Enterovirus 71 Proteins 2A and 3D Antagonize the Antiviral Activity of Gamma Interferon via Signaling Attenuation.

UNLABELLED: Enterovirus 71 (EV71) infection causes severe mortality involving multiple possible mechanisms, including cytokine storm, brain stem encephalitis, and fulminant pulmonary edema. Gamma interferon (IFN-γ) may confer anti-EV71 activity; however, the claim that disease severity is highly correlated to an increase in IFN-γ is controversial and would indicate an immune escape initiated by EV71. This study, investigating the role of IFN-γ in EV71 infection using a murine model, showed that IFN-γ was elevated. Moreover, IFN-γ receptor-deficient mice showed higher mortality rates and more severe disease progression with slower viral clearance than wild-type mice. In vitro results showed that IFN-γ pretreatment reduced EV71 yield, whereas EV71 infection caused IFN-γ resistance with attenuated IFN-γ signaling in IFN regulatory factor 1 (IRF1) gene transactivation. To study the immunoediting ability of EV71 proteins in IFN-γ signaling, 11 viral proteins were stably expressed in cells without cytotoxicity; however, viral proteins 2A and 3D blocked IFN-γ-induced IRF1 transactivation following a loss of signal transducer and activator of transcription 1 (STAT1) nuclear translocation. Viral 3D attenuated IFN-γ signaling accompanied by a STAT1 decrease without interfering with IFN-γ receptor expression. Restoration of STAT1 or blocking 3D activity was able to rescue IFN-γ signaling. Interestingly, viral 2A attenuated IFN-γ signaling using another mechanism by reducing the serine phosphorylation of STAT1 following the inactivation of extracellular signal-regulated kinase without affecting STAT1 expression. These results demonstrate the anti-EV71 ability of IFN-γ and the immunoediting ability by EV71 2A and 3D, which attenuate IFN-γ signaling through different mechanisms. IMPORTANCE: Immunosurveillance by gamma interferon (IFN-γ) may confer anti-enterovirus 71 (anti-EV71) activity; however, the claim that disease severity is highly correlated to an increase in IFN-γ is controversial and would indicate an immune escape initiated by EV71. IFN-γ receptor-deficient mice showed higher mortality and more severe disease progression, indicating the anti-EV71 property of IFN-γ. However, EV71 infection caused cellular insusceptibility in response to IFN-γ stimulation. We used an in vitro system with viral protein expression to explore the novel IFN-γ inhibitory properties of the EV71 2A and 3D proteins through the different mechanisms. According to this study, targeting either 2A or 3D pharmacologically and/or genetically may sustain a cellular susceptibility in response to IFN-γ, particularly for IFN-γ-mediated anti-EV71 activity.

Autophagy facilitates IFN-gamma-induced Jak2-STAT1 activation and cellular inflammation.

Autophagy is regulated for IFN-gamma-mediated antimicrobial efficacy; however, its molecular effects for IFN-gamma signaling are largely unknown. Here, we show that autophagy facilitates IFN-gamma-activated Jak2-STAT1. IFN-gamma induces autophagy in wild-type but not in autophagy protein 5 (Atg5(-/-))-deficient mouse embryonic fibroblasts (MEFs), and, autophagy-dependently, IFN-gamma induces IFN regulatory factor 1 and cellular inflammatory responses. Pharmacologically inhibiting autophagy using 3-methyladenine, a known inhibitor of class III phosphatidylinositol 3-kinase, confirms these effects. Either Atg5(-/-) or Atg7(-/-) MEFs are, independent of changes in IFN-gamma receptor expression, resistant to IFN-gamma-activated Jak2-STAT1, which suggests that autophagy is important for IFN-gamma signal transduction. Lentivirus-based short hairpin RNA for Atg5 knockdown confirmed the importance of autophagy for IFN-gamma-activated STAT1. Without autophagy, reactive oxygen species increase and cause SHP2 (Src homology-2 domain-containing phosphatase 2)-regulated STAT1 inactivation. Inhibiting SHP2 reversed both cellular inflammation and the IFN-gamma-induced activation of STAT1 in Atg5(-/-) MEFs. Our study provides evidence that there is a link between autophagy and both IFN-gamma signaling and cellular inflammation and that autophagy, because it inhibits the expression of reactive oxygen species and SHP2, is pivotal for Jak2-STAT1 activation.

Lymphocyte and antibody responses reduce enterovirus 71 lethality in mice by decreasing tissue viral loads.

Enterovirus 71 (EV71) infects the central nervous system and causes death and long-term neurological sequelae in hundreds of thousands of young children, but its pathogenesis remains elusive. Immunopathological mechanisms have been suspected to contribute to the pathogenesis of neurological symptoms, so anti-inflammatory agents have been used to treat patients with neurological symptoms. The present study was therefore designed to investigate the functions of lymphocyte and antibody responses in EV71 infection using a mouse model. Immunohistochemical staining analysis revealed virus and three types of lymphocytes, B cells, CD4 T cells, and CD8 T cells, in the spinal cord of an EV71-infected patient who died. A study of mice showed that the levels of virus and lymphocytes in brains and antibody titers in sera were elevated during the time when the mice succumbed to death in a phenomenon analogous to that observed in patients. Further studies demonstrated that after infection, the disease severity, mortality, and tissue viral loads of mice deficient in B, CD4 T, or CD8 T cells were significantly higher than those of wild-type mice. In addition, treatment with a virus-specific antibody, but not a control antibody, before or after infection significantly reduced the disease severity, mortality, and tissue viral loads of mice deficient in B cells. Our results show that both lymphocyte and antibody responses protect mice from EV71 infection. Our study suggests the use of vaccines and virus-specific antibodies to control fatal outbreaks and raises caution over the use of corticosteroids to treat EV71-infected patients with neurological symptoms.