African Swine Fever Virus Causes Microtubule-Dependent Dispersal of thetrans-Golgi Network and Slows Delivery of Membrane Protein to the PlasmaMembrane

ABSTRACT Viral interference with secretory cargo is a common mechanism for pathogen immune evasion. Selective down regulation of critical immune system molecules such as major histocompatibility complex (MHC) proteins enables pathogens to mask themselves from their host. African swine fever virus (ASFV) disrupts the trans-Golgi network (TGN) by altering the localization of TGN46, an organelle marker for the distal secretory pathway. Reorganization of membrane transport components may provide a mechanism whereby ASFV can disrupt the correct secretion and/or cell surface expression of host proteins. In the study reported here, we used the tsO45 temperature-sensitive mutant of the G protein of vesicular stomatitis virus to show that ASFV significantly reduces the rate at which the protein is delivered to the plasma membrane. This is linked to a general reorganization of the secretory pathway during infection and a specific, microtubule-dependent disruption of structural components of the TGN. Golgin p230 and TGN46 are separated into distinct vesicles, whereupon TGN46 is depleted. These data suggest that disruption of the TGN by ASFV can slow membrane traffic during viral infection. This may be functionally important because infection of macrophages with virulent isolates of ASFV increased the expression of MHC class I genes, but there was no parallel increase in MHC class I molecule delivery to the plasma membrane.

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The trans Golgi Network Is Lost from Cells Infected with African Swine Fever Virus

Journal of Virology ◽

10.1128/jvi.75.23.11755-11765.2001 ◽

2001 ◽

Vol 75 (23) ◽

pp. 11755-11765 ◽

Cited By ~ 17

Author(s):

Mari McCrossan ◽

Miriam Windsor ◽

Sreenivasan Ponnambalam ◽

John Armstrong ◽

Thomas Wileman

Keyword(s):

Membrane Proteins ◽

Secretory Pathway ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Host Proteins ◽

Viral Membrane ◽

Membrane Compartments ◽

Hydrophobic Amino Acids ◽

Golgi Network

ABSTRACT The cellular secretory pathway is important during the assembly and envelopment of viruses and also controls the transport of host proteins, such as cytokines and major histocompatibility proteins, that function during the elimination of viruses by the immune system. African swine fever virus (ASFV) encodes at least 26 proteins with stretches of hydrophobic amino acids suggesting entry into the secretory pathway (R. J. Yanez, J. M. Rodriguez, M. L. Nogal, L. Yuste, C. Enriquez, J. F. Rodriguez, and E. Vinuela, Virology 208:249–278, 1995). To predict how and where these potential membrane proteins function, we have studied the integrity of the secretory pathway in cells infected with ASFV. Remarkably, ASFV caused complete loss of immunofluorescence signal for the trans Golgi network (TGN) marker protein TGN46 and dispersed the AP1 TGN adapter complex. Loss of TGN46 signal was not due to degradation of TGN46, suggesting redistribution of TGN46 to other membrane compartments. ASFV markedly slowed transport of cathepsin D to lysosomes, demonstrating that loss of TGN structure correlated with loss of TGN function. ASFV shows a tropism for macrophages, and it is possible that ASFV compromises TGN function to augment the activity of viral membrane proteins or to suppress the function of host immunoregulatory proteins.

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The African swine fever virus lectin EP153R modulates the surface membrane expression of MHC class I antigens

Archives of Virology ◽

10.1007/s00705-010-0846-2 ◽

2010 ◽

Vol 156 (2) ◽

pp. 219-234 ◽

Cited By ~ 18

Author(s):

Carolina Hurtado ◽

Maria José Bustos ◽

Aitor G. Granja ◽

Patricia de León ◽

Prado Sabina ◽

...

Keyword(s):

Mhc Class I ◽

African Swine Fever Virus ◽

Surface Membrane ◽

African Swine Fever ◽

Fever Virus ◽

Class I ◽

Membrane Expression ◽

Mhc Class I Antigens

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Polymorphism analysis and supertype definition of swine leukocyte antigen class I molecules in three-way crossbred (Landrace, Duroc, and Yorkshire) pigs: implications for the vaccine development of African swine fever virus

Science China Life Sciences ◽

10.1007/s11427-019-1696-7 ◽

2020 ◽

Vol 63 (10) ◽

pp. 1604-1607

Author(s):

Limin Ba ◽

Zhenbao Wang ◽

William J Liu ◽

Dongxun Wu ◽

Wangzhen Xiang ◽

...

Keyword(s):

Vaccine Development ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Class I ◽

Polymorphism Analysis ◽

Swine Leukocyte Antigen ◽

Leukocyte Antigen ◽

Yorkshire Pigs ◽

Definition Of

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African Swine Fever Virus Structural Protein pE120R Is Essential for Virus Transport from Assembly Sites to Plasma Membrane but Not for Infectivity

Journal of Virology ◽

10.1128/jvi.75.15.6758-6768.2001 ◽

2001 ◽

Vol 75 (15) ◽

pp. 6758-6768 ◽

Cited By ~ 40

Author(s):

Germán Andrés ◽

Ramón Garcı́a-Escudero ◽

Eladio Viñuela ◽

Marı́a L. Salas ◽

Javier M. Rodrı́guez

Keyword(s):

Plasma Membrane ◽

African Swine Fever Virus ◽

Growth Curves ◽

African Swine Fever ◽

Fever Virus ◽

Virus Yield ◽

Virus Infectivity ◽

Structural Protein ◽

Virus Growth ◽

Extracellular Virus

ABSTRACT This report examines the role of African swine fever virus (ASFV) structural protein pE120R in virus replication. Immunoelectron microscopy revealed that protein pE120R localizes at the surface of the intracellular virions. Consistent with this, coimmunoprecipitation assays showed that protein pE120R binds to the major capsid protein p72. Moreover, it was found that, in cells infected with an ASFV recombinant that inducibly expresses protein p72, the incorporation of pE120R into the virus particle is dependent on p72 expression. Protein pE120R was also studied using an ASFV recombinant in which E120R gene expression is regulated by the Escherichia coli lacrepressor-operator system. In the absence of inducer, pE120R expression was reduced about 100-fold compared to that obtained with the parental virus or the recombinant virus grown under permissive conditions. One-step virus growth curves showed that, under conditions that repress pE120R expression, the titer of intracellular progeny was similar to the total virus yield obtained under permissive conditions, whereas the extracellular virus yield was about 100-fold lower than in control infections. Immunofluorescence and electron microscopy demonstrated that, under restrictive conditions, intracellular mature virions are properly assembled but remain confined to the replication areas. Altogether, these results indicate that pE120R is necessary for virus dissemination but not for virus infectivity. The data also suggest that protein pE120R might be involved in the microtubule-mediated transport of ASFV particles from the viral factories to the plasma membrane.

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

Journal of Virology ◽

10.1128/jvi.78.15.7990-8001.2004 ◽

2004 ◽

Vol 78 (15) ◽

pp. 7990-8001 ◽

Cited By ~ 66

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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African Swine Fever Virus Protein pE296R Is a DNA Repair Apurinic/Apyrimidinic Endonuclease Required for Virus Growth in Swine Macrophages

Journal of Virology ◽

10.1128/jvi.80.10.4847-4857.2006 ◽

2006 ◽

Vol 80 (10) ◽

pp. 4847-4857 ◽

Cited By ~ 18

Author(s):

Modesto Redrejo-Rodríguez ◽

Ramón García-Escudero ◽

Rafael J. Yáñez-Muñoz ◽

María L. Salas ◽

José Salas

Keyword(s):

Excision Repair ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Dimensional Structure ◽

Common Mechanism ◽

Virus Protein ◽

Repair System ◽

Ap Endonuclease ◽

Virus Growth

ABSTRACT We show here that the African swine fever virus (ASFV) protein pE296R, predicted to be a class II apurinic/apyrimidinic (AP) endonuclease, possesses endonucleolytic activity specific for AP sites. Biochemical characterization of the purified recombinant enzyme indicated that the Km and catalytic efficiency values for the endonucleolytic reaction are in the range of those reported for Escherichia coli endonuclease IV (endo IV) and human Ape1. In addition to endonuclease activity, the ASFV enzyme has a proofreading 3′→5′ exonuclease activity that is considerably more efficient in the elimination of a mismatch than in that of a correctly paired base. The three-dimensional structure predicted for the pE296R protein underscores the structural similarities between endo IV and the viral protein, supporting a common mechanism for the cleavage reaction. During infection, the protein is expressed at early times and accumulates at later times. The early enzyme is localized in the nucleus and the cytoplasm, while the late protein is found only in the cytoplasm. ASFV carries two other proteins, DNA polymerase X and ligase, that, together with the viral AP endonuclease, could act as a viral base excision repair system to protect the virus genome in the highly oxidative environment of the swine macrophage, the virus host cell. Using an ASFV deletion mutant lacking the E296R gene, we have determined that the viral endonuclease is required for virus growth in macrophages but not in Vero cells. This finding supports the existence of a viral reparative system to maintain virus viability in the infected macrophage.

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Structure and Biochemical Characteristic of the Methyltransferase (MTase) Domain of RNA Capping Enzyme from African Swine Fever Virus

Journal of Virology ◽

10.1128/jvi.02029-20 ◽

2020 ◽

Author(s):

Xuejian Du ◽

Zeng-Qiang Gao ◽

Zhi Geng ◽

Yu-Hui Dong ◽

Heng Zhang

Keyword(s):

Crystal Structure ◽

Active Site ◽

Mrna Stability ◽

African Swine Fever Virus ◽

Substrate Recognition ◽

African Swine Fever ◽

Fever Virus ◽

Class I ◽

End Capping ◽

Rna Capping

African swine fever virus (ASFV) is a complex nucleocytoplasmic large DNA virus (NCLDV) that causes a devastating swine disease and it is urgently needed to develop effective anti-ASFV vaccines and drugs. The process of mRNA 5′-end capping is a common characteristic in eukaryotes and many viruses, and the cap structure is required for mRNA stability and efficient translation. The ASFV protein pNP868R was found to have guanylyltransferase (GTase) activity involved in mRNA capping. Here we report the crystal structure of pNP868R methyltransferase (MTase) domain (referred as pNP868RMT) in complex with S-adenosyl-L-methionine (AdoMet). The structure shows the characteristic core fold of the class I MTase family and the AdoMet is bound in a negative-deep groove. Remarkably, the N-terminal extension of pNP868RMT is ordered and keeps away from the AdoMet-binding site, distinct from the close conformation over the active site of poxvirus RNA capping D1 subunit or the largely disordered conformation in most cellular RNA capping MTases. Structure-based mutagenesis studies based on the pNP868RMT-cap analog complex model revealed essential residues involved in substrate recognition and binding. Functional studies suggest the N-terminal extension may play an essential role in substrate recognition instead of AdoMet-binding. A positively charged path stretching from the N-terminal extension to the region around the active site was suggested to provide a favorable electrostatic environment for the binding and approaching of substrate RNA into the active site. Our structure and biochemical studies provide novel insights into the methyltransfer process of mRNA cap catalyzed by pNP868R. IMPORTANCE African swine fever (ASF) is a highly contagious hemorrhagic viral disease in pigs that is caused by African swine fever virus (ASFV). There are no effective drugs or vaccines for protection against ASFV infection till now. The protein pNP868R was predicted to be responsible for process of mRNA 5′-end capping in ASFV, which is essential for mRNA stability and efficient translation. Here, we solved the high-resolution crystal structure of the methyltransferase (MTase) domain of pNP868R. The MTase domain structure shows a canonical class I MTase family fold and the AdoMet binds into a negative pocket. Structure-based mutagenesis studies revealed critical and conserved residues involved in AdoMet-binding and substrate RNA-binding. Notably, both the conformation and the role in MTase activities of the N-terminal extension are distinct from those of previously characterized poxvirus MTase domain. Our structure-function studies provide the basis for potential anti-ASFV inhibitor design targeting the critical enzyme.

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Mooring stone-like Arg 114 pulls diverse bulged peptides: first insight into African swine fever virus-derived T cell epitopes presented by swine MHC class I

Journal of Virology ◽

10.1128/jvi.01378-21 ◽

2021 ◽

Author(s):

Can Yue ◽

Wangzhen Xiang ◽

Xiaowen Huang ◽

Yuan Sun ◽

Jin Xiao ◽

...

Keyword(s):

T Cells ◽

Mhc Class I ◽

African Swine Fever Virus ◽

Peptide Binding ◽

African Swine Fever ◽

Fever Virus ◽

Swine Leukocyte Antigen ◽

Mhc I ◽

Pork Industry ◽

Leukocyte Antigen

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), which is a devastating pig disease threatening the global pork industry. However, currently no commercial vaccines are available. During the immune response, major histocompatibility complex (MHC) class I molecules select viral peptide epitopes and present them to host cytotoxic T lymphocytes, thereby playing critical roles in eliminating viral infections. Here we screened peptides derived from ASFV and determined the molecular basis of ASFV-derived peptides presented by the swine leukocyte antigen (SLA)-1*0101. We found that peptide binding in SLA-1*0101 differs from the traditional mammalian binding patterns. Unlike the typical B and F pockets used by the common MHC-I molecule, SLA-1*0101 uses the D and F pockets as major peptide anchor pockets. Furthermore, the conformationally stable Arg 114 residue located in the peptide-binding groove (PBG) was highly selective for the peptides. Arg 114 draws negatively charged residues at positions P5 to P7 of the peptides, which led to multiple bulged conformations of different peptides binding to SLA-1*0101 and creating diversity for T cells receptor docking. Thus, the solid Arg 114 residue acts as a “mooring stone” and pulls the peptides into the PBG of SLA-1*0101. Notably, the T cells recognition and activation of p72-derived peptides were verified by SLA-1*0101 tetramer-based flow cytometry in peripheral blood mononuclear cells (PBMCs) of the donor pigs. These results refresh our understanding of MHC I molecular anchor peptides, and provide new insights into vaccine development for the prevention and control of ASF. IMPORTANCE The spread of African swine fever virus (ASFV) has caused enormous losses to the pork industry worldwide. Here, a series of ASFV-derived peptides were identified, which could bind to swine leukocyte antigen SLA-1*0101, a prevalent SLA allele among Yorkshire pigs. The crystal structure of four ASFV-derived peptides and one foot-and-mouth disease virus (FMDV)-derived peptide complexed with SLA-1*0101 revealed an unusual peptide anchoring mode of SLA-1*0101 with D and F pockets as anchoring pockets. Negatively-charged residues are preferred within the middle portion of SLA-1*0101-binding peptides. Notably, we determined an unexpected role of Arg 114 of SLA-1*0101 as a “mooring stone” which pulls the peptide anchoring into the PBG in diverse “M” or “n” shaped conformation. Furthermore, T cells from donor pigs could activate through the recognition of ASFV-derived peptides. Our study sheds light on the uncommon presentation of ASFV peptides by swine MHC I and benefits the development of ASF vaccines.

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