Picornavirus 2A protease regulates stress granule formation to facilitate viral translation

Stress granules (SGs) contain stalled messenger ribonucleoprotein complexes and are related to the regulation of mRNA translation. Picornavirus infection can interfere with the formation of SGs. However, the detailed molecular mechanisms and functions of picornavirus-mediated regulation of SG formation are not clear. Here, we found that the 2A protease of a picornavirus, EV71, induced atypical stress granule (aSG), but not typical stress granule (tSG), formation via cleavage of eIF4GI. Furthermore, 2A was required and sufficient to inhibit tSGs induced by EV71 infection, sodium arsenite, or heat shock. Infection of 2A protease activity-inactivated recombinant EV71 (EV71-2AC110S) failed to induce aSG formation and only induced tSG formation, which is PKR and eIF2α phosphorylation-dependent. By using a Renilla luciferase mRNA reporter system and RNA fluorescence in situ hybridization assay, we found that EV71-induced aSGs were beneficial to viral translation through sequestering only cellular mRNAs, but not viral mRNAs. In addition, we found that the 2A protease of other picornaviruses such as poliovirus and coxsackievirus also induced aSG formation and blocked tSG formation. Taken together, our results demonstrate that, on one hand, EV71 infection induces tSG formation via the PKR-eIF2α pathway, and on the other hand, 2A, but not 3C, blocks tSG formation. Instead, 2A induces aSG formation by cleaving eIF4GI to sequester cellular mRNA but release viral mRNA, thereby facilitating viral translation.Author summary: When cellular translation initiation is stalled, translation initiation complexes aggregate in cytoplasm. We call these aggregations stress granules (SGs), and they can be marked by components such as TIA-1. SGs are always considered to be antiviral structures during viral infection, but viruses also regulate SG formation to facilitate their survival. Here, we show that the 2A protease of EV71 induced TIA-1 foci formation, and we analyzed these TIA-1 foci and found that they were different from typical stress granules (tSGs); thus, we named them atypical stress granules (aSGs). 2A alone could block tSG formation, and we found that protease activity of 2A was required for aSG induction and tSG blockage, but functioned in different ways. When the protease activity of 2A in EV71 was blocked (EV71-2AC110S), the tSGs but not aSGs appeared in infected cells. These tSGs contained cellular and viral mRNAs and translation initiation factors to inhibit viral translation, but aSGs contained only cellular mRNAs to promote viral translation. We propose a model revealing that EV71 escapes cellular antiviral response by manipulating SG formation: 2A transforms the overall translation shutdown system to a selective virally beneficial system by switching from tSGs to aSGs.