage T7 RNA polymerase. After membrane integration, M2 was resistant to alkaline extraction and was converted to an Mr approximately equal to 7,000 membrane-protected fragment after digestion with trypsin. In vitro integration of M2 requires the cotranslational presence of the signal recognition particle. Deletion of as few as two residues from the hydrophobic segment of M2 markedly decreases the efficiency of membrane integration, whereas deletion of six residues completely eliminates integration. M2 proteins containing deletions that eliminate stable membrane anchoring are apparently not recognized by signal recognition particles, as these polypeptides remain sensitive to protease digestion, indicating that in addition they do not have a functional signal sequence. These data thus indicate that the signal sequence that initiates membrane integration of M2 resides within the transmembrane spanning segment of the polypeptide.
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Affiliation(s)
- J D Hull
- Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208
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47
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Sklyanskaya EI, Shie M, Komarov YS, Yamnikova SS, Kaverin NV. Formation of mixed hemagglutinin trimers in the course of double infection with influenza viruses belonging to different subtypes. Virus Res 1988; 10:153-65. [PMID: 3414181 DOI: 10.1016/0168-1702(88)90012-3] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Abstract
Chick embryo primary cultured cells were infected with influenza viruses belonging to H1, H2, H3, H5 or H7 subtypes of hemagglutinin. The cells were subjected to a single or a double infection, labelled with 14C-amino acids from 2 to 6 hours postinfection, lysed with a mixture of ionic and non-ionic detergents, and the lysates were clarified by low-speed centrifugation. The clarified lysates contained 14C-labelled hemagglutinin mostly in the form of 9S trimers, as shown by velocity sedimentation in sucrose gradients with polyacrylamide gel electrophoresis (PAGE) analysis of the gradient fractions. The lysates were immunoprecipitated with antihemagglutinin antibodies specific for one of the co-infecting viruses. The immunoprecipitates were analysed by PAGE. Cells infected separately with each virus and mixed before lysis were used as a control sample in every experiment. In the lysates of cells doubly infected with H2 and H5 influenza viruses the analysis revealed the presence of structures containing HA monomers of both viruses, whereas no such structures were revealed in the lysate of a mixture of separately infected cells. Mixed structures (most likely HA trimers containing monomers of the two co-infecting viruses) were also found in the lysates of cells doubly infected with strains belonging to H1 and H2 subtypes. No such structures were revealed when the cells were co-infected with viruses belonging to H1 and H3 subtypes or H3 and H7 subtypes. The results suggest an extensive formation of mixed HA trimers in the course of double infection with viruses belonging to closely related subtypes, whereas the formation of mixed trimers by more distantly related HA monomers does not occur or is very scarce. The identity of the mixed structures as HA trimers was confirmed by immunoprecipitation experiments with 9S structures.
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Affiliation(s)
- E I Sklyanskaya
- D.I. Ivanovsky Institute of Virology, Academy of Medical Sciences, Moscow, U.S.S.R
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48
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Sithanandam G, Rapp UR. A single point mutation in the envelope gene is responsible for replication and XC fusion deficiency of the endogenous ecotropic C3H/He murine leukemia virus and for its repair in culture. J Virol 1988; 62:932-43. [PMID: 2828688 PMCID: PMC253652 DOI: 10.1128/jvi.62.3.932-943.1988] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
The molecular basis has been determined for differences in infectivity and XC phenotype of endogenous ecotropic murine leukemia virus of the low-leukemia mouse strain C3H/He, its relative in the high-leukemia mouse strain AKR, and highly infectious, XC-positive C3H virus variants selected in vitro. Endogenous ecotropic type C virus induced by iododeoxyuridine from the nontransformed C3H/10T1/2 cell line is XC negative and replication deficient. In contrast, viruses produced late after iododeoxyuridine induction in chemically transformed C3H/10T1/2 cells (MCA5) are XC positive and infectious. XC-negative viruses can be converted to XC-positive viruses by being grown in certain transformed cell lines. We have cloned the endogenous ecotropic provirus of C3H/He from MCA5 cells, which is XC negative and replication deficient, as well as two XC-positive C3H proviruses derived by in vitro conversion. Fragment exchange between the XC-negative molecular clone p110 and the XC-positive AKR virus clone p623 revealed that the defect in p110 lies 3' of the SalI site located in the pol region. Nucleotide sequencing established that the C3H p110 provirus was integrated within the R region of an endogenous VL30 long terminal repeat (LTR) in reverse orientation and that the virus differed from the infectious AKR p623 provirus by a point mutation, substituting Lys for Arg at the potential precursor cleavage site for gp70 and p15E. In vitro-converted XC-positive C3H proviral clones 3211 and 4211 are identical to XC-negative C3H p110, except that they have Arg at this site and the normal cleavage site is thus regenerated in these clones. The XC-negative C3H p110 was blocked in processing of Pr85env, whereas clones 3211 and 4211 had normal cleavage of the env precursor into gp70. Both the XC-negative C3H provirus and the in vitro-converted XC-positive C3H proviruses had a single copy of a 99-base-pair enhancer element in the LTR, whereas two copies of this sequence are present in the AKR proviral LTR. Substitution of Arg for Lys at the envelope precursor processing site of C3H p110 by site-directed mutagenesis is sufficient by itself to convert the virus to the XC-positive replication-competent phenotype. Thus, we have established that a single point mutation at the processing site of the envelope precursor protein Pr85 is responsible for the difference in the infectivity and XC phenotype of endogenous ecotropic murine leukemia virus from C3H/He and AKR mice and that the basis for in vitro conversion is a mutation at this site.
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Affiliation(s)
- G Sithanandam
- Laboratory of Viral Carcinogenesis, National Cancer Institute, Frederick, Maryland 21701
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49
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Abstract
It is an accepted concept that the pathogenicity of a virus is of polygenic nature. Because of their segmented genome, influenza viruses provide a suitable system to prove this concept. The studies employing virus mutants and reassortants have indicated that the pathogenicity depends on the functional integrity of each gene and on a gene constellation optimal for the infection of a given host. As a consequence, virtually every gene product of influenza virus has been reported to contribute to pathogenicity, but evidence is steadily growing that a key role has to be assigned to hemagglutinin. As the initiator of infection, hemagglutinin has a double function: (1) promotion of adsorption of the virus to the cell surface, and (2) penetration of the viral genome through a fusion process among viral and cellular membranes. Adsorption is based on the binding to neuraminic acid-containing receptors, and different virus strains display a distinct preference for specific oligosaccharides. Fusion capacity depends on proteolytic cleavage by host proteases, and variations in amino acid sequence at the cleavage site determine whether hemagglutinin is activated in a given cell. Differences in cleavability and presumably also in receptor specificity are important determinants for host tropism, spread of infection, and pathogenicity. The concept that proteolytic activation is a determinant for pathogenicity was originally derived from studies on avian influenza viruses, but there is now evidence that it may also be relevant for the disease in humans because bacterial proteases have been found to promote the development of influenza pneumonia in mammals.
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Affiliation(s)
- H D Klenk
- Institut für Virologie, Philipps-Universität Marburg, Federal Republic of Germany
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50
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Nestorowicz A, Kawaoka Y, Bean WJ, Webster RG. Molecular analysis of the hemagglutinin genes of Australian H7N7 influenza viruses: role of passerine birds in maintenance or transmission? Virology 1987; 160:411-8. [PMID: 3660587 DOI: 10.1016/0042-6822(87)90012-2] [Citation(s) in RCA: 66] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
In 1985 a fowl plague-like disease occurred in chickens in Lockwood, Victoria, Australia and caused high mortality. An H7N7 influenza virus was isolated from the chickens (A/Chicken/Victoria/1/85); additionally, an antigenically similar virus was isolated from starlings (A/Starling/Victoria/5156/85) and serological evidence of H7N7 virus infection was found in sparrows. Antigenic analysis with monoclonal antibodies to H7, oligonucleotide mapping of total vRNA, and sequence analysis of the HA genes established that the chicken and starling influenza viruses were closely related and probably came from the same source. There was high nucleotide sequence homology (95.3%) between the HA genes of A/Chick/Vic/85 and a fowl plague-like virus isolated from chickens in Victoria 9 years earlier [A/Fowl/Vic/76 (H7N7)]. The sequence homologies indicated that the A/Chick/Vic/85 and A/Fowl/Vic/76 were derived from a common recent ancestor, while another recent H7N7 virus, Seal/Mass/1/80 originated from a different evolutionary lineage. Experimental infection of chickens and starlings with A/Chick/Vic/1/85 (H7N7) was associated with high mortality (100%), transmission to contact birds of the same species, and virus in all organs. In sparrows one-third of the birds died after infection and virus was isolated from most organs; transmission to contact sparrows did not occur. In contrast, the H7N7 virus replicated in ducks and spread to contact ducks but caused no mortality. These studies establish that the host species plays a role in determining the virulence of avian influenza viruses, and provide the first evidence for transmission of virulent influenza viruses between domestic poultry and passerine birds. They support the hypothesis that potentially virulent H7N7 influenza viruses could be maintained in ducks where they cause no apparent disease and may sometimes spread to other wild birds and domestic poultry.
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Affiliation(s)
- A Nestorowicz
- Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101
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