scholarly journals African Swine Fever Virus Is Wrapped by the Endoplasmic Reticulum

1998 ◽  
Vol 72 (3) ◽  
pp. 2373-2387 ◽  
Author(s):  
Isabelle Rouiller ◽  
Sharon M. Brookes ◽  
Alex D. Hyatt ◽  
Miriam Windsor ◽  
Thomas Wileman
Keyword(s):  
Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Virus Assembly ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Viral Envelope ◽  
Immunogold Electron Microscopy ◽  
Icosahedral Symmetry ◽  
Structural Protein ◽  
Intracellular Membrane

ABSTRACT African swine fever (ASF) virus is a large DNA virus that shares the striking icosahedral symmetry of iridoviruses and the genomic organization of poxviruses. Both groups of viruses have a complex envelope structure. In this study, the mechanism of formation of the inner envelope of ASF virus was investigated. Examination of thin cryosections by electron microscopy showed two internal membranes in mature intracellular virions and all structural intermediates. These membranes were in continuity with intracellular membrane compartments, suggesting that the virus gained two membranes from intracellular membrane cisternae. Immunogold electron microscopy showed the viral structural protein p17 and resident membrane proteins of the endoplasmic reticulum (ER) within virus assembly sites, virus assembly intermediates, and mature virions. Resident ER proteins were also detected by Western blotting of isolated virions. The data suggested the ASF virus was wrapped by the ER. Analysis of the published sequence of ASF virus (R. J. Yanez et al., Virology 208:249–278, 1995) revealed a reading frame, XP124L, that encoded a protein predicted to translocate into the lumen of the ER. Pulse-chase immunoprecipitation and glycosylation analysis of pXP124L, the product of the XP124L gene, showed that pXP124L was retained in the ER lumen after synthesis. When analyzed by immunogold electron microscopy, pXP124L localized to virus assembly intermediates and fully assembled virions. Western blot analysis detected pXP124L in virions isolated from Percoll gradients. The packaging of pXP124L from the lumen of the ER into the virion is consistent with ASF virus being wrapped by ER cisternae: a mechanism which explains the presence of two membranes in the viral envelope.

scholarly journals Generation of Filamentous Instead of Icosahedral Particles by Repression of African Swine Fever Virus Structural Protein pB438L

2006 ◽  
Vol 80 (23) ◽  
pp. 11456-11466 ◽  
Author(s):  
Carolina Epifano ◽  
Jacomine Krijnse-Locker ◽  
María L. Salas ◽  
José Salas ◽  
Javier M. Rodríguez
Keyword(s):  
Virus Assembly ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Integral Membrane Protein ◽  
Fever Virus ◽  
Icosahedral Symmetry ◽  
Structural Protein ◽  
Viral Origin ◽  
Virus Particles ◽  
Capsid Formation

ABSTRACT The mechanisms involved in the construction of the icosahedral capsid of the African swine fever virus (ASFV) particle are not well understood at present. Capsid formation requires protein p72, the major capsid component, but other viral proteins are likely to play also a role in this process. We have examined the function of the ASFV structural protein pB438L, encoded by gene B438L, in virus morphogenesis. We show that protein pB438L associates with membranes during the infection, behaving as an integral membrane protein. Using a recombinant ASFV that inducibly expresses protein pB438L, we have determined that this structural protein is essential for the formation of infectious virus particles. In the absence of the protein, the virus assembly sites contain, instead of icosahedral particles, large aberrant tubular structures of viral origin as well as bilobulate forms that present morphological similarities with the tubules. The filamentous particles, which possess an aberrant core shell domain and an inner envelope, are covered by a capsid-like layer that, although containing the major capsid protein p72, does not acquire icosahedral morphology. This capsid, however, is to some extent functional, as the filamentous particles can move from the virus assembly sites to the plasma membrane and exit the cell by budding. The finding that, in the absence of protein pB438L, the viral particles formed have a tubular structure in which the icosahedral symmetry is lost supports a role for this protein in the construction or stabilization of the icosahedral vertices of the virus particle.

scholarly journals African Swine Fever Virus pB119L Protein Is a Flavin Adenine Dinucleotide-Linked Sulfhydryl Oxidase

2006 ◽  
Vol 80 (7) ◽  
pp. 3157-3166 ◽  
Author(s):  
Irene Rodríguez ◽  
Modesto Redrejo-Rodríguez ◽  
Javier M. Rodríguez ◽  
Alí Alejo ◽  
José Salas ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Flavin Adenine Dinucleotide ◽  
Virus Assembly ◽  
Nonstructural Protein ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Structural Protein ◽  
Sulfhydryl Oxidase ◽  
Infected Cells

ABSTRACT Protein pB119L of African swine fever virus belongs to the Erv1p/Alrp family of sulfhydryl oxidases and has been described as a late nonstructural protein required for correct virus assembly. To further our knowledge of the function of protein pB119L during the virus life cycle, we have investigated whether this protein possesses sulfhydryl oxidase activity, using a purified recombinant protein. We show that the purified protein contains bound flavin adenine dinucleotide and is capable of catalyzing the formation of disulfide bonds both in a protein substrate and in the small molecule dithiothreitol, the catalytic activity being comparable to that of the Erv1p protein. Furthermore, protein pB119L contains the cysteines of its active-site motif CXXC, predominantly in an oxidized state, and forms noncovalently bound dimers in infected cells. We also show in coimmunoprecipitation experiments that protein pB119L interacts with the viral protein pA151R, which contains a CXXC motif similar to that present in thioredoxins. Protein pA151R, in turn, was found to interact with the viral structural protein pE248R, which contains disulfide bridges and belongs to a class of myristoylated proteins related to vaccinia virus L1R, one of the substrates of the redox pathway encoded by this virus. These results suggest the existence in African swine fever virus of a system for the formation of disulfide bonds constituted at least by proteins pB119L and pA151R and identify protein pE248R as a possible final substrate of this pathway.

scholarly journals Comparison of the genome sequences of non-pathogenic and pathogenic African swine fever virus isolates

2008 ◽  
Vol 89 (2) ◽  
pp. 397-408 ◽  
Author(s):  
David A. G. Chapman ◽  
Vasily Tcherepanov ◽  
Chris Upton ◽  
Linda K. Dixon
Keyword(s):  
Virus Assembly ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Open Reading Frames ◽  
Structural Protein ◽  
Genome Sequences ◽  
Virus Genotype ◽  
Pathogenic Isolate ◽  
Virus Isolates

The genomic coding sequences, apart from the inverted terminal repeats and cross-links, have been determined for two African swine fever virus (ASFV) isolates from the same virus genotype, a non-pathogenic isolate from Portugal, OURT88/3, and a highly pathogenic isolate from West Africa, Benin 97/1. These genome sequences were annotated and compared with that of a tissue culture-adapted isolate, BA71V. The genomes range in length between 170 and 182 kbp and encode between 151 and 157 open reading frames (ORFs). Compared to the Benin 97/1 isolate, the OURT88/3 and BA71V isolates have deletions of 8–10 kbp that encode six copies of the multigene family (MGF) 360 and either one MGF 505/530 copy in the BA71V or two copies in the OURT88/3 isolate. The BA71V isolate has a deletion, close to the right end of the genome, of 3 kbp compared with the other isolates. The five ORFs in this region include an additional copy of an ORF similar to that encoding the p22 virus structural protein. The OURT88/3 isolate has interruptions in ORFs that encode a CD2-like and a C-type lectin protein. Variation between the genomes is observed in the number of copies of five different MGFs. The 109 non-duplicated ORFs conserved in the three genomes encode proteins involved in virus replication, virus assembly and modulation of the host's defences. These results provide information concerning the genetic variability of African swine fever virus isolates that differ in pathogenicity.

scholarly journals African Swine Fever Virus Protein pE199L Mediates Virus Entry by Enabling Membrane Fusion and Core Penetration

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Tania Matamoros ◽  
Alí Alejo ◽  
Javier María Rodríguez ◽  
Bruno Hernáez ◽  
Milagros Guerra ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Host Cell ◽  
Virus Assembly ◽  
African Swine Fever Virus ◽  
Virus Entry ◽  
African Swine Fever ◽  
Viral Envelope ◽  
Fever Virus ◽  
Virus Protein ◽  
Hemorrhagic Disease

ABSTRACT African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) causing a lethal hemorrhagic disease that currently threatens the global pig industry. Despite its relevance in the infectious cycle, very little is known about the internalization of ASFV in the host cell. Here, we report the characterization of ASFV protein pE199L, a cysteine-rich structural polypeptide with similarity to proteins A16, G9, and J5 of the entry fusion complex (EFC) of poxviruses. Using biochemical and immunomicroscopic approaches, we found that, like the corresponding poxviral proteins, pE199L localizes to the inner viral envelope and behaves as an integral transmembrane polypeptide with cytosolic intramolecular disulfide bonds. Using an ASFV recombinant that inducibly expresses the E199L gene, we found that protein pE199L is not required for virus assembly and egress or for virus-cell binding and endocytosis but is required for membrane fusion and core penetration. Interestingly, similar results have been previously reported for ASFV protein pE248R, an inner membrane virion component related to the poxviral L1 and F9 EFC proteins. Taken together, these findings indicate that ASFV entry relies on a form of fusion machinery comprising proteins pE248R and pE199L that displays some similarities to the unconventional fusion apparatus of poxviruses. Also, these results provide novel targets for the development of strategies that block the first stages of ASFV replication. IMPORTANCE African swine fever virus (ASFV) causes a highly lethal swine disease that is currently present in many countries of Eastern Europe, the Russian Federation, and Southeast Asia, severely affecting the pig industry. Despite extensive research, effective vaccines or antiviral strategies are still lacking and relevant gaps in knowledge of the fundamental biology of the viral infection cycle exist. In this study, we identified pE199L, a protein of the inner viral membrane that is required for virus entry. More specifically, pE199L is necessary for the fusion event that leads to the penetration of the genome-containing core in the host cell. Our results significantly increase our knowledge of the process of internalization of African swine fever virus, which may instruct future research on antiviral strategies.

scholarly journals The Major Structural Protein of African Swine Fever Virus, p73, Is Packaged into Large Structures, Indicative of Viral Capsid or Matrix Precursors, on the Endoplasmic Reticulum

1998 ◽  
Vol 72 (6) ◽  
pp. 5215-5223 ◽  
Author(s):  
Christian Cobbold ◽  
Thomas Wileman
Keyword(s):  
Endoplasmic Reticulum ◽  
Complex Formation ◽  
Inner Core ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
In Vitro Translation ◽  
Structural Protein ◽  
Major Structural Protein ◽  
Outer Capsid

ABSTRACT African swine fever virus (ASFV) is a large enveloped DNA virus that shares the striking icosahedral symmetry of iridoviruses. To understand the mechanism of assembly of ASFV, we have been studying the biosynthesis and subcellular distribution of p73, the major structural protein of ASFV. Sucrose density sedimentation of lysates prepared from infected cells showed that newly synthesized p73 was incorporated into a complex with a size of 150 to 250 kDa. p73 synthesized by in vitro translation migrated at 70 kDa, suggesting that cellular and/or viral proteins are required for the formation of the 150- to 250-kDa complex. During a 2-h chase, approximately 50% of the newly synthesized pool of p73 bound to the endoplasmic reticulum (ER). During this period, the membrane-bound pool of p73, but not the cytosolic pool, formed large complexes of approximately 50,000 kDa. The complexes were formed via assembly intermediates, and the entire membrane-associated pool of p73 was incorporated into the 50,000-kDa complex within 2 h. The 50,000-kDa complexes containing p73 were also detected in virions secreted from cells. Immunoprecipitation of sucrose gradients with sera taken from hyperimmune pigs suggested that p73 was the major component of the 50,000-kDa complex. It is possible, therefore, that the complex contains between 600 and 700 copies of p73. The kinetics of complex formation and envelopment of p73 were similar, and complex formation and envelopment were both reversibly inhibited by cycloheximide, suggesting a functional link between complex assembly and ASFV envelopment. A protease protection assay detected 50,000-kDa complexes on the inside and outside of the membranes forming the viral envelope. The identification of a complex containing p73 beneath the envelope of ASFV suggests that p73 may be a component of the inner core shell or matrix of ASFV. The outer pool may represent p73 within the outer capsid layer of the virus. In summary, the data suggest that the assembly of the inner core matrix and outer capsid of ASFV takes place on the ER membrane during envelopment and that these structures are not preassembled in the cytosol.

scholarly journals African Swine Fever Virus Structural Protein p54 Is Essential for the Recruitment of Envelope Precursors to Assembly Sites

2004 ◽  
Vol 78 (8) ◽  
pp. 4299-4313 ◽  
Author(s):  
Javier M. Rodríguez ◽  
Ramón García-Escudero ◽  
María L. Salas ◽  
Germán Andrés
Keyword(s):  
Transmembrane Domain ◽  
Early Stage ◽  
African Swine Fever Virus ◽  
Close Association ◽  
African Swine Fever ◽  
Viral Envelope ◽  
Fever Virus ◽  
Type I ◽  
Structural Protein ◽  
Virus Morphogenesis

ABSTRACT The assembly of African swine fever virus (ASFV) at the cytoplasmic virus factories commences with the formation of precursor membranous structures, which are thought to be collapsed cisternal domains recruited from the surrounding endoplasmic reticulum (ER). This report analyzes the role in virus morphogenesis of the structural protein p54, a 25-kDa polypeptide encoded by the E183L gene that contains a putative transmembrane domain and localizes at the ER-derived envelope precursors. We show that protein p54 behaves in vitro and in infected cells as a type I membrane-anchored protein that forms disulfide-linked homodimers through its unique luminal cysteine. Moreover, p54 is targeted to the ER membranes when it is transiently expressed in transfected cells. Using a lethal conditional recombinant, vE183Li, we also demonstrate that the repression of p54 synthesis arrests virus morphogenesis at a very early stage, even prior to the formation of the precursor membranes. Under restrictive conditions, the virus factories appeared as discrete electron-lucent areas essentially free of viral structures. In contrast, outside the assembly sites, large amounts of aberrant zipper-like structures formed by the unprocessed core polyproteins pp220 and pp62 were produced in close association to ER cisternae. Altogether, these results indicate that the transmembrane structural protein p54 is critical for the recruitment and transformation of the ER membranes into the precursors of the viral envelope.

scholarly journals Enigmatic origin of the poxvirus membrane from the endoplasmic reticulum shown by 3D imaging of vaccinia virus assembly mutants

2017 ◽  
Vol 114 (51) ◽  
pp. E11001-E11009 ◽  
Author(s):  
Andrea S. Weisberg ◽  
Liliana Maruri-Avidal ◽  
Himani Bisht ◽  
Bryan T. Hansen ◽  
Cindi L. Schwartz ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Endoplasmic Reticulum ◽  
Vaccinia Virus ◽  
Virus Assembly ◽  
Transmembrane Protein ◽  
Membrane Dynamics ◽  
Deletion Mutants ◽  
Viral Membrane ◽  
Transmission Electron

The long-standing inability to visualize connections between poxvirus membranes and cellular organelles has led to uncertainty regarding the origin of the viral membrane. Indeed, there has been speculation that viral membranes form de novo in cytoplasmic factories. Another possibility, that the connections are too short-lived to be captured by microscopy during a normal infection, motivated us to identify and characterize virus mutants that are arrested in assembly. Five conserved vaccinia virus proteins, referred to as Viral Membrane Assembly Proteins (VMAPs), that are necessary for formation of immature virions were found. Transmission electron microscopy studies of two VMAP deletion mutants had suggested retention of connections between viral membranes and the endoplasmic reticulum (ER). We now analyzed cells infected with each of the five VMAP deletion mutants by electron tomography, which is necessary to validate membrane continuity, in addition to conventional transmission electron microscopy. In all cases, connections between the ER and viral membranes were demonstrated by 3D reconstructions, supporting a role for the VMAPs in creating and/or stabilizing membrane scissions. Furthermore, coexpression of the viral reticulon-like transmembrane protein A17 and the capsid-like scaffold protein D13 was sufficient to form similar ER-associated viral structures in the absence of other major virion proteins. Determination of the mechanism of ER disruption during a normal VACV infection and the likely participation of both viral and cell proteins in this process may provide important insights into membrane dynamics.

scholarly journals Bilateral ureteral obstruction is rapidly accompanied by ER stress and activation of autophagic degradation of IMCD proteins, including AQP2

2020 ◽  
Vol 318 (1) ◽  
pp. F135-F147
Author(s):  
Poorichaya Somparn ◽  
Chatikorn Boonkrai ◽  
Komgrid Charngkaew ◽  
Nusara Chomanee ◽  
Kenneth G. Hodge ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Mass Spectrometry ◽  
Endoplasmic Reticulum ◽  
Ureteral Obstruction ◽  
Collecting Duct ◽  
Immunoblot Analysis ◽  
Immunogold Electron Microscopy ◽  
Sham Operation ◽  
Autophagic Degradation ◽  
Cell Cell

After the release of bilateral ureteral obstruction (BUO), postobstructive diuresis from an impaired urine concentration mechanism is associated with reduced aquaporin 2 (AQP2) abundance in the inner medullary collecting duct (IMCD). However, the underlying molecular mechanism of this AQP2 reduction is incompletely understood. To elucidate the mechanisms responsible for this phenomenon, we studied molecular changes in IMCDs isolated from rats with 4-h BUO or sham operation at the early onset of AQP2 downregulation using mass spectrometry-based proteomic analysis. Two-hundred fifteen proteins had significant changes in abundances, with 65% of them downregulated in the IMCD of 4-h BUO rats compared with sham rats. Bioinformatic analysis revealed that significantly changed proteins were associated with functional Gene Ontology terms, including “cell-cell adhesion,” “cell-cell adherens junction,” “mitochondrial inner membrane,” “endoplasmic reticulum chaperone complex,” and the KEGG pathway of glycolysis/gluconeogenesis. Targeted liquid chromatography-tandem mass spectrometry or immunoblot analysis confirmed the changes in 19 proteins representative of each predominant cluster, including AQP2. Electron microscopy demonstrated disrupted tight junctions, disorganized adherens junctions, swollen mitochondria, enlargement of the endoplasmic reticulum lumen, and numerous autophagosomes/lysosomes in the IMCD of rats with 4-h BUO. AQP2 and seven proteins chosen as representative of the significantly altered clusters had a significant increase in immunofluorescence-based colocalization with autophagosomes/lysosomes. Immunogold electron microscopy confirmed colocalization of AQP2 with the autophagosome marker microtubule-associated protein 1A/1B-light chain 3 and the lysosomal marker cathepsin D in IMCD cells of rats with 4-h BUO. We conclude that enhanced autophagic degradation of AQP2 and other critical proteins, as well as endoplasmic reticulum stress in the IMCD, are initiated shortly after BUO.

scholarly journals African Swine Fever Virus Structural Protein pE120R Is Essential for Virus Transport from Assembly Sites to Plasma Membrane but Not for Infectivity

2001 ◽  
Vol 75 (15) ◽  
pp. 6758-6768 ◽  
Author(s):  
Germán Andrés ◽  
Ramón Garcı́a-Escudero ◽  
Eladio Viñuela ◽  
Marı́a L. Salas ◽  
Javier M. Rodrı́guez
Keyword(s):  
Plasma Membrane ◽  
African Swine Fever Virus ◽  
Growth Curves ◽  
African Swine Fever ◽  
Fever Virus ◽  
Virus Yield ◽  
Virus Infectivity ◽  
Structural Protein ◽  
Virus Growth ◽  
Extracellular Virus

ABSTRACT This report examines the role of African swine fever virus (ASFV) structural protein pE120R in virus replication. Immunoelectron microscopy revealed that protein pE120R localizes at the surface of the intracellular virions. Consistent with this, coimmunoprecipitation assays showed that protein pE120R binds to the major capsid protein p72. Moreover, it was found that, in cells infected with an ASFV recombinant that inducibly expresses protein p72, the incorporation of pE120R into the virus particle is dependent on p72 expression. Protein pE120R was also studied using an ASFV recombinant in which E120R gene expression is regulated by the Escherichia coli lacrepressor-operator system. In the absence of inducer, pE120R expression was reduced about 100-fold compared to that obtained with the parental virus or the recombinant virus grown under permissive conditions. One-step virus growth curves showed that, under conditions that repress pE120R expression, the titer of intracellular progeny was similar to the total virus yield obtained under permissive conditions, whereas the extracellular virus yield was about 100-fold lower than in control infections. Immunofluorescence and electron microscopy demonstrated that, under restrictive conditions, intracellular mature virions are properly assembled but remain confined to the replication areas. Altogether, these results indicate that pE120R is necessary for virus dissemination but not for virus infectivity. The data also suggest that protein pE120R might be involved in the microtubule-mediated transport of ASFV particles from the viral factories to the plasma membrane.

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