真核翻译起始因子eIF4E可以通过在丝氨酸209上的磷酸化来调节细胞翻译。在最近的一项研究中,通过两轮TMT相对定量蛋白质组学,我们发现磷酸化的eIF4E(p-eIF4E)有利于选择的mRNA的翻译,编码的蛋白质主要参与ECM受体,病灶粘连,和PI3K-Akt信号。本文主要研究p-eIF4E与下游宿主细胞蛋白的关系,以及它们对PEDV有效进入的推测效果。我们发现膜居住因子TSPAN3,CD63和ITGB2的消耗显着抑制PEDV的病毒侵袭,减少了假型粒子PEDV-pp的进入,SARS-CoV-pp,和SARS-CoV-2-pp。TSPAN3,CD63和ITGB2的特异性抗体阻断了PEDV在宿主细胞中的吸附。此外,我们检测到eIF4E磷酸化在PEDV感染后1小时增加,与TSPAN3、CD63和ITGB2的表达一致。在PEDV攻击的早期阶段,仔猪的肠道也出现了类似的趋势。与Vero细胞相比,其中eIF4E不能被磷酸化的S209A-Vero细胞显示侵袭性PEDV病毒体的减少。MNK激酶抑制剂阻断PEDV侵袭,以及减少TSPAN3,CD63和ITGB2的积累。进一步的研究表明,ERK-MNK途径负责调节PEDV诱导的eIF4E早期磷酸化。本文首次证明了p-eIF4E刺激与膜居住宿主因子之间的联系。我们的发现还丰富了对磷酸化eIF4E在病毒生命周期中的生物学功能的理解。重要意义真核翻译起始因子eIF4E可以通过磷酸化调节细胞翻译。在我们之前的研究中,发现对高水平p-eIF4E敏感的几种宿主因素有助于冠状病毒PEDV的病毒感染。当前的论文集中在细胞膜居住因素上,参与对磷酸化eIF4E敏感的信号通路。我们发现ERK-MNK通路被激活,这导致在早期PEDV感染中eIF4E磷酸化的刺激。磷酸化eIF4E通过在翻译水平而不是转录水平上上调宿主因子TSPAN3,CD63和ITGB2的表达来促进PEDV的病毒入侵。此外,TSPAN3,CD63或ITGB2促进冠状病毒SARS-CoV的有效进入,SARS-CoV-2和HCoV-OC43。我们的发现拓宽了我们对病毒生命周期中eIF4E动态磷酸化的见解,并提供进一步的证据表明磷酸化eIF4E调节宿主mRNA的选择性翻译。
The eukaryotic translation initiation factor eIF4E can regulate cellular translation via phosphorylation on serine 209. In a recent study, by two rounds of TMT relative quantitative proteomics, we found that phosphorylated eIF4E (p-eIF4E) favors the translation of selected mRNAs, and the encoded proteins are mainly involved in ECM-receptor, focal adhesion, and PI3K-Akt signaling. The current paper is focused on the relationship between p-eIF4E and the downstream host cell proteins, and their presumed effect on efficient entry of PEDV. We found that the depletion of membrane-residential factor TSPAN3,
CD63, and ITGB2 significantly inhibited viral invasion of PEDV, and reduced the entry of pseudotyped particles PEDV-pp, SARS-CoV-pp, and SARS-CoV-2-pp. The specific antibodies of TSPAN3,
CD63, and ITGB2 blocked the adsorption of PEDV into host cells. Moreover, we detected that eIF4E phosphorylation was increased at 1 h after PEDV infection, in accordance with the expression of TSPAN3,
CD63, and ITGB2. Similar trends appeared in the intestines of piglets in the early stage of PEDV challenge. Compared with Vero cells, S209A-Vero cells in which eIF4E cannot be phosphorylated showed a decrease of invading PEDV virions. MNK kinase inhibitor blocked PEDV invasion, as well as reduced the accumulation of TSPAN3,
CD63, and ITGB2. Further study showed that the ERK-MNK pathway was responsible for the regulation of PEDV-induced early phosphorylation of eIF4E. This paper demonstrates for the first time the connections among p-eIF4E stimulation and membrane-residential host factors. Our findings also enrich the understanding of the biological function of phosphorylated eIF4E during the viral life cycle.IMPORTANCEThe eukaryotic translation initiation factor eIF4E can regulate cellular translation via phosphorylation. In our previous study, several host factors susceptible to a high level of p-eIF4E were found to be conducive to viral infection by coronavirus PEDV. The current paper is focused on cell membrane-residential factors, which are involved in signal pathways that are sensitive to phosphorylated eIF4E. We found that the ERK-MNK pathway was activated, which resulted in the stimulation of phosphorylation of eIF4E in early PEDV infection. Phospho-eIF4E promoted the viral invasion of PEDV by upregulating the expression of host factors TSPAN3, CD63, and ITGB2 at the translation level rather than at the transcription level. Moreover, TSPAN3,
CD63, or ITGB2 facilitates the efficient entry of coronavirus SARS-CoV, SARS-CoV-2, and HCoV-OC43. Our findings broaden our insights into the dynamic phosphorylation of eIF4E during the viral life cycle, and provide further evidence that phosphorylated eIF4E regulates selective translation of host mRNA.