Intraflagellar transport 46 (IFT46) is essential for trafficking IFT proteins between cilia and cytoplasm in Paramecium.

Intraflagellar transport (IFT) is a bi-directional process by which particles are carried within the cilia or flagella. This process is essential for ciliary growth and functional maintenance. The IFT complex B (IFTB) is linked to a kinesin motor for anterograde transport towards the ciliary tip. The IFT complex A (IFTA) is connected to a dynein motor for retrograde transport towards the ciliary basis. This study focuses on IFT46, an IFTB member that participates in this process. In Paramecium, a GFP-labelled IFT46 protein was found in basal bodies and in some cilia, mostly those undergoing biogenesis. RNA interference against IFT46 in Paramecium triggered severe defects in ciliary growth and architecture, including a decreased cilia number and shortened cilia length. This result differed from that obtained from the cells that were depleted of IFT80, another IFTB protein. Moreover, IFT57-GFP fusion protein abnormally accumulated in the cortex and cytoplasm in IFT46-depleted cells compared with the control. Furthermore, transcriptomic analysis showed that IFT46 depletion induced the abnormal expression of several genes that encodeding kinesin and dynein chains. These findings together indicate that IFT46 plays important roles in trafficking IFT proteins between the cytoplasm and cilia of Paramecium.

Insulin receptor substrate-4 interacts with ubiquitin-specific protease 18 to activate the Jak/STAT signaling pathway.

Ubiquitin-specific protease 18 (USP18) as a negative regulator of the Jak/STAT signaling pathway plays an important role in the host innate immune response. USP18 has been shown to bind to the type I interferon receptor subunit 2 (IFNAR2) to down-regulate the Jak/STAT signaling. In this study, we showed that insulin receptor substrate (IRS)-4 functioned as a novel USP18-binding protein. Co-precipitation assays revealed that two regions (amino acids 335-400 and 1094-1257) of IRS4 were related to bind to the C- terminal region of USP18. IRS4 binding to USP18 diminished the inhibitory effect of USP18 on Jak/STAT signaling. IRS4 over-expression enhanced while IRS4 knock-down suppressed the Jak/STAT signaling in the presence of IFN-a stimulation. As such, IRS4 increased IFN-a-mediated anti-HCV activity. Mechanistically, IRS4 promoted the IFN-a-induced Jak/STAT signaling by interact with USP18. These results suggested that IRS4 binds to USP18 to diminish the blunting effect of USP18 on IFN-a-induced Jak/STAT signaling. Our findings indicated that IRS4 is a novel USP18-binding protein that can be used to boost the host innate immunity to control HCV, and potentially other viruses that are sensitive to IFN-a.

MxA is a positive regulator of type I IFN signaling in HCV infection.

Type I interferons (IFNs) are a family of primordial cytokines that respond to various pathogen infections including Hepatitis C virus (HCV). Type I IFNs signal through Jak/STAT pathway leading to the production of a few hundred interferon stimulated genes (ISGs). The aim of this study was to explore the role of one of these ISGs, MxA in HCV infection and type I IFN production. Plasmid encoding MxA was cloned into PcDNA3.1-3×tag vector and MxA expression was confirmed both at mRNA (RT-PCR) and protein (Western blot, WB) levels. IFNα and IFNß productions were quantified by RT-PCR from cell lysate and by ELISA kit from culture medium following MxA over-expression in Huh7.5.1 cells. The activation status of Jak/STAT signaling pathway was examined at three levels: p-STAT1 (WB), interferon sensitive response element (ISRE) activity (dual luciferase reporter gene assay), and levels of ISG expression (RT-qPCR). J6/JFH1 HCV culture system was used to study the role of MxA in HCV replication. Our findings indicated that MxA over-expression inhibited HCV replication and potentiated the IFNα-mediated anti-HCV activity; MxA stimulated the production of IFNα, IFNß, and enhanced IFNα-induced activation of Jak-STAT signaling pathway. We concluded that MxA is a positive regulator of type I IFN signaling in HCV infection.

ISG12a inhibits HCV replication and potentiates the anti-HCV activity of IFN-α through activation of the Jak/STAT signaling pathway independent of autophagy and apoptosis.

Interferon stimulated (sensitive) genes (ISGs) are the effector molecules downstream of type I/III interferon (IFN) signaling pathways in host innate immunity. ISG12a can be induced by IFN-α. Although ISG12a has been reported to inhibit the replication of HCV, the exact mechanism remains to be determined. In this study, we investigated the possible mechanisms of ISG12a anti- HCV property by exploring the production of type I IFN and the activation of Janus kinase/signal transducer and activator of transcription (Jak/STAT) signaling pathway, apoptosis and autophagy in Huh7.5.1 cells transiently transfected with ISG12a over-expression plasmid. Interestingly, we found that ISG12a inhibited HCV replication in both Con1b replicon and the HCV JFH1-based cell culture system and potentiated the anti-HCV activity of IFN-α. ISG12a promoted the production of IFN α/ß and activated the type I IFN signaling pathway as shown by increased p-STAT1 level, higher Interferon sensitive response element (ISRE) activity and up-regulated ISG levels. However, ISG12a over-expression did not affect cell autophagy and apoptosis. Data from our current study collectively indicated that ISG12a inhibited HCV replication and potentiated the anti-HCV activity of IFN-α possibly through induced production of type I IFNs and activation of Jak/STAT signaling pathway independent of autophagy and cell apoptosis.