The glycine locating at random coil of picornaviruses VP3 enhances viral pathogenicity by targeting p53 to promote apoptosis and autophagy

Picornaviruses, comprising important and widespread pathogens of humans and animals, have evolved to control apoptosis and autophagy for their replication and spread. However, the underlying mechanism of the association between apoptosis/autophage and viral pathogenicity remains unclear. In the present study, VP3 of picornaviruses was demonstrated to induce apoptosis and autophagy. Foot-and-mouth disease virus (FMDV), which served as a research model here, can strongly induce both apoptosis and autophagy in the skin lesions. By directly interacting with p53, FMDV-VP3 facilitates its phosphorylation and translocation, resulting in Bcl-2 family-mediated apoptosis and LC3-dependent autophagy. The single residue Gly129 of FMDV-VP3 plays a crucial role in apoptosis and autophagy induction and the interaction with p53. Consistently, the comparison of rescued FMDV with mutated Gly129 and parental virus showed that the Gly129 is indispensable for viral replication and pathogenicity. More importantly, the Gly129 locates at a bend region of random coil structure, the mutation of Gly to Ala remarkably shrunk the volume of viral cavity. Coincidentally, the Gly is conserved in the similarly location of other picornaviruses, including poliovirus (PV), enterovirus 71 (EV71), coxsackievirus (CV) and seneca valley virus (SVA). This study demonstrates that picornaviruses induce apoptosis and autophagy to facilitate its pathogenicity and the Gly is functional site, providing novel insights into picornavirus biology.


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Viral pathogenicity is frequently associated with the ability of the virus to kill host cells.

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Apoptosis, type Ⅰ cell death and the main and most typical pattern of cell death, can be

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Predicting of FMDV, PV and SVA proteins identifies VP3 as inducer of apoptosis and 282 autophagy.

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The function of proteins to regulate cell death is often related to its hydrophobic regions. The

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Coincidentally, the mutation of Gly129 to Ala remarkably reduced FMDV-VP3 induced LC3-II 376 upregulation. The results indicate that the Gly129 also is a key site of FMDV-VP3 induced 377 autophagy ( Figure 4F).

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The location of Gly129 at picornavirus VP3 was analyzed using SWISS-MODEL tool,

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PYMOL software and PDB data bank. We find that the Gly129 is located at a bend region of

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Then, a VP3-induced increase in p53 protein levels was detected in FMDV-VP3 but not 449 empty vector transfected hTERT-BTY cells ( Figure 5F). We next explored which   Previous studies suggested that p53 translocates from the nucleus into to cytoplasm (41).

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To assess the downstream events following the interaction between FMDV-VP3 with p53, a 506 series of co-immunoprecipitation experiments were performed to screen for downstream 507 proteins. hTERT-BTY cells were co-transfected with the Flag-FMDV-VP3-expressing plasmid 508 or p53-HA/pCMV vector. The results showed that only Bad was pulled down by p53-HA, but 509 not with the pCMV-HA vector ( Figure 6A).

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To assess whether VP3 expression is essential for the interaction between p53 and Bad,

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hTERT-BTY cells were transfected with Flag-FMDV-VP3 or the vector. As shown in Figure 6B,

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FMDV-specific antibody titers were measured. There was no significant difference in 609 antibody levels between comparable dose challenge groups, with the exception of the -1 group,

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To assess the histopathology of animals challenged with the two FMDV strains, H&E

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The data indicated that the viral load in rVP3-129/FMDV-challenged guinea pigs was 673 substantially lower than in rVP3/FMDV-challenged animals ( Figure 8C).

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Next, we assessed the rates of apoptosis over time in lesion sites of guinea pigs challenged 675 by the two FMDV strains. There was no significant difference in apoptotic rate between the two 676 groups at the early stage (3 d.p.c.). In contrast, at the mid and late stages (7-15 d.p.c.),

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Finally, apoptosis and autophagy facilitate viral replication and enhance pathogenicity.

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However, whether other picornaviruses proteins regulate cell death and the association of cell 712 death with viral pathogenicity remains unclear. In the present study, to explore novel cell death 713 regulation proteins of picornaviruses, bioinformatics prediction approaches were performed.

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The functions of proteins to regulate cell death are often related to its hydrophobic regions.

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We also found that p53 levels were increased in case of VP3 expression, and

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In summary, the FMDV VP3 protein directly interacts with p53 during infection, and 835 promotes p53 translocation to the mitochondria and its interaction with Bad. This then triggers

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The apoptosis and autophagy occurred at the mid and late stages of FMDV infection, thereby