Roles Of Enterovirs 71 In Evasion Of Innate Immunity

Enterovirus 71 (EV71) is a single-stranded positive-sense RNA virus andthe main causative agent for hand-foot-and-mouth disease (HFMD) in young children and infants. Since first isolated in California, USA,1969, EV71 has caused outbreaks worldwide but the majoroutbreaks with higher morbidity and mortality rates occurredmainly in the Asia-Pacific region. Most of the cases are self-limited and manifested as hand foot and mouth disease, which is characterized by rash on the skin, fatigue, and exanthema. Insevere cases, however, neurological symptoms could occur, which icludeviral meningitis, poliomyelitis-like acute flaccid paralysis, brainstem encephalitis, even the pulmonary oedema and cardiorespiratory collapse, usually fatal. For now, no vaccine or clinical antiviral drugs are available for the treatment of EV71 infection. Supportive treatmentson symptoms and signs are still the main strategy clinicallyfor EV71 patients.Virus infections tend to trigger production of interferons (IFNs) to block their spread. Type I IFNs serve as first line defense in innate immunity, implicated for the induction of hundreds of interferon-stimulated genes (ISGs) that play a key role in host resistance to viral infections. Viruses have evolved various strategies to counteract IFN-mediated host antiviral immunity. Generally viruses circumvent the IFN responses by either inhibiting the induction of IFNs, blocking IFN stimulation to induce ISGs, or both. Results from recent studies showed that EV71 can not only block the production of IFN, but also could inhibit the action of IFN. However, the exact mechanism remains obscure, which needs further investigation.Few gastrointestinal symptoms or signs are observed clinically, although EV71 is shown to replicate initially in the gastrointestinal tract before spreading to muscle and the central nervous system, suggesting the presence of a unique immunity to EV71 in the gut. Therefore, host responses, especially the antiviral response induced in EV71 infection in the intestinal epithelium, may play a critical role in viral pathogenesis and clinical prognosis. We have previously reported that, unlike in RD or HeLa cells in which antiviral IFN response was effectively suppressed, robust IFN-β was induced in EV71-infected human intestinal epithelial cells. Here, we showed that not only the IFN-(3, but also the other type I IFNs, including IFN-a,-ε,-κ, and-ε, and type III IFNs, including IFN-λ1,-λ2, and-λ3, were remarkably upregulated and induced to high levels in HT-29 cells compared to those in HeLa and RD cells. A full spectrum of IFNs including all from type I, II, and III, could be induced in response to EV71 in the intestinal epithelial cells. The IFN production is mainly regulated by RLR or TLR signaling pathways. We compared the signaling pathways that mediate IFN response to EV71 infection in HT-29 and RD cells and found that TRIF, the key signaling player in TLR signaling, was not cleaved by viral 3Cpro in HT-29, unlike in RD cells where EV71 infection induced significant degradation of TRIF. Our data showed that TLR appeared to be the essential signaling pathway for IFN response, since MAVS was drastically degraded not only in HeLa or RD cells, but also in HT-29 cells, which may have crippled the RLR signaling. Indeed, in TRIF-silenced HT-29 cells, replication of EV71 increased significantly in parallel to the decrease of all IFNs. Thus, a TLR-TRIF axis appears to be essential and responsible for robust induction of IFNs in HT-29 cells and induction of IFNs in the gastrointestinal tract may explain asymptomatic or mild symptoms.Then we studied on the interaction of EV71 and the action of IFN, which is mediated by the JAK/STAT signaling pathway. Although IFNs serve as effective antiviral therapeutics for many infections of viruses including HIV, HCV, and HBV, IFN treatment had little effect on patients infected with EV71. Thus EV71 is capable of antagonizing IFN for downstream stimulation of induction of ISGs. However, reports thus far on how EV71 resists antiviral effect of IFNs remain controversial. A previous study showed that EV71 disrupted type I IFN signaling by downregulating IFNAR1, and that 2A was responsible for the inhibitory effect on IFN response. However, a recent study reported that EV71 infection did not alter either the total expression level or surface expression of IFNAR1; instead, the infection downregulated the protein level of JAK1. We found that pretreatment with IFN could not inhibit viral replication and cytopathic effect. In this report, we present evidence to show that neither IFNAR1 nor JAK1 levels were significantly affected during EV71 infection, and were unchanged even in an infection with an inoculum as high as 10 m.o.i.. We were unable to detect the change of the expression of STAT1 and STAT2, and tyrosine phosphorylation of STAT1 and STAT2 was also not affected, indicating that the stimulation on IFNAR1 by IFN-β and the activation of Jaks were barely affected during EV71 infection. In this study our data clearly showed that following EV71 infection, p-STAT1 and p-STAT2, in response to IFN-β stimulation, were retained in the cytosol and unable to be translocated into the nucleus even though STAT1 and STAT2 were phosphorylated. We explored the mechanism by which activated STAT1/2 was blocked from entering the nucleus, and identified a disruptive interaction by STAT1 and KPNA1. KPNA1 is a member of the KPNA family, which is involved in nucleo-cytoplasmic trafficking and directly interacts with tyrosine phosphorylated STAT1, facilitating the transport of STAT1 into the nucleus. We further characterized the interaction of STAT1 and KPNA1 and observed that KPNA1 was targeted for degradation during EV71 infection.Proteins are generally degraded by ubiquitin-proteasome dependent degradation, autophagy-lysosomal or caspase-mediated proteolysis. First, we examined whether 2A or 3C can induce the degration of KPNA1 and found that the two viral proteins have no effect on the downregulation of KPNA1 by EV71 infection. Then by using specific inhibitors, we observed that blocking the caspase-mediated proteolysis pathway, but not ubiquitin-proteasome or autophagy-lysosomal signal pathway, resulted in the restoration of KPNA1 levels in EV71-infected cells, suggesting that the degradation of KPNA1 could be apoptosis-or caspase-dependent. We selected a group of caspases for further studies, including caspase-3,-4,-6,-7 and -8, among which caspase-3 and -6 or -7 are executioner caspases, while caspase-4 and -8 are initiator caspases. By using specific synthetic inhibitors for these caspases, we found that only the inhibitor specific for caspase-3 could block the degradation of KPNA1, suggesting that the reduction of KPNA1 irr protein level during EV71 infection was caspase-3 dependent. Here, we also found that EV71 infection could blocked the translocation of IRF9 and inhibited the expression of IRF9 and STAT2 but not STAT1 at the first rapid response phase after IFN-β stimulation, thus inhibited the U-ISGF3 formation and the subsequent prolonged rubust production of antiviral genes. Overexpression of both IRF9 and STAT2 in HEK293 cells had reversed the supression of EV71 on the production of ISGs.Our data indicate that caspase cascade may play a unique role in EV71 viral replication, demonstrating that caspase-3 mediated degradation of KPNA1 and the degration of STAT2 and IRF9 could serve as a novel mechanism for viral evasion of IFN signaling responses by an enterovirus.Taken together, our data suggest that IFNs were induced differentially in human HT-29 cells through an intact TLR/TRIF signaling, which differs from other cell types and may be implicated in viral pathogenesis in EV71 infection. Also our study demonstrates novel mechanisms by which antiviral signaling is suppressed partly through degradation of KPNA1 by activated caspase-3 induced in an enteroviral infection or partly through downregulation of IRF9 and STAT2 induced by IFN-β.