scholarly journals Vimentin Rearrangement during African Swine Fever Virus Infection Involves Retrograde Transport along Microtubules and Phosphorylation of Vimentin by Calcium Calmodulin Kinase II

2005 ◽  
Vol 79 (18) ◽  
pp. 11766-11775 ◽  
Author(s):  
Sandra Stefanovic ◽  
Miriam Windsor ◽  
Koh-ici Nagata ◽  
Masaki Inagaki ◽  
Thomas Wileman
Keyword(s):  
Dna Replication ◽  
Virus Assembly ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Cam Kinase Ii ◽  
Cam Kinase ◽  
Microtubule Organizing Center ◽  
Viral Dna ◽  
Viral Dna Replication

ABSTRACT African swine fever virus (ASFV) infection leads to rearrangement of vimentin into a cage surrounding virus factories. Vimentin rearrangement in cells generally involves phosphorylation of N-terminal domains of vimentin by cellular kinases to facilitate disassembly and transport of vimentin filaments on microtubules. Here, we demonstrate that the first stage in vimentin rearrangement during ASFV infection involves a microtubule-dependent concentration of vimentin into an “aster” within virus assembly sites located close to the microtubule organizing center. The aster may play a structural role early during the formation of the factory. Conversion of the aster into a cage required ASFV DNA replication. Interestingly, viral DNA replication also resulted in the activation of calcium calmodulin-dependent protein kinase II (CaM kinase II) and phosphorylation of the N-terminal domain of vimentin on serine 82. Immunostaining showed that vimentin within the cage was phosphorylated on serine 82. Significantly, both viral DNA replication and Ser 82 phosphorylation were blocked by KN93, an inhibitor of CaM kinase II, suggesting a link between CaM kinase II activation, DNA replication, and late gene expression. Phosphorylation of vimentin on serine 82 may be necessary for cage formation or may simply be a consequence of activation of CaM kinase II by ASFV. The vimentin cage may serve a cytoprotective function and prevent movement of viral components into the cytoplasm and at the same time concentrate late structural proteins at sites of virus assembly.

scholarly journals DNA-Binding Properties of African Swine Fever Virus pA104R, a Histone-Like Protein Involved in Viral Replication and Transcription

2017 ◽  
Vol 91 (12) ◽  
Author(s):  
Gonçalo Frouco ◽  
Ferdinando B. Freitas ◽  
João Coelho ◽  
Alexandre Leitão ◽  
Carlos Martins ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Topoisomerase Ii ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Dna Supercoiling ◽  
Fever Virus ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Replication Sites

ABSTRACT African swine fever virus (ASFV) codes for a putative histone-like protein (pA104R) with extensive sequence homology to bacterial proteins that are implicated in genome replication and packaging. Functional characterization of purified recombinant pA104R revealed that it binds to single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) over a wide range of temperatures, pH values, and salt concentrations and in an ATP-independent manner, with an estimated binding site size of about 14 to 16 nucleotides. Using site-directed mutagenesis, the arginine located in pA104R's DNA-binding domain, at position 69, was found to be relevant for efficient DNA-binding activity. Together, pA104R and ASFV topoisomerase II (pP1192R) display DNA-supercoiling activity, although none of the proteins by themselves do, indicating that the two cooperate in this process. In ASFV-infected cells, A104R transcripts were detected from 2 h postinfection (hpi) onward, reaching a maximum concentration around 16 hpi. pA104R was detected from 12 hpi onward, localizing with viral DNA replication sites and being found exclusively in the Triton-insoluble fraction. Small interfering RNA (siRNA) knockdown experiments revealed that pA104R plays a critical role in viral DNA replication and gene expression, with transfected cells showing lower viral progeny numbers (up to a reduction of 82.0%), lower copy numbers of viral genomes (−78.3%), and reduced transcription of a late viral gene (−47.6%). Taken together, our results strongly suggest that pA104R participates in the modulation of viral DNA topology, probably being involved in viral DNA replication, transcription, and packaging, emphasizing that ASFV mutants lacking the A104R gene could be used as a strategy to develop a vaccine against ASFV. IMPORTANCE Recently reintroduced in Europe, African swine fever virus (ASFV) causes a fatal disease in domestic pigs, causing high economic losses in affected countries, as no vaccine or treatment is currently available. Remarkably, ASFV is the only known mammalian virus that putatively codes for a histone-like protein (pA104R) that shares extensive sequence homology with bacterial histone-like proteins. In this study, we characterized the DNA-binding properties of pA104R, analyzed the functional importance of two conserved residues, and showed that pA104R and ASFV topoisomerase II cooperate and display DNA-supercoiling activity. Moreover, pA104R is expressed during the late phase of infection and accumulates in viral DNA replication sites, and its downregulation revealed that pA104R is required for viral DNA replication and transcription. These results suggest that pA104R participates in the modulation of viral DNA topology and genome packaging, indicating that A104R deletion mutants may be a good strategy for vaccine development against ASFV.

scholarly journals African swine fever virus infection disrupts centrosome assembly and function

2005 ◽  
Vol 86 (3) ◽  
pp. 589-594 ◽  
Author(s):  
Nolwenn Jouvenet ◽  
Thomas Wileman
Keyword(s):  
Virus Infection ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Dna Virus ◽  
Viral Dna ◽  
Microtubule Network ◽  
Viral Dna Replication ◽  
Infected Cells ◽  
And Function

African swine fever virus (ASFV) is a large, enveloped DNA virus that assembles in perinuclear sites located close to the centrosome. It is reported here that the microtubule network becomes disorganized soon after the onset of viral DNA replication and formation of assembly sites. ASFV infection resulted in loss of γ-tubulin and pericentrin at the centrosome; this was due to protein relocalization, but not degradation. ASFV infection also inhibited the ability of the centrosome to nucleate microtubules. The reorganization of microtubules seen in ASFV-infected cells may therefore be mediated by γ-tubulin and pericentrin redistribution, and consequent disruption of centrosome assembly and function.

Genetic variation of african swine fever virus: Variable regions near the ends of the viral DNA

Virology ◽  
1989 ◽  
Vol 173 (1) ◽  
pp. 251-257 ◽  
Author(s):  
Rafael Blasco ◽  
Inmaculada de la Vega ◽  
Fernando Almazan ◽  
Montserrat Aguero ◽  
Eladio Viñuela
Keyword(s):  
Genetic Variation ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Viral Dna ◽  
Variable Regions

scholarly journals A Simple Method for Sample Preparation to Facilitate Efficient Whole-Genome Sequencing of African Swine Fever Virus

Viruses ◽  
2019 ◽  
Vol 11 (12) ◽  
pp. 1129 ◽  
Author(s):  
Ferenc Olasz ◽  
István Mészáros ◽  
Szilvia Marton ◽  
Győző L. Kaján ◽  
Vivien Tamás ◽  
...  
Keyword(s):  
Sample Preparation ◽  
African Swine Fever Virus ◽  
Animal Health ◽  
African Swine Fever ◽  
Fever Virus ◽  
Whole Genome ◽  
Swine Industry ◽  
Viral Dna ◽  
Simple Method ◽  
Generation Sequencing

In the recent years, African swine fever has become the biggest animal health threat to the swine industry. To facilitate quick genetic analysis of its causative agent, the African swine fever virus (ASFV), we developed a simple and efficient method for next generation sequencing of the viral DNA. Execution of the protocol does not demand complicated virus purification steps, enrichment of the virus by ultracentrifugation or of the viral DNA by ASFV-specific PCRs, and minimizes the use of Sanger sequencing. Efficient DNA-se treatment, monitoring of sample preparation by qPCR, and whole genome amplification are the key elements of the method. Through detailed description of sequencing of the first Hungarian ASFV isolate (ASFV_HU_2018), we specify the sensitive steps and supply key reference numbers to assist reproducibility and to facilitate the successful use of the method for other ASFV researchers.

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 Aggresomes Resemble Sites Specialized for Virus Assembly

2001 ◽  
Vol 153 (3) ◽  
pp. 449-456 ◽  
Author(s):  
Colin M. Heath ◽  
Miriam Windsor ◽  
Thomas Wileman
Keyword(s):  
Cellular Response ◽  
Virus Assembly ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Microtubule Organizing Center ◽  
Dna Viruses ◽  
Cytoplasmic Dna Viruses ◽  
Cell Biol ◽  
Cytoplasmic Dna ◽  
Organizing Center

The large cytoplasmic DNA viruses such as poxviruses, iridoviruses, and African swine fever virus (ASFV) assemble in discrete perinuclear foci called viral factories. Factories exclude host proteins, suggesting that they are novel subcellular structures induced by viruses. Novel perinuclear structures, called aggresomes are also formed by cells in response to misfolded protein (Johnston, J.A., C.L. Ward, and R.R. Kopito. 1998. J. Cell Biol. 143:1883–1898; García-Mata, R., Z. Bebök, E.J. Sorscher, and E.S. Sztul. 1999. J. Cell Biol. 146:1239–1254). In this study, we have investigated whether aggresomes and viral factories are related structures. Aggresomes were compared with viral factories produced by ASFV. Aggresomes and viral factories were located close to the microtubule organizing center and required an intact microtubular network for assembly. Both structures caused rearrangement of intermediate filaments and the collapse of vimentin into characteristic cages, and both recruited mitochondria and cellular chaperones. Given that ASFV factories resemble aggresomes, it is possible that a cellular response originally designed to reduce the toxicity of misfolded proteins is exploited by cytoplasmic DNA viruses to concentrate structural proteins at virus assembly sites.

scholarly journals An Isothermal Molecular Point of Care Testing for African Swine Fever Virus Using Recombinase-Aided Amplification and Lateral Flow Assay Without the Need to Extract Nucleic Acids in Blood

2021 ◽  
Vol 11 ◽  
Author(s):  
Yuhang Zhang ◽  
Qingmei Li ◽  
Junqing Guo ◽  
Dongliang Li ◽  
Li Wang ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
Point Of Care ◽  
African Swine Fever ◽  
Lateral Flow ◽  
Fever Virus ◽  
Lateral Flow Assay ◽  
Economic Losses ◽  
Point Of Care Testing ◽  
Viral Dna ◽  
Blood Samples

African swine fever (ASF) is a highly contagious and usually deadly porcine infectious disease listed as a notifiable disease by the World Organization for Animal Health (OIE). It has brought huge economic losses worldwide, especially since 2018, the first outbreak in China. As there are still no effective vaccines available to date, diagnosis of ASF is essential for its surveillance and control, especially in areas far from city with limited resources and poor settings. In this study, a sensitive, specific, rapid, and simple molecular point of care testing for African swine fever virus (ASFV) B646L gene in blood samples was established, including treatment of blood samples with simple dilution and boiling for 5 min, isothermal amplification with recombinase-aided amplification (RAA) at 37°C in a water bath for 10 min, and visual readout with lateral flow assay (LFA) at room temperature for 10–15 min. Without the need to extract viral DNA in blood samples, the intact workflow from sampling to final diagnostic decision can be completed with minimal equipment requirement in 30 min. The detection limit of RAA-LFA for synthesized B646L gene-containing plasmid was 10 copies/μl, which was 10-fold more sensitive than OIE-recommended PCR and quantitative PCR. In addition, no positive readout of RAA-LFA was observed in testing classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine epidemic diarrhea virus, pseudorabies virus and porcine circovirus 2, exhibiting good specificity. Evaluation of clinical blood samples of RAA-LFA showed 100% coincident rate with OIE-recommended PCR, in testing both extracted DNAs and treated bloods. We also found that some components in blood samples greatly inhibited PCR performance, but had little effect on RAA. Inhibitory effect can be eliminated when blood was diluted at least 32–64-fold for direct PCR, while only a 2–4 fold dilution of blood was suitable for direct RAA, indicating RAA is a better choice than PCR when blood is used as detecting sample. Taken together, we established an sensitive, specific, rapid, and simple RAA-LFA for ASFV molecular detection without the need to extract viral DNA, providing a good choice for point of care testing of ASF diagnosis in the future.

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 Polyprotein pp62 Is Essential for Viral Core Development

2009 ◽  
Vol 84 (1) ◽  
pp. 176-187 ◽  
Author(s):  
Cristina Suárez ◽  
María L. Salas ◽  
Javier M. Rodríguez
Keyword(s):  
Gene Expression ◽  
Viral Particle ◽  
Virus Assembly ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Proteolytic Processing ◽  
Fever Virus ◽  
Clear Correlation ◽  
Late Gene Expression ◽  
The Core

ABSTRACT One of the most characteristic features of African swine fever virus gene expression is its use of two polyproteins, pp220 and pp62, to produce several structural proteins that account for approximately 32% of the total protein virion mass. Equimolecular amounts of these proteins are the major components of the core shell, a thick protein layer that lies beneath the inner envelope, surrounding the viral nucleoid. Polyprotein pp220, which is located immediately underneath the internal envelope, is essential for the encapsidation of the core of the viral particle. In its absence, the infection produces essentially coreless particles. In this study we analyzed, by means of an IPTG (isopropyl-β-d-thiogalactopyranoside)-inducible virus, the role of polyprotein pp62 in virus assembly. Polyprotein pp62 is indispensable for viral replication. The repression of polyprotein pp62 expression does not alter late gene expression or the proteolytic processing of the polyprotein pp220. However, it has a profound impact on the subcellular localization of polyprotein pp220. Electron microscopy studies revealed that polyprotein pp62 is necessary for the correct assembly and maturation of the core of the viral particle. Its repression leads to the appearance of a significant fraction of empty particles, to an increase in the number of immature-like particles, and to the accumulation of defective particles. Immunoelectron microscopy analysis showed a clear correlation between the amount of polyprotein pp62, the quantity of polyprotein pp220, and the state of development of the core, suggesting that the complete absence of polyprotein pp62 during morphogenesis would produce a homogenous population of empty particles.

scholarly journals Transport of African Swine Fever Virus from Assembly Sites to the Plasma Membrane Is Dependent on Microtubules and Conventional Kinesin

2004 ◽  
Vol 78 (15) ◽  
pp. 7990-8001 ◽  
Author(s):  
Nolwenn Jouvenet ◽  
Paul Monaghan ◽  
Michael Way ◽  
Thomas Wileman
Keyword(s):  
Plasma Membrane ◽  
Immunofluorescence Microscopy ◽  
African Swine Fever Virus ◽  
Large Fraction ◽  
African Swine Fever ◽  
Fever Virus ◽  
Cell Periphery ◽  
Microtubule Organizing Center ◽  
Microtubule Motor ◽  
Conventional Kinesin

ABSTRACT African swine fever virus (ASFV) is a large DNA virus that assembles in perinuclear viral factories located close to the microtubule organizing center. In this study, we have investigated the mechanism by which ASFV reaches the cell surface from the site of assembly. Immunofluorescence microscopy revealed that at 16 h postinfection, mature virions were aligned along microtubules. Furthermore, virus movement to the cell periphery was inhibited when microtubules were depolymerized by nocodazole. In addition, ASFV infection resulted in the increased acetylation of microtubules as well as their protection against depolymerization by nocodazole. Immunofluorescence microscopy showed that conventional kinesin was recruited to virus factories and to a large fraction of virus particles in the cytoplasm. Consistent with a role for conventional kinesin during ASFV egress to the cell periphery, overexpression of the cargo-binding domain of the kinesin light chain severely inhibited the movement of particles to the plasma membrane. Based on our observations, we propose that ASFV is recognized as cargo by conventional kinesin and uses this plus-end microtubule motor to move from perinuclear assembly sites to the plasma membrane.

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