The African Swine Fever Virus DP71L Protein Recruits the Protein Phosphatase 1 Catalytic Subunit To Dephosphorylate eIF2α and Inhibits CHOP Induction but Is Dispensable for These Activities during Virus Infection

ABSTRACT The African swine fever virus (ASFV) DP71L protein is present in all isolates as either a short form of 70 to 72 amino acids or a long form of about 184 amino acids, and both of these share sequence similarity to the C-terminal domain of the herpes simplex virus ICP34.5 protein and cellular protein GADD34. In the present study we expressed DP71L in different mammalian cells and demonstrated that DP71L causes dephosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α) in resting cells and during chemical-induced endoplasmic reticulum stress and acts to enhance expression of cotransfected reporter genes. We showed that DP71L binds to all the three isoforms (α, β, and γ) of the protein phosphatase 1 catalytic subunit (PP1c) and acts by recruiting PP1c to eIF2α. We also showed that DP71L inhibits the induction of ATF4 and its downstream target, CHOP. We investigated the eIF2α phosphorylation status and induction of CHOP in porcine macrophages infected by two ASFV field isolates, Malawi Lil20/1 and Benin 97/1, and two DP71L deletion mutants, MalawiΔNL and E70ΔNL. Our results showed that deletion of the DP71L gene did not cause an increase in the level of eIF2α phosphorylation or induction of CHOP, indicating that DP71L is not the only factor required by the virus to control the phosphorylation level of eIF2α during infection. We therefore hypothesize that ASFV has other mechanisms to prevent the eIF2α phosphorylation and the subsequent protein synthesis inhibition.

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The MyD116 African Swine Fever Virus Homologue Interacts with the Catalytic Subunit of Protein Phosphatase 1 and Activates Its Phosphatase Activity

Journal of Virology ◽

10.1128/jvi.02077-06 ◽

2007 ◽

Vol 81 (6) ◽

pp. 2923-2929 ◽

Cited By ~ 37

Author(s):

José Rivera ◽

Charles Abrams ◽

Bruno Hernáez ◽

Alberto Alcázar ◽

José M. Escribano ◽

...

Keyword(s):

Protein Phosphatase ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Catalytic Subunit ◽

Protein Phosphatase 1 ◽

Initiation Factor ◽

Wild Type ◽

Α Subunit ◽

Initiation Factor 2

ABSTRACT The DP71L protein of African swine fever virus (ASFV) shares sequence similarity with the herpes simplex virus ICP34.5 protein over a C-terminal domain. We showed that the catalytic subunit of protein phosphatase 1 (PP1) interacts specifically with the ASFV DP71L protein in a yeast two-hybrid screen. The chimeric full-length DP71L protein, from ASFV strain Badajoz 71 (BA71V), fused to glutathione S-transferase (DP71L-GST) was expressed in Escherichia coli and shown to bind specifically to the PP1-α catalytic subunit expressed as a histidine fusion protein (6×His-PP1α) in E. coli. The functional effects of this interaction were investigated by measuring the levels of PP1 and PP2A in ASFV-infected Vero cells. This showed that infection with wild-type ASFV strain BA71V activated PP1 between two- and threefold over that of mock-infected cells. This activation did not occur in cells infected with the BA71V isolate in which the DP71L gene had been deleted, suggesting that expression of DP71L leads to PP1 activation. In contrast, no effect was observed on the activity of PP2A following ASFV infection. We showed that infection of cells with wild-type BA71V virus resulted in decreased phosphorylation of the α subunit of eukaryotic initiation factor 2 (eIF-2α). ICP34.5 recruits PP1 to dephosphorylate the α subunit of eukaryotic translational initiation factor 2 (also known as eIF-2α); possibly the ASFV DP71L protein has a similar function.

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An eIF2α-binding motif in protein phosphatase 1 subunit GADD34 and its viral orthologs is required to promote dephosphorylation of eIF2α

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1501557112 ◽

2015 ◽

Vol 112 (27) ◽

pp. E3466-E3475 ◽

Cited By ~ 35

Author(s):

Margarito Rojas ◽

Gabriel Vasconcelos ◽

Thomas E. Dever

Keyword(s):

Protein Phosphatase ◽

African Swine Fever Virus ◽

Consensus Sequence ◽

Point Mutations ◽

Translation Initiation Factor ◽

Protein Phosphatase 1 ◽

Initiation Factor ◽

Protective Mechanism ◽

Binding Motif ◽

Α Subunit

Transient protein synthesis inhibition, mediated by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α), is an important protective mechanism cells use during stress conditions. Following relief of the stress, the growth arrest and DNA damage-inducible protein GADD34 associates with the broadly acting serine/threonine protein phosphatase 1 (PP1) to dephosphorylate eIF2α. Whereas the PP1-binding motif on GADD34 has been defined, it remains to be determined how GADD34 directs PP1 to specifically dephosphorylate eIF2α. In this report, we map a novel eIF2α-binding motif to the C terminus of GADD34 in a region distinct from where PP1 binds to GADD34. This motif is characterized by the consensus sequence Rx[Gnl]x1–2Wxxx[Arlv]x[Dn][Rg]xRFxx[Rlvk][Ivc], where capital letters are preferred and x is any residue. Point mutations altering the eIF2α-binding motif impair the ability of GADD34 to interact with eIF2α, promote eIF2α dephosphorylation, and suppress PKR toxicity in yeast. Interestingly, this eIF2α-docking motif is conserved among viral orthologs of GADD34, and is necessary for the proteins produced by African swine fever virus, Canarypox virus, and Herpes simplex virus to promote eIF2α dephosphorylation. Taken together, these data indicate that GADD34 and its viral orthologs direct specific dephosphorylation of eIF2α by interacting with both PP1 and eIF2α through independent binding motifs.

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Identification of residues within the African swine fever virus DP71L protein required for dephosphorylation of translation initiation factor eIF2α and inhibiting activation of pro-apoptotic CHOP

Virology ◽

10.1016/j.virol.2017.02.002 ◽

2017 ◽

Vol 504 ◽

pp. 107-113 ◽

Cited By ~ 9

Author(s):

Claire Barber ◽

Chris Netherton ◽

Lynnette Goatley ◽

Alice Moon ◽

Steve Goodbourn ◽

...

Keyword(s):

Translation Initiation ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Fever Virus ◽

Translation Initiation Factor ◽

Initiation Factor

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Interaction of Structural Glycoprotein E2 of Classical Swine Fever Virus with Protein Phosphatase 1 Catalytic Subunit Beta (PPP1CB)

Viruses ◽

10.3390/v11040307 ◽

2019 ◽

Vol 11 (4) ◽

pp. 307 ◽

Cited By ~ 4

Author(s):

Elizabeth Vuono ◽

Elizabeth Ramirez-Medina ◽

Lauren Holinka ◽

Ryan Baker-Branstetter ◽

Manuel Borca ◽

...

Keyword(s):

Classical Swine Fever Virus ◽

Protein Phosphatase ◽

Specific Binding ◽

Classical Swine Fever ◽

Viral Envelope ◽

Fever Virus ◽

Catalytic Subunit ◽

Protein Phosphatase 1 ◽

Proximity Ligation Assay ◽

Negative Effect

Classical swine fever virus (CSFV) E2 protein, the major virus structural glycoprotein, is an essential component of the viral envelope. E2 is involved in virus absorption, induction of a protective immune response and is critical for virulence in swine. Using the yeast two-hybrid system, we identified protein phosphatase 1 catalytic subunit beta (PPP1CB), which is part of the Protein Phosphatase 1 (PP1) complex, as a specific binding host partner for E2. We further confirmed the occurrence of this interaction in CSFV-infected swine cells by using two independent methodologies: Co-immunoprecipitation and Proximity Ligation Assay. In addition, we demonstrated that pharmacological activation of the PP1 pathway has a negative effect on CSFV replication while inhibition of the PP1 pathway or knockdown of PPP1CB by siRNA had no observed effect. Overall, our data suggests that the CSFV E2 and PPP1CB protein interact in infected cells, and that activation of the PP1 pathway decreases virus replication.

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African swine fever virus regulates host energy and amino acid metabolism to promote viral replication

Journal of Virology ◽

10.1128/jvi.01919-21 ◽

2021 ◽

Author(s):

Qiao Xue ◽

Huisheng Liu ◽

Zixiang Zhu ◽

Fan Yang ◽

Yingying Song ◽

...

Keyword(s):

Amino Acids ◽

Immune Responses ◽

African Swine Fever Virus ◽

African Swine Fever ◽

Tca Cycle ◽

Innate Immune ◽

Fever Virus ◽

Innate Immune Responses ◽

First Time ◽

Self Replication

African swine fever is one of the most serious viral diseases caused by African swine fever virus (ASFV). The metabolic changes induced by ASFV infection remain unknown. Here, PAMs infected with ASFV was analyzed by ultra-high-performance liquid chromatography/quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS) in combination with multivariate statistical analysis. A total of 90 metabolites were significantly changed after ASFV infection, and most of them belong to amino acids and TCA cycle intermediates. ASFV infection induced increase of most of amino acids in host during the early stages of infection, and amino acids decreased in the late stages of infection. ASFV infection did not significantly affected glycolysis pathway, whereas it induced the increase of citrate, succinate, α-ketoglutarate, and oxaloacetate levels in the TCA cycle, suggesting that ASFV infection promoted TCA cycle. The activity of aspartate aminotransferase and glutamate production were significantly elevated in ASFV-infected cells and pigs, resulting in reversible transition between TCA cycle and amino acids synthesis. Aspartate, glutamate, and TCA cycle were essential for ASFV replication. In addition, ASFV infection induced an increase in lactate level using lactate dehydrogenase, which led to low expression of IFN-β and increased of ASFV replication. Our data, for the first time, indicated that ASFV infection controls IFN-β production through RIG-I-mediated signaling pathways. These data identified a novel mechanism evolved by ASFV to inhibit host innate immune responses, and will provide insights for development of new preventive or therapeutic strategies targeting the altered metabolic pathways. IMPORTANCE In order to promote viral replication, viruses often cause severe immunosuppression and seize organelles to synthesize a large number of metabolites required for self-replication. African swine fever virus (ASFV) has developed many strategies to evade host innate immune responses. However, the impact of ASFV infection on host cellular metabolism remains unknown. Here, for the first time, we analyzed the metabolomic profiles of ASFV-infected PAMs cells. ASFV infection increased host TCA cycle and amino acids metabolism. Aspartate, glutamate, and TCA cycle promoted ASFV replication. ASFV infection also induced the increase of lactate production to inhibit innate immune responses for self-replication. This study identified novel immune evasion mechanisms utilized by ASFV and provided viewpoints on ASFV-host interactions, which is critical for guiding the design of new prevention strategies against ASFV targeting the altered metabolic pathways.

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