Leilei Wang

Flaviviruses have evolved complex mechanisms to evade the mammalian host immune systems including the RIG-I (retinoic acid-inducible gene I) like receptor (RLR) signaling. Zika virus (ZIKV) is a re-emerging flavivirus that is associated with severe neonatal microcephaly and adult Guillain-Barre syndrome. However, the molecular mechanisms underlying ZIKV pathogenesis remain poorly defined. Here we report that ZIKV non-structural protein 4A (NS4A) impairs the RLR-mitochondrial antiviral-signaling protein (MAVS) interaction and subsequent induction of antiviral immune responses. In human trophoblasts, both RIG-I and melanoma differentiation-associated protein 5 (MDA5) contribute to type I interferon (IFN) induction and control ZIKV replication. Type I IFN induction by ZIKV is almost completely abolished in MAVS-/- cells. NS4A represses RLR-, but not Toll-like receptor-mediated immune responses. NS4A specifically binds the N-terminal caspase activation and recruitment domain (CARD) of MAVS and thus blocks its accessibility by RLRs. Our study provides in-depth understanding of the molecular mechanisms of immune evasion by ZIKV and its pathogenesis.

PURPOSE: Endoplasmic reticulum (ER) stress has been demonstrated to contribute to neurodegeneration in multiple ocular diseases. However, whether ER stress can induce vascular degeneration in the retina remains unknown. We investigated the possible role of ER stress in retinal vascular degeneration in vivo, and the effects of resveratrol on tunicamycin and ischemia and reperfusion (I/R)-induced retinal vascular degeneration. METHODS: Different dosages of tunicamycin, an ER stress inducer, were injected into the vitreous of mouse eyes. Retinal I/R injury was induced by elevating the intraocular pressure for 60 minutes followed by reperfusion in mice. Two dosages of resveratrol (5 and 25 mg/kg body weight per day) were administrated 2 days before retinal I/R injury, while 100 μM resveratrol were injected into the vitreous together with tunicamycin. Formation of acellular capillaries was assessed 7 days after I/R injury and tunicamycin injection, while cell bodies in ganglion cell layer and brain-specific homeobox/POU domain protein 3A (Brn3a) staining on retinal flat-mounts were analyzed 4 days after I/R injury. ER stress markers, including eukaryotic initiation factor 2α (eIF2α), CCAAT enhancer-binding protein homologous protein (CHOP), immunoglobulin binding protein (Bip), inositol requiring enzyme 1α (IRE1α), C-jun N-terminal kinase (JNK)1/2 and Xbp1 splicing, were examined by RT-PCR, or Western blots or immunostaining from retinas 1 or 2 days after tunicamycin injection and I/R injury. RESULTS: Tunicamycin caused ER stress and capillary degeneration in vivo, both of which were inhibited by resveratrol. Pretreatment of high dosage of resveratrol also significantly inhibited retinal I/R injury-induced capillary degeneration; however, neither of the dosages prevented the injury-induced neurodegeneration. Levels of CHOP, phosphorylated eIF2α, IRE1α, phosphorylated JNK1/2, Xbp1 splicing and Bip were elevated after I/R injury. High dosage of resveratrol pretreatment inhibited the injury-induced up-regulation of eIF2α-CHOP and IRE1α-XBP1 pathways. CONCLUSIONS: ER stress is an important contributor to vascular degeneration in retina. Resveratrol suppresses I/R injury and tunicamycin-induced vascular degeneration by inhibiting ER stress.

BACKGROUND: Nitric oxide synthase (NOS) is responsible for synthesizing nitric oxide (NO) from L-arginine, and involved in multiple physiological functions. However, its immunological role in mollusc was seldom reported. METHODOLOGY: In the present study, an NOS (CfNOS) gene was identified from the scallop Chlamys farreri encoding a polypeptide of 1486 amino acids. Its amino acid sequence shared 50.0~54.7, 40.7~47.0 and 42.5~44.5% similarities with vertebrate neuronal (n), endothelial (e) and inducible (i) NOSs, respectively. CfNOS contained PDZ, oxygenase and reductase domains, which resembled those in nNOS. The CfNOS mRNA transcripts expressed in all embryos and larvae after the 2-cell embryo stage, and were detectable in all tested tissues with the highest level in the gonad, and with the immune tissues hepatopancreas and haemocytes included. Moreover, the immunoreactive area of CfNOS distributed over the haemocyte cytoplasm and cell membrane. After LPS, β-glucan and PGN stimulation, the expression level of CfNOS mRNA in haemocytes increased significantly at 3 h (4.0-, 4.8- and 2.7-fold, respectively, P < 0.01), and reached the peak at 12 h (15.3- and 27.6-fold for LPS and β-glucan respectively, P < 0.01) and 24 h (17.3-fold for PGN, P < 0.01). In addition, TNF-α also induced the expression of CfNOS, which started to increase at 1 h (5.2-fold, P < 0.05) and peaked at 6 h (19.9-fold, P < 0.01). The catalytic activity of the native CfNOS protein was 30.3 ± 0.3 U mgprot(-1), and it decreased significantly after the addition of the selective inhibitors of nNOS and iNOS (26.9 ± 0.4 and 29.3 ± 0.1 U mgprot(-1), respectively, P < 0.01). CONCLUSIONS: These results suggested that CfNOS, with identical structure with nNOS and similar enzymatic characteristics to nNOS and iNOS, played the immunological role of iNOS to be involved in the scallop immune defense against PAMPs and TNF-α.