scholarly journals Dynamin- and Clathrin-Dependent Endocytosis in African Swine Fever Virus Entry

2009 ◽  
Vol 84 (4) ◽  
pp. 2100-2109 ◽  
Author(s):  
Bruno Hernaez ◽  
Covadonga Alonso
Keyword(s):  
Actin Polymerization ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Cell Types ◽  
Fever Virus ◽  
Dominant Negative ◽  
Endocytic Pathway ◽  
Host Cells ◽  
Productive Infection ◽  
Cellular Compartments

ABSTRACT African swine fever virus (ASFV) is a large DNA virus that enters host cells after receptor-mediated endocytosis and depends on acidic cellular compartments for productive infection. The exact cellular mechanism, however, is largely unknown. In order to dissect ASFV entry, we have analyzed the major endocytic routes using specific inhibitors and dominant negative mutants and analyzed the consequences for ASFV entry into host cells. Our results indicate that ASFV entry into host cells takes place by clathrin-mediated endocytosis which requires dynamin GTPase activity. Also, the clathrin-coated pit component Eps15 was identified as a relevant cellular factor during infection. The presence of cholesterol in cellular membranes, but not lipid rafts or caveolae, was found to be essential for a productive ASFV infection. In contrast, inhibitors of the Na+/H+ ion channels and actin polymerization inhibition did not significantly modify ASFV infection, suggesting that macropinocytosis does not represent the main entry route for ASFV. These results suggest a dynamin-dependent and clathrin-mediated endocytic pathway of ASFV entry for the cell types and viral strains analyzed.

scholarly journals Short and Long-Read Sequencing Survey of the Dynamic Transcriptomes of African Swine Fever Virus and the Host Cells

2020 ◽  
Vol 11 ◽  
Author(s):  
Ferenc Olasz ◽  
Dóra Tombácz ◽  
Gábor Torma ◽  
Zsolt Csabai ◽  
Norbert Moldován ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Host Cells ◽  
Long Read

scholarly journals Cholesterol Flux Is Required for Endosomal Progression of African Swine Fever Virions during the Initial Establishment of Infection

2015 ◽  
Vol 90 (3) ◽  
pp. 1534-1543 ◽  
Author(s):  
Miguel Ángel Cuesta-Geijo ◽  
Michele Chiappi ◽  
Inmaculada Galindo ◽  
Lucía Barrado-Gil ◽  
Raquel Muñoz-Moreno ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Cholesterol Efflux ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Endocytic Pathway ◽  
Productive Infection ◽  
Effective Vaccine ◽  
The Caucasus ◽  
Late Endosomes ◽  
Adenovirus 5

ABSTRACTAfrican swine fever virus (ASFV) is a major threat for porcine production that has been slowly spreading in Eastern Europe since its first appearance in the Caucasus in 2007. ASFV enters the cell by endocytosis and gains access to the cytosol to start replication from late endosomes and multivesicular bodies. Cholesterol associated with low-density lipoproteins entering the cell by endocytosis also follows a trafficking pathway similar to that of ASFV. Here we show that cholesterol plays an essential role in the establishment of infection as the virus traffics through the endocytic pathway. In contrast to the case for other DNA viruses, such as vaccinia virus or adenovirus 5, cholesterol efflux from endosomes is required for ASFV release/entry to the cytosol. Accumulation of cholesterol in endosomes impairs fusion, resulting in retention of virions inside endosomes. ASFV also remodels intracellular cholesterol by increasing its cellular uptake and redistributes free cholesterol to viral replication sites. Our analysis reveals that ASFV manipulates cholesterol dynamics to ensure an appropriate lipid flux to establish productive infection.IMPORTANCESince its appearance in the Caucasus in 2007, African swine fever (ASF) has been spreading westwards to neighboring European countries, threatening porcine production. Due to the lack of an effective vaccine, ASF control relies on early diagnosis and widespread culling of infected animals. We investigated early stages of ASFV infection to identify potential cellular targets for therapeutic intervention against ASF. The virus enters the cell by endocytosis, and soon thereafter, viral decapsidation occurs in the acid pH of late endosomes. We found that ASFV infection requires and reorganizes the cellular lipid cholesterol. ASFV requires cholesterol to exit the endosome to gain access to the cytoplasm to establish productive replication. Our results indicate that there is a differential requirement for cholesterol efflux for vaccinia virus or adenovirus 5 compared to ASFV.

scholarly journals Endosomal proteins NPC1 and NPC2 at African swine fever virus entry/fusion

2021 ◽  
Author(s):  
Covadonga Alonso ◽  
Miguel Ángel Cuesta-Geijo ◽  
Jesús Urquiza ◽  
Ana Del Puerto ◽  
Isabel García-Dorival ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
African Swine Fever Virus ◽  
Direct Interaction ◽  
African Swine Fever ◽  
Vero Cells ◽  
Fever Virus ◽  
Endocytic Pathway ◽  
Viral Fusion ◽  
Transporter Protein ◽  
Niemann Pick

African swine fever virus (ASFV) infectious cycle starts with the viral adsorption and entry into the host cell. The virus is internalized via clathrin/dynamin mediated endocytosis and macropinocytosis. As several other viruses, ASF virion is then internalized and incorporated into the endocytic pathway. Endosomal maturation entails luminal acidification and the lowering of pH acting on the multi-layered virion structure dissolves the outer capsid. Upon decapsidation, the inner viral membrane is exposed to interact with the limiting membrane of the late endosome for fusion. Egress from endosome is related to cholesterol efflux, but it remains an intriguing process albeit essential for infection, specifically for the viral nucleic acid exit to the cytoplasm for replication. ASFV proteins E248R and E199L, with structural homology to the VACV proteins of the fusion complex, seem to have similar functions in ASFV. A direct interaction between these ASFV proteins with the cholesterol transporter protein NPC1 (Niemann-Pick C type 1) was observed, which was also shared by the E248R homologous protein L1R of VACV. Binding occurs between the transmembrane domain of E248R with the loop C of NPC1 at the same domain than EBOV binding site. These interactions suggest that these ASFV proteins are crucial for membrane fusion. CRISPR NPC1 KO Vero cells lacking NPC1 protein that were resistant to EBOV, reduced ASFV infection levels significantly. Reductions on ASFV infectivity and replication in NPC1 KO cells were accompanied by lesser viral factories of smaller size and lacking the typical cohesive morphology between endosomes and viral proteins.  We observed a compensatory effect in NPC1 KO cells, elevating NPC2 levels while silencing NPC2 in Vero cells with shRNA, also reduced ASFV infection. Our findings pave the way to understand the role of these proteins at the membrane viral fusion step for several viruses.

African swine fever virus infects macrophages, the natural host cells, via clathrin- and cholesterol-dependent endocytosis

2015 ◽  
Vol 200 ◽  
pp. 45-55 ◽  
Author(s):  
Inmaculada Galindo ◽  
Miguel Angel Cuesta-Geijo ◽  
Karolina Hlavova ◽  
Raquel Muñoz-Moreno ◽  
Lucía Barrado-Gil ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Natural Host ◽  
Fever Virus ◽  
Host Cells

scholarly journals African Swine Fever Virus IAP-Like Protein Induces the Activation of Nuclear Factor Kappa B

2002 ◽  
Vol 76 (8) ◽  
pp. 3936-3942 ◽  
Author(s):  
Clara I. Rodríguez ◽  
María L. Nogal ◽  
Angel L. Carrascosa ◽  
María L. Salas ◽  
Manuel Fresno ◽  
...  
Keyword(s):  
Caspase 3 ◽  
African Swine Fever Virus ◽  
Tumor Necrosis Factor Receptor ◽  
African Swine Fever ◽  
Fever Virus ◽  
Dominant Negative ◽  
Jurkat Cells ◽  
Dominant Negative Mutant ◽  
Iκb Kinase ◽  
Necrosis Factor

ABSTRACT African swine fever virus (ASFV) encodes a homologue of the inhibitor of apoptosis (IAP) that promotes cell survival by controlling the activity of caspase-3. Here we show that ASFV IAP is also able to activate the transcription factor NF-κB. Thus, transient transfection of the viral IAP increases the activity of an NF-κB reporter gene in a dose-responsive manner in Jurkat cells. Similarly, stably transfected cells expressing ASFV IAP have elevated basal levels of c-rel, an NF-κB-dependent gene. NF-κB complexes in the nucleus were increased in A224L-expressing cells compared with control cells upon stimulation with phorbol myristate acetate (PMA) plus ionomycin. This resulted in greater NF-κB-dependent promoter activity in ASFV IAP-expressing than in control cells, both in basal conditions and after PMA plus ionophore stimulation. The elevated NF-κB activity seems to be the consequence of higher IκB kinase (IKK) basal activity in these cells. The NF-κB-inducing activity of ASFV IAP was abrogated by an IKK-2 dominant negative mutant and enhanced by expression of tumor necrosis factor receptor-associated factor 2.

scholarly journals African Swine Fever Virus Uses Macropinocytosis to Enter Host Cells

2012 ◽  
Vol 8 (6) ◽  
pp. e1002754 ◽  
Author(s):  
Elena G. Sánchez ◽  
Ana Quintas ◽  
Daniel Pérez-Núñez ◽  
Marisa Nogal ◽  
Susana Barroso ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Host Cells

scholarly journals African Swine Fever Virus dUTPase Is a Highly Specific Enzyme Required for Efficient Replication in Swine Macrophages

1999 ◽  
Vol 73 (11) ◽  
pp. 8934-8943 ◽  
Author(s):  
Mariano Oliveros ◽  
Ramón García-Escudero ◽  
Alí Alejo ◽  
Eladio Viñuela ◽  
María L. Salas ◽  
...  
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Vero Cells ◽  
Fever Virus ◽  
Parental Virus ◽  
Productive Infection ◽  
Genome Replication ◽  
Specific Enzyme ◽  
Efficient Replication ◽  
Dividing Cells

ABSTRACT The African swine fever virus (ASFV) gene E165R, which is homologous to dUTPases, has been characterized. A multiple alignment of dUTPases showed the conservation in ASFV dUTPase of the motifs that define this protein family. A biochemical analysis of the purified recombinant enzyme showed that the virus dUTPase is a trimeric, highly specific enzyme that requires a divalent cation for activity. The enzyme is most probably complexed with Mg2+, the preferred cation, and has an apparent Km for dUTP of 1 μM. Northern and Western blotting, as well as immunofluorescence analyses, indicated that the enzyme is expressed at early and late times of infection and is localized in the cytoplasm of the infected cells. On the other hand, an ASFV dUTPase-deletion mutant (vΔE165R) has been obtained. Growth kinetics showed that vΔE165R replicates as efficiently as parental virus in Vero cells but only to 10% or less of parental virus in swine macrophages. Our results suggest that the dUTPase activity is dispensable for virus replication in dividing cells but is required for productive infection in nondividing swine macrophages, the natural host cell for the virus. The viral dUTPase may play a role in lowering the dUTP concentration in natural infections to minimize misincorporation of deoxyuridine into the viral DNA and ensure the fidelity of genome replication.

scholarly journals African swine fever virus structural protein p17 inhibits cGAS-STING signaling pathway through interacting with STING

2021 ◽  
Author(s):  
Wanglong Zheng ◽  
Nengwen Xia ◽  
Jia Luo ◽  
Sen Jiang ◽  
Jiajia Zhang ◽  
...  
Keyword(s):  
Signaling Pathway ◽  
Transmembrane Domain ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Host Cells ◽  
Structural Protein ◽  
Swine Industry ◽  
Lack Of Information ◽  
Sting Pathway

African swine fever (ASF) is highly contagious, causes high mortality in domestic and feral swine, and has a significant economic impact on the global swine industry due to the lack of a vaccine or an effective treatment. African swine fever virus (ASFV) encodes more than 150 polypeptides, which may have intricate and delicate interactions with the host for the benefit of the virus to evade the host’s defenses. However, currently, there is still a lack of information regarding the roles of the viral proteins in host cells. Here, our data demonstrated that the p17, encoded by D117L gene could suppress porcine cGAS-STING signaling pathway, exhibiting the inhibitions of TBK1 and IRF3 phosphorylations, downstream promoter activities, cellular mRNA transcriptions and ISG56 induction, and antiviral responses. Further, we found that p17 was located in endoplasmic reticulum (ER) and Golgi apparatus, and interacted with STING, perturbing it in the recruitment of TBK1 and IKKϵ Additionally, it appeared that the transmembrane domain (amino acids 39–59) of p17 could be required for interacting with STING and inhibiting cGAS-STING pathway. Taken together, p17 could inhibit the cGAS-STING pathway through its interaction with STING and interference with STING in the recruitment of TBK1 and IKKϵ

scholarly journals Induction of Robust Immune Responses in Swine by Using a Cocktail of Adenovirus-Vectored African Swine Fever Virus Antigens

2016 ◽  
Vol 23 (11) ◽  
pp. 888-900 ◽  
Author(s):  
Shehnaz Lokhandwala ◽  
Suryakant D. Waghela ◽  
Jocelyn Bray ◽  
Cameron L. Martin ◽  
Neha Sangewar ◽  
...  
Keyword(s):  
Immune Responses ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Host Cells ◽  
Hemorrhagic Disease ◽  
Virulent Strains ◽  
Challenge Study ◽  
Cellular Immune

ABSTRACTThe African swine fever virus (ASFV) causes a fatal hemorrhagic disease in domestic swine, and at present no treatment or vaccine is available. Natural and gene-deleted, live attenuated strains protect against closely related virulent strains; however, they are yet to be deployed and evaluated in the field to rule out chronic persistence and a potential for reversion to virulence. Previous studies suggest that antibodies play a role in protection, but induction of cytotoxic T lymphocytes (CTLs) could be the key to complete protection. Hence, generation of an efficacious subunit vaccine depends on identification of CTL targets along with a suitable delivery method that will elicit effector CTLs capable of eliminating ASFV-infected host cells and confer long-term protection. To this end, we evaluated the safety and immunogenicity of an adenovirus-vectored ASFV (Ad-ASFV) multiantigen cocktail formulated in two different adjuvants and at two immunizing doses in swine. Immunization with the cocktail rapidly induced unprecedented ASFV antigen-specific antibody and cellular immune responses against all of the antigens. The robust antibody responses underwent rapid isotype switching within 1 week postpriming, steadily increased over a 2-month period, and underwent rapid recall upon boost. Importantly, the primed antibodies strongly recognized the parental ASFV (Georgia 2007/1) by indirect fluorescence antibody (IFA) assay and Western blotting. Significant antigen-specific gamma interferon-positive (IFN-γ+) responses were detected postpriming and postboosting. Furthermore, this study is the first to demonstrate induction of ASFV antigen-specific CTL responses in commercial swine using Ad-ASFV multiantigens. The relevance of the induced immune responses in regard to protection needs to be evaluated in a challenge study.

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