Titration of African Swine Fever (ASF) Virus

Enjuanes, Luis; Carrascosa, Ángel L.; Moreno, Megan A.; Viñuela, Eladio

doi:10.1099/0022-1317-32-3-471

Cited by 210 publications

(156 citation statements)

References 4 publications

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“…Cells were stimulated by phorbol 12-myristate 13-acetate (PMA; Sigma) at 15 ng/ml and A23187 calcium ionophore (Ion; Sigma) at 1 M. Cyclosporin A (CsA, Sandoz, 100 ng/ml) was added 1 h before the addition of PMA and Ion. The Vero-adapted ASFV strain Ba71V was propagated and titrated by plaque assay on Vero cells as described previously (41).…”

Section: Methodsmentioning

confidence: 99%

The Viral Protein A238L Inhibits Cyclooxygenase-2 Expression through a Nuclear Factor of Activated T Cell-dependent Transactivation Pathway

Granja

Nogal

Hurtado

et al. 2004

Journal of Biological Chemistry

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Cyclooxygenase-2 is transiently induced upon cell activation or viral infections, resulting in inflammation and modulation of the immune response. Here we report that A238L, an African swine fever virus protein, efficiently inhibits cyclooxygenase-2 gene expression in Jurkat T cells and in virus-infected Vero cells. Transfection of Jurkat cells stably expressing A238L with cyclooxygenase-2 promoter-luciferase constructs containing 5 -terminal deletions or mutations in distal or proximal nuclear factor of activated T cell (NFAT) response elements revealed that these sequences are involved in the inhibition induced by A238L. Overexpression of a constitutively active version of the calciumdependent phosphatase calcineurin or NFAT reversed the inhibition mediated by A238L on cyclooxygenase-2 promoter activation, whereas overexpression of p65 NF B had no effect. A238L does not modify the nuclear localization of NFAT after phorbol 12-myristate 13-acetate/calcium ionophore stimulation. Moreover, we show that the mechanism by which the viral protein downregulates cyclooxygenase-2 activity does not involve inhibition of the binding between NFAT and its specific DNA sequences into the cyclooxygenase-2 promoter. Strikingly, A238L dramatically inhibited the transactivation mediated by a GAL4-NFAT fusion protein containing the N-terminal transactivation domain of NFAT1. Taken together, these data indicate that A238L down-regulates cyclooxygenase-2 transcription through the NFAT response elements, being NFAT-dependent transactivation implicated in this down-regulation.Viruses have been known for a long time to use a variety of strategies not only to alter the host metabolism via their signaling proteins but also to hijack cellular signaling pathways and transcription factors to control them to their own advantage. Both the nuclear factor-B (NF B) 1 and the nuclear factor of activated T cells (NFAT) pathways appear to be attractive targets for common viral pathogens, probably due to their ability to promote the expression of numerous proteins involved in adaptative and innate immunity (1, 2). Several viruses, including hepatitis C virus (3), immunodeficiency virus (4), herpes viruses (5), and African swine fever virus (ASFV) (6 -8) have been shown to modulate the activation of NFAT or NF B.NF B is a collective term referring to a class of dimeric transcription factors belonging to the rel family. In resting cells, NF B exists in the cytoplasm as an inactive complex bound to inhibitory proteins of the I B family (9, 10). In response to a variety of stimuli, I B proteins undergo phosphorylation of Ser 32 and Ser 36 (11, 12), followed by ubiquitination and degradation in the proteasome, thus unmasking the nuclear localization sequence of the transactivating heterodimers and allowing translocation of active NF B to the nucleus. Recently, there is accumulating evidence suggesting that another level of NF-〉 regulation independent on I B degradation exists. This second level of regulation relies in the activation of the transcription...

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Section: Methodsmentioning

confidence: 99%

The Viral Protein A238L Inhibits Cyclooxygenase-2 Expression through a Nuclear Factor of Activated T Cell-dependent Transactivation Pathway

Granja

Nogal

Hurtado

et al. 2004

Journal of Biological Chemistry

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“…The Vero-adapted ASFV strain BA71V was propagated and titrated as described previously (21). Highly purified ASFV was prepared as described (22).…”

Section: Methodsmentioning

confidence: 99%

African Swine Fever Virus Protease, a New Viral Member of the SUMO-1-specific Protease Family

Andrés¹,

Alejo²,

Simón‐Mateo³

et al. 2001

Journal of Biological Chemistry

104

107

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African swine fever virus (ASFV) is a complex DNA virus that employs polyprotein processing at Gly-GlyXaa sites as a strategy to produce several major core components of the viral particle. The virus gene S273R encodes a 31-kDa protein that contains a "core domain" with the conserved catalytic residues characteristic of SUMO-1-specific proteases and the adenovirus protease. Using a COS cell expression system, it was found that protein pS273R is capable of cleaving the viral polyproteins pp62 and pp220 in a specific way giving rise to the same intermediates and mature products as those produced in ASFV-infected cells. Furthermore, protein pS273R, like adenovirus protease and SUMO-1-specific enzymes, is a cysteine protease, because its activity is abolished by mutation of the predicted catalytic histidine and cysteine residues and is inhibited by sulfhydryl-blocking reagents. Protein pS273R is expressed late after infection and is localized in the cytoplasmic viral factories, where it is found associated with virus precursors and mature virions. In the virions, the protein is present in the core shell, a domain where the products of the viral polyproteins are also located. The identification of the ASFV protease will allow a better understanding of the role of polyprotein processing in virus assembly and may contribute to our knowledge of the emerging family of SUMO-1-specific proteases.Positive strand RNA viruses and retroviruses encode polyproteins, which are proteolytically cleaved by viral proteases to yield the nonstructural and structural proteins required for replication and morphogenesis (1-3). On the other hand, DNA viruses, such as adenoviruses and poxviruses, synthesize precursor proteins whose maturation by proteolytic removal of terminal peptides plays an essential role in virion formation (1).African swine fever virus (ASFV), 1 a large and complex virus containing a 170-kb double-stranded DNA molecule with 151 potential genes (4, 5), is atypical among DNA viruses in that it encodes two polyproteins, pp220 and pp62, which are cleaved to produce six major structural components of the virus particle (6, 7). These proteins, p150, p37, p34, and p14, derived from polyprotein pp220 and p35 and p15, products of polyprotein pp62, are the major components of the core shell, a thick protein layer that surrounds the DNA-containing central nucleoid and that is enwrapped by the inner lipoprotein envelope and the icosahedral capsid (8).2 All the proteolytic cleavages occur after the second Gly of the consensus sequence Gly-GlyXaa, which is also recognized as a cleavage site in the maturation of adenovirus structural proteins and in some cellular proteins, including polyubiquitin (9) and ubiquitin-like proteins (10). A similar cleavage site (Ala-Gly-Xaa) is used for the maturation of vaccinia virus structural proteins (11,12). Although the adenovirus protease that processes at Gly-Gly-Xaa sites is well characterized (13-15), the enzymes involved in the processing of the ASFV polyproteins or in the cleavage of vaccin...

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“…The ASFV strain BA71V, adapted to grow in Vero cells, and vGUSREP, a BA71V-derived recombinant that expresses the Escherichia coil lac repressor, have been described before (23,26).…”

Section: Cells and Virusesmentioning

confidence: 99%

The African Swine Fever Virus Virion Membrane Protein pE248R Is Required for Virus Infectivity and an Early Postentry Event

Rodrı́guez

Nogal

Redrejo-Rodríguez

et al. 2009

J Virol

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The African swine fever virus (ASFV) protein pE248R, encoded by the gene E248R, is a late structural component of the virus particle. The protein contains intramolecular disulfide bonds and has been previously identified as a substrate of the ASFV-encoded redox system. Its amino acid sequence contains a putative myristoylation site and a hydrophobic transmembrane region near its carboxy terminus. We show here that the protein pE248R is myristoylated during infection and associates with the membrane fraction in infected cells, behaving as an integral membrane protein. Furthermore, the protein localizes at the inner envelope of the virus particles in the cytoplasmic factories. The function of the protein pE248R in ASFV replication was investigated by using a recombinant virus that inducibly expresses the gene E248R. Under repressive conditions, the ASFV polyproteins pp220 and pp62 are normally processed and virus particles with morphology indistinguishable from that of those produced in a wild-type infection or under permissive conditions are generated. Moreover, the mutant virus particles can exit the cell as does the parental virus. However, the infectivity of the pE248R-deficient virions was reduced at least 100-fold. An investigation of the defect of the mutant virus indicated that neither virus binding nor internalization was affected by the absence of the protein pE248R, but a cytopathic effect was not induced and early and late gene expression was impaired, indicating that the protein is required for some early postentry event.

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Titration of African Swine Fever (ASF) Virus

Cited by 210 publications

References 4 publications

The Viral Protein A238L Inhibits Cyclooxygenase-2 Expression through a Nuclear Factor of Activated T Cell-dependent Transactivation Pathway

The Viral Protein A238L Inhibits Cyclooxygenase-2 Expression through a Nuclear Factor of Activated T Cell-dependent Transactivation Pathway

African Swine Fever Virus Protease, a New Viral Member of the SUMO-1-specific Protease Family

The African Swine Fever Virus Virion Membrane Protein pE248R Is Required for Virus Infectivity and an Early Postentry Event

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