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

Abstract

Viral infections result in cellular stress responses, which can trigger protein translation shutoff via phosphorylation of eukaryotic initiation factor 2 alpha (eIF2a). 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 eIF2a, a checkpoint of translation initiation. Meanwhile, the eIF2a kinase, PKR, was upregulated and activated by phosphorylation and another eIF2a kinase, PERK, was phosphorylated and cleaved into two fragments. Pharmacological inhibition experiments revealed that only PKR activity was required for eIF2a phosphorylation, suggesting that recognition of viral dsRNA by PKR was responsible for translation shutoff. High levels of phospho-eIF2a led to preferential translation of the transcription factor ATF4 and an increase in GADD34 expression. Functionally, GADD34, in conjunction with PP1, dephosphorylated eIF2a 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 eIF2a, which is mediated by continual activation of PKR and degradation of PP1.

Newcastle disease virus induces G0/G1 cell cycle arrest in asynchronously growing cells

“…In order to address this question, we determined the activation of PERK/eIF2 α signaling cascade at different time point postinfection. Coincided with our previous report (Liao et al, 2016), NDV infection activated the PERK/ eIF2α signaling cascade by exhibiting increased phosphorylation of PERK and eIF-2α, as well as the increased level of CHOP expression. (Fig.…”

“…A previous report (Liao et al, 2016) from our lab showed that NDV infection activates the PERK/ eIF2α cascade and inhibits protein translation of the host cells, while viral protein NP levels increase. C/ EBP homologous protein (CHOP) is a downstream effector of PKR/ eIF2α/ATF4 and is also known as "growth arrest and DNA damageinducible gene 153" (GADD153).…”

“…The reduced levels of these complexes could then lead to insufficient pRb hyper-phosphorylation, thereby inhibiting cell cycle progression in the G 0 /G 1 phase. Some reports have indicated that NDV infection can result in a general decrease in cellular protein synthesis (Liao et al, 2016) and the inhibition of translation may contribute to the decreased levels of cyclin D1 and cyclin E. Moreover, NDV infection reportedly will activate the PERK-eIF2α-ATF4 cascade and inhibit protein translation in the host cells. As a downstream effector, CHOP plays an important role in the regulation of apoptosis.…”

“…16 h post-transfection, cells were infected with NDV. PXJ40F plasmid was constructed and preserved in out lab (Liao et al, 2016). Specific sets of small interfering RNA (siRNA) for CHOP as well as nonsense sequence used as scrambled siRNA were purchased from GenePharma (Shanghai China).…”

Genetic responses of inbred chicken lines illustrate importance of eIF2 family and immune-related genes in resistance to Newcastle disease virus

“…PKR is another ISG to be activated after viral infection and is been related to the reduction in protein synthesis 46 . In chicken cells challenged by NDV, PKR undergoes autophosphorylation and phosphorylates the eIF2α subunit 10 . Phosphorylation of eIF2α turns it into an inhibitor of the guanine nucleotide exchange factor eIF2B and prevents the formation of the translation initiation complex eIF2:GTP:tRNA Met that is required for the initiation of protein synthesis 15 .…”