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 Computational Analysis of African Swine Fever Virus Protein Space for the Design of an Epitope-Based Vaccine Ensemble

Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1078 ◽  
Author(s):  
Albert Ros-Lucas ◽  
Florencia Correa-Fiz ◽  
Laia Bosch-Camós ◽  
Fernando Rodriguez ◽  
Julio Alonso-Padilla
Keyword(s):  
T Cell ◽  
Vaccine Development ◽  
De Novo ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Economic Losses ◽  
Virus Protein ◽  
Hemorrhagic Disease ◽  
Starting Point

African swine fever virus is the etiological agent of African swine fever, a transmissible severe hemorrhagic disease that affects pigs, causing massive economic losses. There is neither a treatment nor a vaccine available, and the only method to control its spread is by extensive culling of pigs. So far, classical vaccine development approaches have not yielded sufficiently good results in terms of concomitant safety and efficacy. Nowadays, thanks to advances in genomic and proteomic techniques, a reverse vaccinology strategy can be explored to design alternative vaccine formulations. In this study, ASFV protein sequences were analyzed using an in-house pipeline based on publicly available immunoinformatic tools to identify epitopes of interest for a prospective vaccine ensemble. These included experimentally validated sequences from the Immune Epitope Database, as well as de novo predicted sequences. Experimentally validated and predicted epitopes were prioritized following a series of criteria that included evolutionary conservation, presence in the virulent and currently circulating variant Georgia 2007/1, and lack of identity to either the pig proteome or putative proteins from pig gut microbiota. Following this strategy, 29 B-cell, 14 CD4+ T-cell and 6 CD8+ T-cell epitopes were selected, which represent a starting point to investigating the protective capacity of ASFV epitope-based vaccines.

scholarly journals African Swine Fever Virus Blocks the Host Cell Antiviral Inflammatory Response through a Direct Inhibition of PKC-θ-Mediated p300 Transactivation

2008 ◽  
Vol 83 (2) ◽  
pp. 969-980 ◽  
Author(s):  
Aitor G. Granja ◽  
Elena G. Sánchez ◽  
Prado Sabina ◽  
Manuel Fresno ◽  
Yolanda Revilla
Keyword(s):  
Gene Expression ◽  
Inflammatory Response ◽  
Viral Infection ◽  
Host Cell ◽  
Expression Pattern ◽  
African Swine Fever Virus ◽  
Gene Expression Pattern ◽  
African Swine Fever ◽  
Fever Virus ◽  
Amino Terminal

ABSTRACT During a viral infection, reprogramming of the host cell gene expression pattern is required to establish an adequate antiviral response. The transcriptional coactivators p300 and CREB binding protein (CBP) play a central role in this regulation by promoting the assembly of transcription enhancer complexes to specific promoters of immune and proinflammatory genes. Here we show that the protein A238L encoded by African swine fever virus counteracts the host cell inflammatory response through the control of p300 transactivation during the viral infection. We demonstrate that A238L inhibits the expression of the inflammatory regulators cyclooxygenase-2 (COX-2) and tumor necrosis factor alpha (TNF-α) by preventing the recruitment of p300 to the enhanceosomes formed on their promoters. Furthermore, we report that A238L inhibits p300 activity during the viral infection and that its amino-terminal transactivation domain is essential in the A238L-mediated inhibition of the inflammatory response. Importantly, we found that the residue serine 384 of p300 is required for the viral protein to accomplish its inhibitory function and that ectopically expressed PKC-θ completely reverts this inhibition, thus indicating that this signaling pathway is disrupted by A238L during the viral infection. Furthermore, we show here that A238L does not affect PKC-θ enzymatic activity, but the molecular mechanism of this viral inhibition relies on the lack of interaction between PKC-θ and p300. These findings shed new light on how viruses alter the host cell antiviral gene expression pattern through the blockade of the p300 activity, which represents a new and sophisticated viral mechanism to evade the inflammatory and immune defense responses.

Characterization of the African swine fever virus protein p49: a new late structural polypeptide

Microbiology ◽  
2000 ◽  
Vol 81 (1) ◽  
pp. 59-65 ◽  
Author(s):  
Inmaculada Galindo ◽  
Eladio Viñuela ◽  
Angel L. Carrascosa
Keyword(s):  
Cell Attachment ◽  
African Swine Fever Virus ◽  
Rabbit Antiserum ◽  
African Swine Fever ◽  
Virus Genome ◽  
Fever Virus ◽  
Virus Protein ◽  
Significant Similarity ◽  
Reading Frame ◽  
Cell Extracts

The open reading frame B438L, located within the EcoRI B fragment of the African swine fever virus genome, is predicted to encode a protein of 438 amino acids with a molecular mass of 49·3 kDa. It presents a cell attachment RGD (Arg–Gly–Asp) motif but no other significant similarity to protein sequences in databases. Northern blot and primer extension analysis showed that B438L is transcribed only at late times during virus infection. The B438L gene product has been expressed in Escherichia coli, purified and used as an antigen for antibody production. The rabbit antiserum specific for pB438L recognized a protein of about 49 kDa in virus-infected cell extracts. This protein was synthesized late in infection by all the virus strains tested, was located in cytoplasmic virus factories and appeared as a structural component of purified virus particles.

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 Two African Swine Fever Virus Proteins Derived from a Common Precursor Exhibit Different Nucleocytoplasmic Transport Activities

2004 ◽  
Vol 78 (18) ◽  
pp. 9731-9739 ◽  
Author(s):  
A. Eulálio ◽  
I. Nunes-Correia ◽  
A. L. Carvalho ◽  
C. Faro ◽  
V. Citovsky ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Mammalian Cells ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Nucleocytoplasmic Transport ◽  
Proteolytic Processing ◽  
Fever Virus ◽  
Hemorrhagic Disease ◽  
Common Precursor ◽  
Green Fluorescent

ABSTRACT African swine fever virus (ASFV), a large icosahedral deoxyvirus, is the causative agent of an economically relevant hemorrhagic disease that affects domestic pigs. The major purpose of the present study was to investigate the nuclear transport activities of the ASFV p37 and p14 proteins, which result from the proteolytic processing of a common precursor. Experiments were performed by using yeast-based nucleocytoplasmic transport assays and by analysis of the subcellular localization of different green fluorescent and Myc fusion proteins in mammalian cells. The results obtained both in yeast and mammalian cells clearly demonstrated that ASFV p14 protein is imported into the nucleus but not exported to the cytoplasm. The ability of p37 protein to be exported from the nucleus to the cytoplasm of both yeast and mammalian cells was also demonstrated, and the results clearly indicate that p37 nuclear export is dependent on the interaction of the protein with the CRM-1 receptor. In addition, p37 was shown to exhibit nuclear import activity in mammalian cells. The p37 protein nuclear import and export abilities described here constitute the first report of a nucleocytoplasmic shuttling protein encoded by the ASFV genome. Overall, the overlapping results obtained for green fluorescent protein fusions and Myc-tagged proteins undoubtedly demonstrate that ASFV p37 and p14 proteins exhibit nucleocytoplasmic transport activities. These findings are significant for understanding the role these proteins play in the replication cycle of ASFV.

scholarly journals Detection of African Swine Fever Virus Protein VP73 in Tissues of Experimentally and Naturally Infected Pigs

1994 ◽  
Vol 6 (3) ◽  
pp. 360-365 ◽  
Author(s):  
José Pérez ◽  
Francisco Rodríguez ◽  
Antonio Fernández ◽  
Juana Martín de las Mulas ◽  
José Carlos Gómez-Villamandos ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Virus Protein

Pathogenesis of African swine fever virus inOrnithodorosticks

2001 ◽  
Vol 2 (2) ◽  
pp. 121-128 ◽  
Author(s):  
Steven B. Kleiboeker ◽  
Glen A. Scoles
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Sub Saharan Africa ◽  
Hemorrhagic Disease ◽  
Sylvatic Cycle ◽  
Infectious Dose ◽  
Domestic Pigs ◽  
Ornithodoros Porcinus Porcinus ◽  
Sub Saharan

AbstractAfrican swine fever virus (ASFV) is the only known DNA arbovirus and the sole member of the family Asfarviridae. It causes a lethal, hemorrhagic disease in domestic pigs. ASFV is enzootic in sub-Saharan Africa and is maintained in a sylvatic cycle by infecting both wild members of the Suidae (e.g. warthogs) and the argasid tickOrnithodoros porcinus porcinus. The pathogenesis of ASFV inO. porcinus porcinusticks is characterized by a low infectious dose, lifelong infection, efficient transmission to both pigs and ticks, and low mortality until after the first oviposition. ASFV pathogenesis in warthogs is characterized by an inapparent infection with transient, low viremic titers. ThusO. porcinus porcinusticks probably constitute the most important natural vector of ASFV, although both the mammalian and tick hosts are probably required for the maintenance of ASFV in the sylvatic cycle. The mechanism of ASFV transmission from the sylvatic cycle to domestic pigs is probably through infected ticks feeding on pigs. In addition toO. porcinus porcinus, a number of North American, Central American and Caribbean species ofOrnithodoroshave been shown to be potential vectors of ASFV.

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