eIF2α of Litopenaeus vannamei involved in shrimp immune response to WSSV infection

Highlights

• WSSV challenge experiment indicated LveIF2α was involved in antiviral immunity.

• Inhibitor treatment suggested LveIF2α may facilitate WSSV infection.

• The upregulated expression of Bip demonstrated WSSV infection can induce ER stress.

• The transcription of LvPERK was induced by WSSV infection.

• WSSV infection activated the PERK-eIF2α pathway in shrimp.

Abstract

The alpha subunit of Eukaryotic Initiation Factor 2 (eIF2α) is a key translation regulator that plays an important role in cellular stress responses, which including virus infection. To investigate whether WSSV infection can activate the PERK-eIF2α pathway, the eIF2α in shrimp Litopenaeus vannamei, designed as LveIF2α, was analyzed. The LveIF2α, a 332-amino acid polypeptide, shares a high degree of similarity with eIF2α from other species, having two eIF2α protein signatures at the 13–88 aa and 192–243 aa. The WSSV challenge experiment showed that the protein level of the total LveIF2α was decreased after infection, while the phosphorylation of LveIF2α has no significant change, which indicated that the phosphorylation ratio of LveIF2α was increased after infection. Furthermore, inhibitor treatment led to a significant decrease of WSSV loads. Moreover, the Binding immunoglobulin protein (BiP), an endoplasmic reticulum (ER) stress sensor, and PERK were also investigated during virus infection and it was shown that they were both up-regulated. Taken together, these results suggested that WSSV infection can induce ER stress and activated the unfolded protein response (UPR), and the PERK-eIF2α pathway is important for innate immune during WSSV infection in shrimp.

Introduction

Eukaryotic Initiation Factor 2 (eIF2) is an essential factor for initiation of protein synthesis. It is a heterotrimer that is composed of three subunits, namely α, β and γ. Its activity is regulated by a mechanism involving both guanine nucleotide exchange and phosphorylation. eIF2α is the main target for phosphorylation, which contains a serine at position 51. Therefore, the α subunit can be considered to be the regulatory subunit of the trimer, whose activity is regulated by phosphorylation [1]. The phosphorylation of eIF2α can result in a severe decline in protein synthesis, which is an important strategy in the cell's armory against stressful insults including viral infection, the accumulation of misfolded proteins, and starvation [2].

There are four well-characterized serine–threonine kinases, double-stranded RNA-activated protein kinase (PKR), PKR-like ER kinase (PERK), general control non-derepressible-2 (GCN2) and heme-regulated inhibitor (HRI), were reported to phosphorylate the eIF2α under different stresses [3], [4], [5], [6]. Among them, PKR can be activated by the dsRNA stimulation, and it's well known to play an important role in innate antiviral immunity [7]. And PERK is a sensor of endoplasmic reticulum (ER) stress. The endoplasmic reticulum is the place for the folding of proteins, and viral infection [8] or the over-expression of proteins [9] can lead to endoplasmic reticulum stress response. Unfolded protein response (UPR) is a cellular stress response related to the ER, and the studies indicated that virus infection could induce the ER stress and activate the UPR. Herpes simplex virus (HSV) infection induced phosphorylation of an endoplasmic reticulum (ER) resident kinase PERK [10], and African swine fever virus (ASFV) infection also induced transcription factor 6 signaling pathway of the UPR. The UPR was regulated by the virus to prevent the early apoptosis and facilitate viral replication [11].

Litopenaeus vannamei, as the main species of cultured shrimp, has been threatened by white spot syndrome virus (WSSV) [12]. Many studies on the innate immunity of shrimp have been carried out, and several immune-related genes in shrimp, such as hemocyanin [13], [14], lectin [15], [16] and antimicrobial peptide [17], [18] have been identified and characterized. Moreover, to understand the mechanisms involved in WSSV pathogenesis in shrimp, more and more studies had been focused on the signal transduction and some pathways have also been reported to be involved in the interaction between WSSV and shrimp [19], [20], [21], [22], [23]. Previous studies have indicated that there is a complex relationship between WSSV and UPR [24], [25], [26]. Activating transcription factor 4 of L. vannamei (LvATF4), a transcription factor of shrimp UPR, was activated under WSSV challenge [26], which may be regulated by the PERK/eIF2α.

To further understand the role of the UPR pathway between host and virus, the eIF2α of L. vannamei, designed as LveIF2α, was investigated under WSSV challenge in this study. The result suggested that LveIF2α was involved in WSSV infection.