scholarly journals Crystal Structure of African Swine Fever Virus A179L with the Autophagy Regulator Beclin

2019 ◽  
Author(s):  
Suresh Banjara ◽  
Gareth Shimmon ◽  
Linda Dixon ◽  
Christopher Netherton ◽  
Mark Hinds ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cell Death ◽  
Ligand Binding ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
B Cell Lymphoma ◽  
Vero Cells ◽  
Fever Virus ◽  
Site Directed Mutagenesis ◽  
Binding Groove

Subversion of programmed cell death-based host defence systems is a prominent feature of infections by large DNA viruses. African swine fever virus (ASFV) is a large DNA virus and sole member of the Asfarviridae family that harbors the B-cell lymphoma 2 or Bcl-2 homolog A179L. A179L has been shown to bind to a range of cell death inducing host proteins including pro-apoptotic Bcl-2 proteins as well as the autophagy regulator Beclin. Here we report the crystal structure of A179L bound to the Beclin BH3 motif. A179L engages Beclin using the same canonical ligand binding groove that is utilized to bind to pro-apoptotic Bcl-2 proteins. The mode of binding of Beclin to A179L mirrors that of Beclin binding to human Bcl-2 and Bcl-xL as well as murine gamma-herpesvirus 68. Introduction of bulky hydrophobic residues into the A179L ligand binding groove via site directed mutagenesis ablates binding of Beclin to A179L, leading to a loss of ability of A179L to modulate autophagosome formation in Vero cells during starvation. Our findings provide a mechanistic understanding for the potent autophagy inhibitory activity of A179L and serve as a platform for more detailed investigations into the role of autophagy during ASFV infection.

scholarly journals Crystal Structure of African Swine Fever Virus A179L with the Autophagy Regulator Beclin

Viruses ◽  
2019 ◽  
Vol 11 (9) ◽  
pp. 789 ◽  
Author(s):  
Suresh Banjara ◽  
Gareth L. Shimmon ◽  
Linda K. Dixon ◽  
Christopher L. Netherton ◽  
Mark G. Hinds ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cell Death ◽  
Ligand Binding ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
B Cell Lymphoma ◽  
Vero Cells ◽  
Fever Virus ◽  
Site Directed Mutagenesis ◽  
Binding Groove

Subversion of programmed cell death-based host defence systems is a prominent feature of infections by large DNA viruses. African swine fever virus (ASFV) is a large DNA virus and sole member of the Asfarviridae family that harbours the B-cell lymphoma 2 or Bcl-2 homolog A179L. A179L has been shown to bind to a range of cell death-inducing host proteins, including pro-apoptotic Bcl-2 proteins as well as the autophagy regulator Beclin. Here we report the crystal structure of A179L bound to the Beclin BH3 motif. A179L engages Beclin using the same canonical ligand-binding groove that is utilized to bind to pro-apoptotic Bcl-2 proteins. The mode of binding of Beclin to A179L mirrors that of Beclin binding to human Bcl-2 and Bcl-xL as well as murine γ-herpesvirus 68. The introduction of bulky hydrophobic residues into the A179L ligand-binding groove via site-directed mutagenesis ablates binding of Beclin to A179L, leading to a loss of the ability of A179L to modulate autophagosome formation in Vero cells during starvation. Our findings provide a mechanistic understanding for the potent autophagy inhibitory activity of A179L and serve as a platform for more detailed investigations into the role of autophagy during ASFV infection.

scholarly journals African Swine Fever Virus IAP Homologue Inhibits Caspase Activation and Promotes Cell Survival in Mammalian Cells

2001 ◽  
Vol 75 (6) ◽  
pp. 2535-2543 ◽  
Author(s):  
Marı́a L. Nogal ◽  
Gonzalo González de Buitrago ◽  
Clara Rodrı́guez ◽  
Beatriz Cubelos ◽  
Angel L. Carrascosa ◽  
...  
Keyword(s):  
Cell Death ◽  
Caspase 3 ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Vero Cells ◽  
Proteolytic Processing ◽  
Fever Virus ◽  
Caspase Activation ◽  
Apoptosis Protein ◽  
Infected Cells

ABSTRACT African swine fever virus (ASFV) A224L is a member of the inhibitor of apoptosis protein (IAP) family. We have investigated the antiapoptotic function of the viral IAP both in stably transfected cells and in ASFV-infected cells. A224L was able to substantially inhibit caspase activity and cell death induced by treatment with tumor necrosis factor alpha and cycloheximide or staurosporine when overexpressed in Vero cells by gene transfection. We have also observed that ASFV infection induces caspase activation and apoptosis in Vero cells. Furthermore, using a deletion mutant of ASFV lacking the A224L gene, we have shown that the viral IAP modulates the proteolytic processing of the effector cell death protease caspase-3 and the apoptosis which are induced in the infected cells. Our findings indicate that A224L interacts with the proteolytic fragment of caspase-3 and inhibits the activity of this protease during ASFV infection. These observations could indicate a conserved mechanism of action for ASFV IAP and other IAP family members to suppress apoptosis.

scholarly journals Crystal Structure of African Swine Fever Virus pS273R Protease and Implications for Inhibitor Design

2020 ◽  
Vol 94 (10) ◽  
Author(s):  
Guobang Li ◽  
Xiaoxia Liu ◽  
Mengyuan Yang ◽  
Guangshun Zhang ◽  
Zhengyang Wang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Structural Information ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Hydrolytic Activity ◽  
Viral Disease ◽  
Fever Virus ◽  
Dna Virus ◽  
Core Domain ◽  
The Core

ABSTRACT African swine fever (ASF) is a highly contagious hemorrhagic viral disease of domestic and wild pigs that is responsible for serious economic and production losses. It is caused by the African swine fever virus (ASFV), a large and complex icosahedral DNA virus of the Asfarviridae family. Currently, there is no effective treatment or approved vaccine against the ASFV. pS273R, a specific SUMO-1 cysteine protease, catalyzes the maturation of the pp220 and pp62 polyprotein precursors into core-shell proteins. Here, we present the crystal structure of the ASFV pS273R protease at a resolution of 2.3 Å. The overall structure of the pS273R protease is represented by two domains named the “core domain” and the N-terminal “arm domain.” The “arm domain” contains the residues from M1 to N83, and the “core domain” contains the residues from N84 to A273. A structure analysis reveals that the “core domain” shares a high degree of structural similarity with chlamydial deubiquitinating enzyme, sentrin-specific protease, and adenovirus protease, while the “arm domain” is unique to ASFV. Further, experiments indicated that the “arm domain” plays an important role in maintaining the enzyme activity of ASFV pS273R. Moreover, based on the structural information of pS273R, we designed and synthesized several peptidomimetic aldehyde compounds at a submolar 50% inhibitory concentration, which paves the way for the design of inhibitors to target this severe pathogen. IMPORTANCE African swine fever virus, a large and complex icosahedral DNA virus, causes a deadly infection in domestic pigs. In addition to Africa and Europe, countries in Asia, including China, Vietnam, and Mongolia, were negatively affected by the hazards posed by ASFV outbreaks in 2018 and 2019, at which time more than 30 million pigs were culled. Until now, there has been no vaccine for protection against ASFV infection or effective treatments to cure ASF. Here, we solved the high-resolution crystal structure of the ASFV pS273R protease. The pS273R protease has a two-domain structure that distinguishes it from other members of the SUMO protease family, while the unique “arm domain” has been proven to be essential for its hydrolytic activity. Moreover, the peptidomimetic aldehyde compounds designed to target the substrate binding pocket exert prominent inhibitory effects and can thus be used in a potential lead for anti-ASFV drug development.

Saturable binding sites mediate the entry of African swine fever virus into VERO cells

Virology ◽  
1989 ◽  
Vol 168 (2) ◽  
pp. 393-398 ◽  
Author(s):  
Antonio Alcamí ◽  
Angel L. Carrascosa ◽  
Eladio Viñuela
Keyword(s):  
Binding Sites ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Vero Cells ◽  
Fever Virus ◽  
Saturable Binding

scholarly journals Identification of the principal serological immunodeterminants of African swine fever virus by screening a virus cDNA library with antibody

2002 ◽  
Vol 83 (6) ◽  
pp. 1331-1342 ◽  
Author(s):  
S. D. Kollnberger ◽  
B. Gutierrez-Castañeda ◽  
M. Foster-Cuevas ◽  
A. Corteyn ◽  
R. M. E. Parkhouse
Keyword(s):  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Vero Cells ◽  
Polyclonal Antiserum ◽  
Fever Virus ◽  
Open Reading Frames ◽  
Cdna Expression Library ◽  
Lambda Phage ◽  
Cellular Mechanisms ◽  
Expression Library

Protective immunity to African swine fever virus (ASFV) may involve a combination of both serological and cellular mechanisms. This work is focused on the identification of the possible relevant serological immunodeterminants of immunity. Thus, 14 serological immunodeterminants of ASFV have been characterized by exhaustive screening of a representative lambda phage cDNA expression library of the tissue culture-adapted Ba71V strain of ASFV. The library was constructed using RNA extracted from Vero cells infected for 3, 6, 9 and 12 h. A total of 150 clones was selected arbitrarily by antibody screening of the library with a polyclonal antiserum from a domestic pig surviving infection with the virulent Malta isolate of ASFV. Sequencing of these clones permitted identification of 14 independent viral proteins that stimulated an antibody response. These included six proteins encoded by previously unassigned open reading frames (ORFs) (B602L, C44L, CP312R, E184L, K145R and K205R) as well as some of the more well-studied structural (A104R, p10, p32, p54 and p73) and non-structural proteins (RNA reductase, DNA ligase and thymidine kinase). Immunogenicity of these proteins was confirmed by demonstrating the corresponding antibodies in sera from pigs infected either with the Malta isolate or with the OURT88/3–OURT88/1 isolate combination. Furthermore, the majority of these ORFs were also recognized by immune antiserum from the natural host, the bush pig, following secondary challenge with the virulent Malawi (SINT90/1) isolate of ASFV. Thus, it is possible that some of these determinants may be important in protection against virus infection.

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.

scholarly journals Crystal structure of the African swine fever virus structural protein p35 reveals its role for core shell assembly

2020 ◽  
Vol 11 (8) ◽  
pp. 600-605
Author(s):  
Guobang Li ◽  
Dan Fu ◽  
Guangshun Zhang ◽  
Dongming Zhao ◽  
Mingyu Li ◽  
...  
Keyword(s):  
Crystal Structure ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Fever Virus ◽  
Core Shell ◽  
Structural Protein
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