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Phosphorylation of enteroviral 2Apro at Ser/Thr125 benefits its proteolytic activity and viral pathogenesis - PubMed

Phosphorylation of enteroviral 2Apro at Ser/Thr125 benefits its proteolytic activity and viral pathogenesis

Yuya Wang et al. J Med Virol. 2023 Jan.

Abstract

Enteroviral 2A proteinase (2Apro ), a well-established and important viral functional protein, plays a key role in shutting down cellular cap-dependent translation, mainly via its proteolytic activity, and creating optimal conditions for Enterovirus survival. Accumulated data show that viruses take advantage of various signaling cascades for their life cycle; studies performed by us and others have demonstrated that the extracellular signal-regulated kinase (ERK) pathway is essential for enterovirus A71 (EV-A71) and other viruses replication. We recently showed that ERK1/2 is required for the proteolytic activity of viral 2Apro ; however, the mechanism underlying the regulation of 2Apro remains unknown. Here, we demonstrated that the 125th residue Ser125 of EV-A71 2Apro or Thr125 of coxsackievirus B3 2Apro , which is highly conserved in the Enterovirus, was phosphorylated by ERK1/2. Importantly, 2Apro with phosphor-Ser/Thr125 had much stronger proteolytic activity toward eukaryotic initiation factor 4GI and rendered the virus more efficient for multiplication and pathogenesis in hSCARB2 knock-in mice than that in nonphospho-Ser/Thr125A (S/T125A) mutants. Notably, phosphorylation-mimic mutations caused deleterious changes in 2Apro catalytic function (S/T125D/E) and in viral propagation (S125D). Crystal structure simulation analysis showed that Ser125 phosphorylation in EV-A71 2Apro enabled catalytic Cys to adopt an optimal conformation in the catalytic triad His-Asp-Cys, which enhances 2Apro proteolysis. Therefore, we are the first to report Ser/Thr125 phosphorylation of 2Apro increases enteroviral adaptation to the host to ensure enteroviral multiplication, causing pathogenicity. Additionally, weakened viruses containing a S/T125A mutation could be a general strategy to develop attenuated Enterovirus vaccines.

Keywords: eIF4GI; enteroviral 2Apro; phosphorylation; proteolytic activity; viral pathogenesis.

© 2022 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1

Effect of ERK phosphorylation on 2A‐mediated eIF4GI cleavage of EV‐A71 and CVB3. (A) Human embryonic kidney (HEK) 293 cells transfected with 2 μg of A71‐2A‐wt, B3‐2A‐wt, or vector as a control were harvested and lysed at 12, 24, and 36 h posttransfection for WB analysis. (B) HEK 293 cells transfected with A71‐2A‐mut, B3‐2A‐mut, or vector were harvested and lysed at 24 h posttransfection for WB analysis. (C) RD cells preinfected with 1 multiplicity of infection of EV‐A71 or CVB3 for 2 h were incubated with 10, 20, and 30 μmol/L U0 for 24 h and lysed for WB analysis. (D) HEK 293 cells transfected with 2 μg A71‐2A‐wt or B3‐2A‐wt were incubated with U0 at different concentrations and lysed for WB analysis. (E) HEK 293 cells pretransfected with siNC, msiERK1+2, or siERK1+2 for 12 h to knock down ERK1/2 were transfected with A71‐2A‐wt or B3‐2A‐wt and then lysed for WB analysis. 50 μg protein was used for 2A's (EGFP‐2A‐His) examination. The ratio of eIF4GI (or VP1/p‐ERKs/t‐ERKs) to the respective vector or NC group signals shown in Arabic numeric in (A–E) was determined using densitometric scanning. The experiments were repeated three times. A71‐2A‐wt, eukaryotic protein expression plasmid of EV‐A71 2Apro wild‐type; B3‐2A‐wt, eukaryotic protein expression plasmid of CVB3‐2Apro wild‐type; vector, eukaryotic protein expression empty plasmid; A71‐2A‐mut, plasmid A71‐2Apro catalytic triad mutant; B3‐2A‐mut, plasmid CVB3‐2Apro catalytic triad mutant. CP, cleavage products; CVB3, coxsackievirus B3; eIF4GⅠ, eukaryotic translation initiation factor 4γ I; ERK, extracellular‐signal regulated kinase; EV‐A71, enterovirus A71; RD, rhabdomyosarcoma; U0, U0126; WB, western blotting.

Figure 2
Figure 2

Identification of EV‐A71 2A phosphorylation at Ser125 by activated ERK1/2. (A) HEK 293 cells transfected with 2 μg of A71‐2A‐mut or vector were lysed at 48 h posttransfection and collected by pull down for WB analysis. (B) HEK 293 cells transfected with A71‐2A‐mut or vector were harvested with U0126 (30 μM) or Sorafenib (2 μM) for 48 h and collected by His pull‐down for WB analysis. (C) HEK 293 cells transfected with A71‐2A‐mut or vector were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X‐100, and stained with anti‐p‐ERK (red); A71‐2A‐mut was stained with EGFP fusion protein (green), and nuclear DNA with DAPI (blue). The cells were observed using confocal microscopy (LSM 700; Zeiss). Scale bar = 50 μm. Yellow arrows indicate target proteins. (D) HEK 293 cells transfected with A71‐2A‐mut and GST‐ERK2 were lysed after 48 h, and proteins were purified and mixed with buffer with activated ERK2 or kinase‐dead ERK2 in vitro. (E) Distribution of 13 individual Ser residues on A71‐2A. (F) HEK 293 cells transfected with S‐to‐A mutations were collected after 48 h for WB analysis. (G) HEK 293 and RD cells transfected with A71‐2A‐mut or A71‐2A‐mut‐S125A for 24 h were fixed, permeabilized, and stained, as shown in (C), and were observed using confocal microscopy. Scale bar = 10 μm. Yellow arrows indicate target protein interactions. (H) Phosphorylation in vitro was conducted as shown in (D) with either A71‐2A‐mut or A71‐2A‐mut‐S125A. The ratios of the p‐Ser to the respective 2A‐His signals shown in Arabic numbers in (A, B, D) were determined using densitometric scanning. The experiments were repeated three times. A71‐2A‐mut, plasmid A71‐2Apro catalytic triad mutant; A71‐2A‐wt, eukaryotic protein expression plasmid of EV‐A71 2Apro wild‐type; vector, eukaryotic protein expression empty plasmid; GST‐ERK2, eukaryotic expression plasmid for the fusion proteins ERK2 and GST. A71‐2A‐mut‐S125A, plasmids carrying the 125 site mutation of the A71‐2Apro catalytic triad mutant. ERK, extracellular‐signal regulated kinase; EV‐A71, enterovirus A71; HEK 293, human embryonic kidney 293; RD, rhabdomyosarcoma; WB, western blotting.

Figure 3
Figure 3

Ser125 phosphorylation favors EV‐A71 2A trans‐cleavage activity and viral propagation. (A) HEK 293 cells, RD, and HeLa cells transfected with A71‐2A‐wt and A71‐2A‐S125A were collected and lysed at 48 h for WB analysis. (B) HEK 293 cells transfected with A71‐2A‐wt and A71‐2A‐S125A were treated with epidermal growth Factor (10 ng/ml) for 24 h and lysed for WB analysis. (C) HEK 293 cells transfected with A71‐2A‐wt and A71‐2A‐S125D were treated with U0126 (30 μM) for 24 h and lysed for WB analysis. (D) HEK 293 cells pretransfected with or without siERK1+2 (30 nM) for 12 h were treated with A71‐2A‐wt or A71‐2A‐S125D for 24 h and lysed for WB analysis. The ratio of the eIFGI (or p‐ERKs/t‐ERKs) to the respective vector group signals shown in Arabic numbers in (A–D) was determined using densitometric scanning. The experiments were repeated three times. (E, F) Cis‐cleavage activity by wt, S125A, or S125D mutation of P1‐2A protein was observed from the pET21a‐P1‐2A set in the bacterial system (E) and RNAs in vitro transcribed from the pIRES P1‐2A set in the eukaryotic system (F). (G, H) wt, S125A, and S125D EV‐A71 genomic RNAs transcribed in vitro were transfected at 3 μg into RD cells. Cytopathic effects were observed at 12, 24, 36, and 48 h (×200 magnification) (G), and cell lysis was detected using WB (H). (I) Intracellular levels of viral particles and the particle levels in the supernatant were measured at 48 h postinfection using absolute. (J) Intracellular viral particles were measured at different time points in RD cells infected with rescued progeny viruses (MOI = 1) from wt, S125A, and S125D EV‐A71 RNA transcripts. Data are presented as mean ± SD (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 versus respective controls, as determined by Student's t test. ERK, extracellular‐signal regulated kinase; EV‐A71, enterovirus A71; HEK 293, human embryonic kidney 293; MOI, multiplicity of infection; RT‐qPCR, reverse‐transcription polymerase chain reaction; WB, western blotting.

Figure 4
Figure 4

2A Ser125 phosphorylation favors EV‐A71 pathogenesis. (A) Survival rate of 4‐week‐old hSCARB2 knock‐in mice (n = 7) intravenously inoculated with wt and S125A EV‐A71 Isehara (Ise‐EV‐A71) (107 TCID50/ml). (B, C) Body weight and scores of clinical symptoms of infected hSCARB2 knock‐in mice. (D) Viral loads of the hind‐limb skeletal muscle and brain at 7 d.p.i. by absolute RT‐qPCR. Data are presented as mean ± standard deviation (n ≥ 3). *** p < 0.001 as determined by Student's t test. (E, F) Waxed section (3 μm) of brain and hind‐limb muscle at 7 d.p.i. (n = 3) for immunofluorescence (LSM700; Zeiss, 50 μm) (E) and hematoxylin and eosin staining (×200). Lymphocytic infiltrations with yellow arrows (F). d.p.i., day postinfection; EV‐A71, enterovirus A71; RT‐qPCR, reverse‐transcription polymerase chain reaction; wt, wild‐type.

Figure 5
Figure 5

Structural conformation of the EV‐A71 2A set. (A) Molecular dynamics simulation of EV‐A71 2A wild‐type (A71‐2A‐wt) (RMSD of 0.943 Å) derived from A71‐2A‐C110A (gray, PBD code: 4FVD). (B) The crystal structure simulation of A71‐2A‐wt with Ser125 phosphorylation (A71‐2A‐pSer125) on the basis of 4FVD. The RMSD of conformational changes between A71‐2A‐wt and A71‐2A‐pSer125 was only 1.444 Å. (C–F) The crystal structure simulation of S‐to‐A mutants A71‐2A‐S125A and S‐to‐D mutants A71‐2A‐S125D on the basis of 4FVD. The HB networks formed by Ser125 (A71‐2A‐wt), phospho‐Ser125 (A71‐2A‐pSer125), A125 (A71‐2A‐S125A), and D125 (A71‐2A‐S125D) and residues in the catalytic site within the structural conformation. The distances were measured among the Asp‐His‐Cys, phospho‐Ser125, A125, and D125 (magenta lines). EV‐A71, enterovirus A71; RMSD, root mean square deviation.

Figure 6
Figure 6

2A phosphorylation at Ser/Thr125 in proteolytic activity is highly conserved in the genus Enterovirus. (A) The amino acid sequences of enterovirus 2A protein were obtained from GenBank and constructed into phylogenetic trees using the neighbor‐joining method. The numbers at the nodes represent bootstrap values. HPeV‐1 (parechovirus‐like), FMDV (aptovirus‐like), EMCV (cardiovirus), and HAV (hepatovirus A) were used as controls. (B) Sequences of enterovirus 2A protein of EV A–L and RV A–C were aligned using MEGA (version 5.05). The relevant sequences of Aphthovirus (family Picornaviridae) were used as controls. The conserved Ser/Thr125 residues are marked in red. (C) HEK 293 cells transfected with 2 μg of B3‐2A‐mut or vector were lysed at 48 h posttransfection and collected by pull down for WB analysis. The ratio of the p‐Thr to 2A‐His signals shown in Arabic numerals was determined using densitometric scanning. (D) Phosphorylation was conducted in vitro as shown in Figure 2D with either B3‐2A‐mut or B3‐2A‐mut‐T125A. (E, F) HEK 293 cells transfected with B3‐2A‐wt and B3‐2A‐T125A or B3‐2A‐T125E were treated with EGF (10 ng/ml) (E) or U0126 (30 μM) (F) for 24 h and lysed for WB analysis. The ratio of the eIF4GI (or p‐ERK) to the respective vector group signals shown in Arabic numerals in (E, F) was determined using densitometric scanning. The experiments were repeated three times. (G, H) RD cells transfected with 3 μg wt or T125A CVB3 genomic RNAs transcribed in vitro were lysed for WB analysis (G), and intracellular levels of viral particles and the particle levels in the supernatant were measured at 48 h post infection. using absolute RT‐qPCR (H). (I) HEK 293 cells transfected with 2 μg plasmid of S/T‐to‐A on 2 A of EV‐A71, CVB3, PV, EV‐D68, and HRV were harvested and lysed at 24 h for WB analysis. Data are presented as mean ± SD (n ≥ 3). *p < 0.05, **p < 0.01, ***p < 0.001 as determined by Student's t test. CVB3, coxsackievirus B3; EGF, epidermal growth factor; HEK 293, human embryonic kidney 293; NJ method, neighbor‐joining method; RT‐qPCR, reverse‐transcription polymerase chain reaction; WB, western blotting.

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