The 3C Protein of Enterovirus 71 Inhibits Retinoid Acid-Inducible Gene I-Mediated Interferon Regulatory Factor 3 Activation and Type I Interferon Responses

Enterovirus 71 (EV71) is a human pathogen that induces hand, foot, and mouth disease and fatal neurological diseases. Immature or impaired immunity is thought to associate with increased morbidity and mortality. In a murine model, EV71 does not facilitate the production of type I interferon (IFN) that plays a critical role in the first-line defense against viral infection. Administration of a neutralizing antibody to IFN-α/β exacerbates the virus-induced disease. However, the molecular events governing this process remain elusive. Here, we report that EV71 suppresses the induction of antiviral immunity by targeting the cytosolic receptor retinoid acid-inducible gene I (RIG-I). In infected cells, EV71 inhibits the expression of IFN-β, IFN-stimulated gene 54 (ISG54), ISG56, and tumor necrosis factor alpha. Among structural and nonstructural proteins encoded by EV71, the 3C protein is capable of inhibiting IFN-β activation by virus and RIG-I. Nevertheless, EV71 3C exhibits no inhibitory activity on MDA5. Remarkably, when expressed in mammalian cells, EV71 3C associates with RIG-I via the caspase recruitment domain. This precludes the recruitment of an adaptor IPS-1 by RIG-I and subsequent nuclear translocation of interferon regulatory factor 3. An R84Q or V154S substitution in the RNA binding motifs has no effect. An H40D substitution is detrimental, but the protease activity associated with 3C is dispensable. Together, these results suggest that inhibition of RIG-I-mediated type I IFN responses by the 3C protein may contribute to the pathogenesis of EV71 infection.Enterovirus 71 (EV71) is a single-stranded, positive-sense RNA virus belonging to the Picornaviridae family. The viral genome is approximately 7,500 nucleotides in length, with a single open reading frame that encodes a large precursor protein. Upon infection, this protein precursor is processed into four structural (VP1, VP2, VP3, and VP4) and seven nonstructural (2A, 2B, 2C, 3A, 3B, 3C, and 3D) proteins (32). EV71 infection manifests most frequently as the childhood exanthema known as hand, foot, and mouth disease (HFMD). Additionally, EV71 infection may cause neurological diseases, which include aseptic meningitis, brain stem and/or cerebellar encephalitis, and acute flaccid paralysis (32). Young children and infants are especially susceptible to EV71 infection. Since the initial recognition of EV71 in the United States, outbreaks have been reported in Southeast Asia, Europe, and Australia (1-3, 11, 14, 24, 30-32). Recently, large epidemics of HFMD occurred in the mainland of China (26, 42, 52).The mechanism of EV71 pathogenesis remains obscure. It is believed that immature or impaired immunity, upon EV71 infection, is associated with increased morbidity and mortality (7, 14, 17). In a murine infection model, lymphocyte as well as antibody responses reduce tissue viral loads and EV71 lethality (28). Notably, EV71 induces skin rash at the early stage and hind limb paralysis or death at the late stage. Oral infection leads to initial replication in the intestine and subsequent spread to various organs such as the spinal cord and the brain stem (8). Intriguingly, EV71 does not facilitate the production of type I interferon (IFN), a family of cytokines involved in first-line defense against virus infection. Indeed, administration of neutralizing antibody to IFN-α/β increases tissue viral loads and exacerbates the virus-induced disease (29).Type I IFN is produced in response to viral infections (22). For example, Toll-like receptor 3 (TLR3) in the endosome recognizes double-stranded RNA (dsRNA), where it recruits the adaptor Toll/interleukin-1 receptor (TIR) domain-containing adaptor inducing IFN-β (TRIF) (22). TRIF, together with tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3), then activates the two IKK-related kinases, TANK-binding kinase 1 (TBK1) and inducible IκB kinase (IKKi), both of which phosphorylate interferon regulatory factor 3/7 (IRF3/7) (10, 13, 36, 45). IRF3 or IRF7, in turn, stimulates the expression of target genes, such as IFN-α/β (33, 37, 39, 51). In parallel, TRIF also induces NF-κB activation via TRAF6 (18, 19). In addition, alternative mechanisms exist in host cells to detect cytosolic nucleic acids. Two RNA helicases, retinoid acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5), recognize viral RNA present in the cytoplasm and subsequently recruit the adaptor IFN promoter-stimulating factor 1 (IPS-1; also called Cardif, MAVS, and VISA) (22, 23, 54). The interaction of IPS-1, TRAF3, and TBK1/IKKi activates IRF3/IRF7 and induces the expression of IFN-α/β while the interaction of IPS-1 with the Fas-associated protein-containing death domain (FADD) leads to NF-κB activation. It has been shown that MDA5 recognizes long double-stranded RNAs, such as in cells infected with picornaviruses, whereas RIG-I senses 5′ triphosphate single-stranded RNA with poly(U/A) motifs and short dsRNA in cells infected with a variety of RNA viruses (16, 20, 40, 43).The objective of this study was to investigate the interaction of EV71 with the type I IFN system. We demonstrate that, unlike Sendai virus or double-stranded RNA, EV71 does not stimulate the expression of antiviral genes in mammalian cells. Among structural and nonstructural proteins encoded by EV71, the 3C protein is able to inhibit virus-induced activation of the IFN-β promoter. We provide evidence that when expressed in mammalian cells, the 3C protein suppresses RIG-I signaling by disruption of the RIG-I-IPS-1 complex and IRF3 nuclear translocation. While H40, KFRDI, and VGK motifs are involved, the protease and RNA binding activities are dispensable. Collectively, these results suggest that control of RIG-I by the 3C protein impairs type I IFN responses, which may contribute to the pathogenesis of EV71 infection.     

A Critical Regulatory Role for Macrophage Migration Inhibitory Factor in Hyperoxia-Induced Injury in the Developing Murine Lung

Background

The role and mechanism of action of MIF in hyperoxia-induced acute lung injury (HALI) in the newborn lung are not known. We hypothesized that MIF is a critical regulatory molecule in HALI in the developing lung.

Methodology

We studied newborn wild type (WT), MIF knockout (MIFKO), and MIF lung transgenic (MIFTG) mice in room air and hyperoxia exposure for 7 postnatal (PN) days. Lung morphometry was performed and mRNA and protein expression of vascular mediators were analyzed.

Results

MIF mRNA and protein expression were significantly increased in WT lungs at PN7 of hyperoxia exposure. The pattern of expression of Angiopoietin 2 protein (in MIFKO>WT>MIFTG) was similar to the mortality pattern (MIFKO>WT>MIFTG) in hyperoxia at PN7. In room air, MIFKO and MIFTG had modest but significant increases in chord length, compared to WT. This was associated with decreased expression of Angiopoietin 1 and Tie 2 proteins in the MIFKO and MIFTG, as compared to the WT control lungs in room air. However, on hyperoxia exposure, while the chord length was increased from their respective room air controls, there were no differences between the 3 genotypes.

Conclusion

These data point to the potential roles of Angiopoietins 1, 2 and their receptor Tie2 in the MIF-regulated response in room air and upon hyperoxia exposure in the neonatal lung.     

Role of Interferon Regulatory Factor 7 in T Cell Responses during Acute Lymphocytic Choriomeningitis Virus Infection

Type I interferons (IFNs), predominantly IFN-α and -β, play critical roles in both innate and adaptive immune responses against viral infections. Interferon regulatory factor 7 (IRF7), a key innate immune molecule in the type I IFN signaling pathway, is essential for the type I IFN response to many viruses, including lymphocytic choriomeningitis virus (LCMV). Here, we show that although IRF7 knockout (KO) mice failed to control the replication of LCMV in the early stages of infection, they were capable of clearing LCMV infection. Despite the lack of type I IFN production, IRF7 KO mice generated normal CD4⁺ T cell responses, and the expansion of naïve CD8⁺ T cells into primary CD8⁺ T cells specific for LCMV GP_33–41 was relatively normal. In contrast, the expansion of the LCMV NP₃₉₆-specific CD8⁺ T cells was severely impaired in IRF7 KO mice. We demonstrated that this defective CD8⁺ T cell response is due neither to an impaired antigen-presenting system nor to any intrinsic role of IRF7 in CD8⁺ T cells. The lack of a type I IFN response in IRF7 KO mice did not affect the formation of memory CD8⁺ T cells. Thus, the present study provides new insight into the impact of the innate immune system on viral pathogenesis and demonstrates the critical contribution of innate immunity in controlling virus replication in the early stages of infection, which may shape the quality of CD8⁺ T cell responses.     

Intracellular Signaling Molecules Activated by Epstein-Barr Virus for Induction of Interferon Regulatory Factor 7

Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) is the principal oncogenic protein in the EBV transformation process. LMP-1 induces the expression of interferon regulatory factor 7 (IRF-7) and activates IRF-7 protein by phosphorylation and nuclear translocation. LMP-1 is an integral membrane protein with two regions in its C terminus that initiate signaling processes, the C-terminal activator regions 1 (CTAR-1) and CTAR-2. Here, genetic analysis of LMP-1 has determined that the PXQXT motif that governs the interaction between LMP-1 CTAR-1 and tumor necrosis factor receptor-associated factors (TRAFs) is needed to induce the expression of IRF-7. Mutations in the PXQXT motif in CTAR-1 that disrupt the interaction between LMP-1 and TRAFs abolished the induction of IRF-7. Also, dominant-negative mutants of TRAFs inhibited the induction of IRF-7 by CTAR-1. The last three amino acids (YYD) of CTAR-2 are also important for the induction of IRF-7. When both PXQXT and YYD were mutated (LMP-DM), the LMP-1 mutant failed to induce IRF-7. Also, LMP-DM blocked the induction of IRF-7 by wild-type LMP-1. These data strongly suggest that both CTAR-1 and CTAR-2 of LMP-1 independently induce the expression of IRF-7. In addition, NF-kappaB is involved in the induction of IRF-7. A superrepressor of IkappaB (sr-IkappaB) could block the induction of IRF-7 by LMP-1, and overexpression of NF-kappaB (p65 plus p50) could induce the expression of IRF-7. In addition, we have found that human IRF-7 is a stable protein, and sodium butyrate, a modifier of chromatin structure, induces IRF-7.     

Murine Norovirus 1 (MNV1) Replication Induces Translational Control of the Host by Regulating eIF4E Activity during Infection

Protein synthesis is a tightly controlled process responding to several stimuli, including viral infection. As obligate intracellular parasites, viruses depend on the translation machinery of the host and can manipulate it by affecting the availability and function of specific eukaryotic initiation factors (eIFs). Human norovirus is a member of the Caliciviridae family and is responsible for gastroenteritis outbreaks. Previous studies on feline calicivirus and murine norovirus 1 (MNV1) demonstrated that the viral protein, genome-linked (VPg), acts to direct translation by hijacking the host protein synthesis machinery. Here we report that MNV1 infection modulates the MAPK pathway to activate eIF4E phosphorylation. Our results show that the activation of p38 and Mnk during MNV1 infection is important for MNV1 replication. Furthermore, phosphorylated eIF4E relocates to the polysomes, and this contributes to changes in the translational state of specific host mRNAs. We propose that global translational control of the host by eIF4E phosphorylation is a key component of the host-pathogen interaction.