Regulation of de novo translation of host cells by manipulation of PERK/PKR and GADD34-PP1 activity during Newcastle disease virus infection

Viral infections result in cellular stress responses, which can trigger protein translation shutoff via phosphorylation of eukaryotic initiation factor 2 alpha (eIF2 alpha). Newcastle disease virus (NDV) causes severe disease in poultry and selectively kills human tumour cells. In this report, we determined that infection of HeLa human cervical cancer cells and DF-1 chicken fibroblast cells with NDV maintained protein at early infection times, 0-12 h post-infection (p.i.), and gradually inhibited global protein translation at late infection times, 12-24 h p.i. Mechanistic studies showed that translation inhibition at late infection times was accompanied by phosphorylation of eIF2 alpha, a checkpoint of translation initiation. Meanwhile, the eIF2 alpha kinase, PKR, was upregulated and activated by phosphorylation and another eIF2 alpha kinase, PERK, was phosphorylated and cleaved into two fragments. Pharmacological inhibition experiments revealed that only PKR activity was required for eIF2 alpha phosphorylation, suggesting that recognition of viral dsRNA by PKR was responsible for translation shutoff. High levels of phospho-eIF2 alpha led to preferential translation of the transcription factor ATF4 and an increase in GADD34 expression. Functionally, GADD34, in conjunction with PP1, dephosphorylated eIF2 alpha and restored protein translation, benefiting virus protein synthesis. However, PP1 was degraded at late infection times, functionally counteracting the upregulation of GADD34. Taken together, our data support that NDV-induced translation shutoff at late infection times was attributed to sustaining phosphorylation of eIF2 alpha, which is mediated by continual activation of PKR and degradation of PP1.