Apoptosis is characterized by DNA fragmentation, chromatin condensation, membrane blebbing, cell shrinkage and cell death. An increasing number of viruses can induce apoptosis at the late stage of infection. This process may be important for the spread of progeny viruses to the neighbor cells and for the protection of progeny viruses against host enzymes and antibodies. There are three major apoptotic pathways including death receptor (extrinsic), mitochondrial (intrinsic) and endoplasmic reticulum pathways, respectively. All of these pathways are observed in the cytoplasm [21, 22]. The mechanisms of host defense can limit virus replication, enable infected cells apoptosis, release and exclude immature virus particles through lysosome or proteases. However, the viruses survive through a variety of ways which can interfere with the apoptosis of infected cells to evade or delay the early onset of apoptosis [16, 23].
In order to confirm the pathophysiology of EV71 infection and host cellular responses, especially for apoptotic responses, we have employed PCR to analyze the expression changes of cellular genes during the infection process. The expression profiles of 84 pro- and anti-apoptotic genes revealed a significant difference in various time points. With the onset of EV71 replication, the genes such as ACIN1, Akt, APAF1, caspase, CIDEB, DAPK, NF-κB1, STAT1 and p53 associated with apoptosis were interrupted at 8 h postinfection by apoptosis-suppressing effect of EV71. Although many RNA viruses do not encode anti-apoptotic genes, the apoptosis-suppressing mechanisms are still unclear. EV71 infection at 20 h postinfection could stimulate the up-regulation of genes such as FasL, CD40L, TNF-α, TNFRSF10A (DR4), Fas, caspase-10, -8, -7, -3 and XIAP involved in TNFR1 pathway in RD cells. FasL is a type II membrane protein in the TNF-α receptor family and Fas receptor binding with FasL can induce cell apoptosis [18, 24]. Interestingly, the expressions of Fas, FasL and DR4 revealed an enhancement by 3.26, 7.53 and 2.03 fold in RD cells at 20 h postinfection, suggesting that the proteins involved in Fas receptor-mediated pathway were activated in EV71-infected RD cells. Furthermore, downstream proteins in the Fas pathway such as caspase-10, -8, -7, -3 and CIDEB were also significantly up-regulated. The activation of TNFR1, CD95 and DR4/DR5 by TNF-α, FasL and TRAIL can trigger the activation of caspase-10, -8, -7, and -3 involved in cell death. The activated caspase-3 can cleave its substrates such as CIDE-B to trigger chromosomal DNA fragmentation. However, the expressions of caspase-12, -9, Bcl-2, and APAF1 were unchanged in EV71-infected cells. Previous studies reported that the death receptor pathway for apoptosis was the major pathway during the EV71 infection of RD cells [22, 25]. In this study, differential changes of apoptotic gene expressions may be attributed to different conditions such as MOI, infection duration and EV71 strains.
CD40L is described originally in T lymphocytes. However, its expression has been observed in a wide variety of cells including platelets, mast cells, macrophages, basophils, NK cells, B lymphocytes as well as non-haematopoietic cells . As a member of the TNF-α receptor family, CD40 relies on interaction with TRAF proteins to mediate an intracellular signal in response to CD40L binding [27, 28]. The downstream protein of TRAF2/3 can activate consecutive initiation of MEK4 and MEK7, resulting in the activation of stress-activated protein kinases JNK1/2 . Subsequently, transcription factor activator of proteins (c-Fos and c-Jun) and NF-κB were activated through a kinase pathway involving in map kinases, NIK (NF-κB inducing kinase) and I-κB kinase, thus co-stimulating the proliferation of activated T-cells accompanied by the production of IFN-γ, TNF-α, and IL-2 . Interestingly, the expressions of CD40L, MEK4 and MEK7 were induced by 5.44, 2.54, and 3.05-fold in RD cells at 20 h postinfection, whereas JNK1/2 was highly phosphorylated. Therefore, we postulated that CD40L/CD40 signaling pathway was also activated in EV71-infected RD cells.
The serine/threonine kinase AKT, also known as protein kinase B (PKB), has become a major focus because of its critical regulatory role in cellular processes including cancer progression . AKT cascades are activated by receptor tyrosine kinases, integrins, B- and T-cell receptors, cytokine receptors, G-protein coupled receptors and other stimuli for inducing the accumulation of phosphatidylinositol 3,4,5-triphosphates by phosphoinositide 3-kinase (PI3K) . Three AKT isoforms (AKT1, AKT2 and AKT3) can mediate many downstream events regulated by PI3K. At 8 h postinfection, the expression of AKT2 was significantly down-regulated by 3.36 fold, while PI3K and AKT2 were enhanced by 5.18 fold and 2.66 fold at 20 h postinfection, respectively. The phosphorylation of AKT2 was highly elevated. As previously reported, EV71 can activate PI3K/AKT to trigger the anti-apoptotic pathways at the early phase during infection [32, 33]. In this study, the activation of PI3K/AKT may be associated with EV71 strain virulence and virus titer.
Transcription factor NF-κB p65 (NF-κB3) is a protein encoded RELA gene in human. Activated NF-κB p65 translocates into the nucleus and binds DNA at kappa-B-binding motifs, thus being involved in the production of cytokines including IL-2, IL-6, TNF-α, IFN-β and IL-1β [34, 35]. While STAT1 is a member of the signal transducers and activators of transcription family and involved in the up-regulation of the genes through type I, type II or type III interferon . Both NF-κB and STAT1 are rapidly activated in response to various stimuli including viral infection and cytokines, thus controlling the expressions of anti-apoptotic, pro-proliferative and immune response genes. In this study, NF-κB p65 and STAT1 were upregulated by 2.63 and 4.04 fold, respectively. Meanwhile, NF-κB p65 was significantly phosphorylated at 20 h postinfection. However, NF-κBIA was up-regulated by 2.96 fold, which can block NF-κB to bind to DNA. IL-2, IL-4, IL-10 and TNF-α exhibited the enhancement by 12.15, 2.39, 12.15 and 2.19 fold at 20 h after EV71 infection. Compared with EV71-uninfected controls, the expression levels of IL-4, IL-10 and TNF-α in EV71-infected RD cells revealed a significant increase at 20 and 32 h postinfection (P < 0.01,or P < 0.001).
Eight human IAPs such as NAIP (BIRC1), c-IAP1 (BIRC2), c-IAP2 (BIRC3), X-linked IAP (XIAP, BIRC4), Survivin (BIRC5), Apollon (BRUCE, BIRC6), Livin/ML-IAP (BIRC7) and IAP-like protein 2 (BIRC8) have been identified [37, 38]. BIRC3 can participate in TNF-α-mediated NF-κB activation, while XIAP can bind to the active site of effector caspases including caspase-3, caspase-7 and caspase-9 for preventing substrate binding and subsequent catalysis through its BIR2 domain with N-terminal linker . BIRC3 and XIAP were significantly up-regulated by 3.19 fold and 2.59 fold at 20 h postinfection, which may be related to the apoptosis inhibition of RD cells. Additionally, the up-regulation of CD5, CD24, CD70, CD226 and PDCD1 (PD-1) that usually express on the surface of T cells, B cells, dendritic cells, NK cells, and tumor cells have also been observed [40–44]. Interestingly, these genes revealed a significant up-regulation in EV71-infected RD cells and may be associated with cell signal pathway and apoptosis. Therefore, further study is needed to be undertaken for these gene expressions.