3.1 RD-VP4 cells Validation
RD-Vector and RD-VP4 cells were saved in our Lab. To explore the function of EV71 VP4 protein. RD-Vector and RD-VP4 cells were collected to extract the total RNAs and proteins, followed by detecting VP4 levels. After reverse transcription and PCR amplification, gene transcription of VP4 was visualized in Fig. 1A. As expected, a fusion protein VP4 at 16 KD was detected by Western blot (Fig. 1B). It suggested that the constructed RD-VP4 cells could be used in the following experiments. But there were two nonspecific bands at 15 KD and 20 KD respectively, we inferred that they were caused by polyclonal antibody (pAb).
3.2 DEGs analysis and validation after Transcriptome sequencing
We thereafter performed transcription sequencing in RD-VP4 and the negative control RD-Vector cells, a total of 5,708 DEGs with 2-fold changes (|Log2fold change|>1 and p < 0.05) were screened out, including 3,232 upregulated and 2,476 downregulated. In Fig. 2A, the Y-axis represented the adjusted p-value (padj). A smaller ordinate value suggested a more significant difference and a larger corresponding -log10 value (padj). Therefore, plots in the upper left and the upper right corner very significantly downregulated and upregulated genes, respectively.
DEGs were subjected to GO analysis (http://www.geneontology.org/). GO analysis for categorizing the cellular component (CC), molecular function (MF) and biological process (BP). The top 10 CC, MF and BP were show in Fig. 2B, and it showed that DEGs were mostly associated to protein binding functions.
DEGs were also carried out KEGG analysis (https://www.kegg.jp/). In this analysis, it showed that DEGs were in up-regulated ten pathways (Fig. 2C) and in down-regulated two pathways (Fig. 2D). It was revealed that DEGs were mainly up-regulated enriched in Focal Adhesion, PI3K/AKT and MAPK pathway and down-regulated enriched in phospholipase D signal pathway.
Previous studies showed that VP4 protein band to cell membrane during virus entry [13, 14, 28], so we selected differential genes related to focal adhesion pathway and cell surface receptors for verification by RT-qPCR (Fig. 3). Using housekeeping gene 18s rRNA to do normalized quotient, a total of 8 genes were upregulated and 5 were downregulated. Integrin receptors ITGA4/5/7 and ITGB1, caveolin and some receptor tyrosine kinases (RTKs, EGF and FGF) were upregulated, while FPR3 and some other RTKs (VEGF and PDGF) were downregulated. These results were consistent with those in bioinformatics analysis. Then, according to reading literatures, we founded that ITGA5 and ITGB1 composited ITGA5-ITGB1 complex, which located in cell membrane[29] and could activate PI3K/AKT and MAPK pathway[30]. Thus, we selected the ITGA5 and ITGB1 to research the molecular mechanism of VP4 protein.
3.3 EV71 VP4 activation the PI3K/AKT and MAPK pathways and promote viral replication
VP4 protein band to cell membrane during virus entry [13, 14, 28], ITGA5/ITGB1 complex were up-regulated in RD-VP4 cell, and ITGA5-ITGB1 complex could activate PI3K/AKT and MAPK pathway[30], so we speculated that VP4 protein interact with the ITGA5 or ITGB1. Thus, the co-immuneprecipitated (Co-IP) assay was designed to validate the interaction between VP4 and ITGA5 or ITGB1. In RD-VP4 cells, WB result showed that both VP4 and ITGA5 existed after IP from the Fig. 4A, while there was only VP4 and did not exist ITGB1 after IP from Fig. 4B, suggesting that VP4 not interaction with ITGB1 but interaction with the ITGA5.
To explore the role of VP4-ITGA5/ITGB1 complex in the virus replication process, two ITGA5 shRNAs were used to lentiviruses packaging and knockdown the ITGA5 expression. The ITGA5 protein was downregulated effectively after shITGA5-1 and sh-ITGA5-2 lentivirus transduction RD cells (Fig. 5A). Notably, knockdown of ITGA5, phosphorylated-AKT and MEK1 proteins (p-AKT and p-MEK1) were downregulated and phosphorylated-Elk1 (p-Elk1) protein was upregulated in PI3K/AKT and MAPK pathway (Fig. 5B), suggesting that the PI3K/AKT and MAPK pathway were activated by ITGA5. Knockdown ITGA5 in RD cells, the copies of EV71, which was detected by RT-qPCR, was significantly downregulated in EV71 infection at 24 hpi and 48 hpi (Fig. 5C) respectively. This result showed that ITGA5 knockdown inhibited EV71 virus replication. Thus, we concluded that EV71 VP4 protein probably promote viral replication.