The CP312R protein of African swine fever virus inhibits host protein translation via the BiP/PERK/eIF2α pathway

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  Figure 1. ASFV pCP312R inhibits de novo protein synthesis. A and B ASFV infection inhibits cellular protein synthesis in PAMs and HEK293T cells. PAMs infected with ASFV Pig/HLJ/18 (ASFV-WT) (MOI = 1) were pulsed with puromycin at 18, 24, or 36 h post-infection (hpi) and then subjected to western blotting analysis using anti-p54, anti-puromycin, or anti-β-actin antibodies (A). The inhibition effect was observed in ASFV-P61-infected HEK293T cells (B). C pCP312R inhibits the expression of Renilla luciferase (Rluc). HEK293T cells were cotransfected with pRL-SV40 (0.01 μg) and pCA-CP312R-Flag (0, 0.1, 0.2, or 0.5 μg). At 24 h post-transfection (hpt), the cells were examined by the Rluc assay system. D pCP312R is highly conserved among various ASFV isolates. Multiple sequence alignment analysis of pCP312R of 13 ASFV isolates was conducted on the Jalview software (v2.11.1.4) using the ClustalW algorithm. The 13 ASFV isolates are as follows: genotype I strains (Benin 97/1, E75, L60, BA71, OURT 88/3, and NHV), genotype II strains (Pig/HLJ/18, Georgia_2007/1, and 47/Ss/2008), genotype IX strain (Ken06.Bus), and genotype X strain (Ken05/Tk1). E The transcriptional dynamics of the CP312R gene. PAMs…

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  Figure 2. The ASFV pCP312R inhibits cellular protein synthesis at the translation stage. A and B pCP312R inhibits de novo protein synthesis. HEK293T cells were transfected with an increasing amount of pCA-CP312R-Flag (0, 0.5, 1, 1.5, 2, or 2.5 μg). At 18 h post-transfection (hpt), the cells were pulsed with 3 μM puromycin for 30 min and then subjected to western blotting analysis using anti-Flag, anti-puromycin, or anti-β-actin antibodies (A). HEK293T cells were transfected with pCAGGS or pCA-CP312R-Flag (1 μg each). The cells were pulsed with 3 μM puromycin for 30 min at 6, 12, or 24 hpt and then subjected to western blotting analysis using anti-Flag, anti-puromycin, or anti-β-actin antibodies (B). C Knockdown of CP312R partially restores cellular protein synthesis upon ASFV infection. PAMs were transfected with the siCP312R or a siNC. At 6 hpt, the cells were infected with ASFV-WT (MOI = 0.1). At 12 h post-infection, the cells were pulsed with 3 μM puromycin for 30 min and subjected to western blotting analysis using anti-CP312R, anti-puromycin, or anti-β-actin antibodies. *nonspecific protein band. D pCP312R does not alter mRNA transcription of the Rluc gene. The effects of the…

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  Figure 3. The RNA polymerase-like domain of pCP312R is responsible for the inhibition of cellular protein synthesis. A Schematic diagram of the pCP312R domains. The domains of pCP312R were analyzed by SWISS-MODEL [28], which revealed that the amino acids (aa) 59–163 on pCP312R display an RNA polymerase-like domain (left panel), and the three-dimensional structure diagram of pCP312R (based on PDB:7YEQ) was generated using the ChimeraX software [44] (right panel). The pCP312R-derived mutants, pCP312R-∆R (the mutant pCP312R lacking the RNA polymerase-like domain) and pCP312R-R (harboring the RNA polymerase-like domain of pCP312R), were each fused with a Flag tag in the C-terminus of the target genes. B and C The RNA polymerase-like domain of pCP312R exerts its inhibition of cellular protein synthesis. HEK293T cells were cotransfected with the pRL-SV40 plasmid (0.01 μg) harboring the Rluc gene controlled by the SV40 promoter and each of the following plasmids: pCAGGS, pCA-CP312R-Flag, pCA-CP312R-R-Flag, or pCA-CP312R-∆R-Flag (0.5 μg each). At 24 h post-transfection (hpt), the cell lysates were subjected to Rluc assay (B). HEK293T cells were transfected with pCAGGS, pCA-CP312R-Flag,…

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  Figure 4. The ectopically expressed pCP312R inhibits the expression of BiP. A pCP312R is localized in the endoplasmic reticulum (ER) of the cell. HEK293T cells cotransfected with pCA-CP312R-Flag (0.2 μg) and pDsRed-calnexin (expressing calnexin, an ER marker) (0.5 μg). At 18 h post-transfection (hpt), the cells were analyzed by laser confocal microscopy. Scale bars: 2 μm. B pCP312R is localized in the ER in the ASFV-infected PAMs. PAMs were infected with ASFV-WT (MOI = 1). At 12 h post-infection (hpi), the cells were immunostained with anti-pCP312R and anti-KDEL antibodies. Cell nuclei were stained with DAPI. Scale bars: 2 μm. C ASFV infection induces in the differential expression of BiP. PAMs were infected with ASFV-WT (MOI = 1). At 12, 18, and 24 hpi, the cells were collected for western blotting analysis using anti-BiP, anti-pCP312R, or anti-β-actin antibodies. D The ectopic expression of pCP312R leads to a reduction in BiP expression. HEK293T cells were transfected with an increasing amount of pCA-CP312R-Flag (0, 0.5, 1, 1.5, or 2 μg). At 18 hpt, the cells were collected for western blotting analysis using anti-BiP, anti-Flag, or anti-β-actin antibodies. E Knockdown of CP312R…

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  Figure 5. pCP312R promotes the phosphorylation of PERK and eIF2α. A pCP312R promotes the phosphorylation of PERK in a dose-dependent manner. HEK293T cells were transfected with an increasing amount of pCA-CP312R-Flag (0, 0.5, 1, 1.5, or 2 μg). At 24 h post-transfection (hpt), the cells were collected for western blotting analysis using anti-Flag, anti-p-PERK (Thr982), anti-PERK, or anti-β-actin antibodies. B pCP312R promotes eIF2α phosphorylation in a dose-dependent manner. HEK293T cells were transfected with an increasing amount of pCA-CP312R-Flag (0, 0.5, 1, 1.5, or 2 μg). At 12 hpt, the cells were incubated with 300 nM thapsigargin (Tg) for 8 h, and then the cells were collected for western blotting analysis using anti-Flag, anti-p-eIF2α, anti-eIF2α, or anti-β-actin antibodies. C Inhibition of the cellular protein synthesis by pCP312R is attenuated by ISRIB. HEK293T cells were transfected with an increasing amount of pCA-CP312R-Flag (0, 1, 1.5, 2, or 2.5 μg). At 12 hpt, the cells were treated with ISRIB (0.5 μM) for another 12 h. The cells were pulsed with 3 μM puromycin for 30 min, followed by western blotting analysis using anti-Flag, anti-puromycin, or anti-β-actin antibodies.

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  Figure 6. A schematic model illustrating the inhibition of cellular protein synthesis by the ASFV pCP312R. In addition to interaction with RPS27A [27], pCP312R downregulates the expression of heavy-chain-binding protein (BiP) and promotes the phosphorylation of the PKR-like endoplasmic reticulum kinase (PERK) and the eukaryotic initiation factor 2α (eIF2α), thereby inhibiting cellular protein synthesis.