Differential unfolded protein response during Chikungunya and Sindbis virus infection: CHIKV nsP4 suppresses eIF2α phosphorylation.

Virol J. 2013 Jan 28;10:36.

Rathore AP, Ng ML, Vasudevan SG.

Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, 8-College Road, Singapore 169857, Singapore.

 

Abstract

Chikungunya (CHIKV) and Sindbis (SINV) are arboviruses belonging to the alphavirus genus within the Togaviridae family. They cause frequent epidemics of febrile illness and long-term arthralgic sequelae that affect millions of people each year. Both viruses replicate prodigiously in infected patients and in vitro in mammalian cells, suggesting some level of control over the host cellular translational machinery that senses and appropriately directs the cell’s fate through the unfolded protein response (UPR). The mammalian UPR involves BIP (or GRP78), the master sensor in the endoplasmic reticulum (ER) together with the three downstream effector branches: inositol-requiring ser/thr protein kinase/endonuclease (IRE-1), PKR-like ER resident kinase (PERK) and activating transcription factor 6 (ATF-6). Through careful analysis of CHIKV and SINV infections in cell culture we found that the former selectively activates ATF-6 and IRE-1 branches of UPR and suppresses the PERK pathway. By separately expressing each of the CHIKV proteins as GFP-fusion proteins, we found that non-structural protein 4 (nsP4), which is a RNA-dependent-RNA polymerase, suppresses the serine-51 phosphorylation of eukaryotic translation initiation factor, alpha subunit (eIF2α), which in turn regulates the PERK pathway. This study provides insight into a mechanism by which CHIKV replication responds to overcome the host UPR machinery.

 

Supplementary

The study by Rathore et al. described the cellular Unfolded Protein Response (UPR) signaling during CHIKV and SINV infections and showed that the gene/protein expression responses in the pathway are differentially modulated between the two closely related viruses.

In this study Rathore et al. identified that the UPR machinery is tightly regulated during CHIKV infection. Activation of the ATF-6 and IRE1 signaling arms of the UPR provides a balanced homeostatic environment, during translational stress caused by CHIKV infection. In response to virus protein translation, BIP activates ATF-6 to auto-proteolyse and induce the transcription of ER chaperone genes such as Grp-78 (BIP) and Grp-94 (HSP-90). Similarly the induction of the IRE1 signaling during CHIKV infection is revealed through probing for spliced XBP-1 gene transcript.  The spliced XBP-1 product is known to activate the transcription of pro-survival genes such as EDEM and BCL-2 family proteins. The third arm of UPR signaling, PERK, is activated during CHIKV infection by self-dimerization and phosphorylation. Activated PERK phosphorylates eIF2α at serine-51 and leads to general protein synthesis arrest. PERK activation also induces the activation of C/EBP homologous protein (CHOP) and growth arrest and DNA damage-inducible protein GADD34. CHOP is responsible for apoptosis mediated cell death whilst GADD34 and its binding partner protein phosphatase-1 catalytic subunit (PP1c) are involved in eIF2α de-phosphorylation.

The main thrust of our finding is that in the early phase of CHIKV infection (3-24h) the PERK signaling is suppressed by inhibition of the phosphorylation of eIF2α. The use of UPR specific drugs such as thapsigargin or tunicamycin, the well known strong inducers of PERK and eIF2α phosphorylation, clearly demonstrate that eIF2α phosphorylation in the cell is suppressed at the early stages (3-24h) of CHIKV infection, so as to allow high and sustained viral protein production without accumulated ER stress. At the later phase of CHIKV infection (48h) the phosphorylation of eIF2α is quite prominent. However, at this time, GADD34, a negative regulator of PERK that mediates the de-phosphorylation of phospho-eIF2α, and p58IPK, a chaperone that suppresses the PERK mediated phosphorylation of eIF2α, is also induced. This suggests that even when the cell tries to overcome its control by CHIKV infection, negative loop transcripts like GADD34 and p58IPK are activated in order to rescue viral protein synthesis. Using the GADD34 inhibitor salubrinal, its importance in mediating the CHIKV induced suppression of eIF2α-phosphorylation is demonstrated.

The viral factor responsible for the suppression in the phosphorylation of eIF2α during the early stage of CHIKV infection is investigated in Rathore et al. using the eGFP-tagged clones of seven CHIKV proteins. The results show that the observed phenotype in the PERK pathway (i.e. suppression of the phosphorylation of eIF2α) is mediated by CHIKV nsP4 protein, which contains the RNA-dependent-RNA polymerase activity. An interesting conjunction to this finding is that nsP4 protein of alphavirus is the first non-structural protein to be cleaved from the nsP1-4 polyprotein and this cleavage as well as its enzymatic activity play a critical role in the synthesis of minus strand viral RNA. Furthermore, it is also well known that the alphavirus nsP4 is unstable, short-lived and degrades rapidly in the infected cell. This instability of nsP4 may possibly explain why infected cells recover some degree of eIF2α phosphorylation in the late phase of infection (48h). The early suppression of UPR showed by Rathore et al. provides a mechanistic explanation for robust CHIKV replication. This study opens up the possibility to explore in detail the interplay of CHIKV nsP4 protein in establishing the infection and raises the possibility of nsP4 as a therapeutic target for antiviral intervention.

Subhash Vasudevan-2

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