Xiaojuan Chi

Suppression of Interferon Lambda Signaling by SOCS-1 Results in Their Excessive Production during Influenza Virus Infection

Innate cytokine response provides the first line of defense against influenza virus infection. However, excessive production of cytokines appears to be critical in the pathogenesis of influenza virus. Interferon lambdas (IFN-λ) have been shown to be overproduced during influenza virus infection, but the precise pathogenic processes of IFN-λ production have yet to be characterized. In this report, we observed that influenza virus induced robust expression of IFN-λ in alveolar epithelial cells (A549) mainly through a RIG-I-dependent pathway, but IFN-λ-induced phosphorylation of the signal transducer and activator of transcription protein 1 (STAT1) was dramatically inhibited in the infected cells. Remarkably, influenza virus infection induced robust expression of suppressor of cytokine signaling-1 (SOCS-1), leading to inhibition of STAT1 activation. Interestingly, the virus-induced SOCS-1 expression was cytokine-independent at early stage of infection both in vitro and in vivo. Using transgenic mouse model and distinct approaches altering the expression of SOCS-1 or activation of STAT signaling, we demonstrated that disruption of the SOCS-1 expression or expression of constitutively active STAT1 significantly reduced the production of IFN-λ during influenza virus infection. Furthermore, we revealed that disruption of IFN-λ signaling pathway by increased SOCS-1 protein resulted in the activation of NF-κB and thereby enhanced the IFN-λ expression. Together, these data imply that suppression of IFN-λ signaling by virus-induced SOCS-1 causes an adaptive increase in IFN-λ expression by host to protect cells against the viral infection, as a consequence, leading to excessive production of IFN-λ with impaired antiviral response.

Roles of Rho GTPases in Intracellular Transport and Cellular Transformation

Rho family GTPases belong to the Ras GTPase superfamily and transduce intracellular signals known to regulate a variety of cellular processes, including cell polarity, morphogenesis, migration, apoptosis, vesicle trafficking, viral transport and cellular transformation. The three best-characterized Rho family members are Cdc42, RhoA and Rac1. Cdc42 regulates endocytosis, the transport between the endoplasmic reticulum and Golgi apparatus, post-Golgi transport and exocytosis. Cdc42 influences trafficking through interaction with Wiskott-Aldrich syndrome protein (N-WASP) and the Arp2/3 complex, leading to changes in actin dynamics. Rac1 mediates endocytic and exocytic vesicle trafficking by interaction with its effectors, PI3kinase, synaptojanin 2, IQGAP1 and phospholipase D1. RhoA participates in the regulation of endocytosis through controlling its downstream target, Rho kinase. Interestingly, these GTPases play important roles at different stages of viral protein and genome transport in infected host cells. Importantly, dysregulation of Cdc42, Rac1 and RhoA leads to numerous disorders, including malignant transformation. In some cases, hyperactivation of Rho GTPases is required for cellular transformation. In this article, we review a number of findings related to Rho GTPase function in intracellular transport and cellular transformation.

Influenza A virus-induced degradation of eukaryotic translation initiation factor 4B contributes to viral replication by suppressing IFITM3 protein expression.

ABSTRACT Although alteration in host cellular translation machinery occurs in virus-infected cells, the role of such alteration and the precise pathogenic processes are not well understood. Influenza A virus (IAV) infection shuts off host cell gene expression at transcriptional and translational levels. Here, we found that the protein level of eukaryotic translation initiation factor 4B (eIF4B), an integral component of the translation initiation apparatus, was dramatically reduced in A549 cells as well as in the lung, spleen, and thymus of mice infected with IAV. The decrease in eIF4B level was attributed to lysosomal degradation of eIF4B, which was induced by viral NS1 protein. Silencing eIF4B expression in A549 cells significantly promoted IAV replication, and conversely, overexpression of eIF4B markedly inhibited the viral replication. Importantly, we observed that eIF4B knockdown transgenic mice were more susceptible to IAV infection, exhibiting faster weight loss, shorter survival time, and more-severe organ damage. Furthermore, we demonstrated that eIF4B regulated the expression of interferon-induced transmembrane protein 3 (IFITM3), a critical protein involved in immune defense against a variety of RNA viruses, including influenza virus. Taken together, our findings reveal that eIF4B plays an important role in host defense against IAV infection at least by regulating the expression of IFITM3, which restricts viral entry and thereby blocks early stages of viral production. These data also indicate that influenza virus has evolved a strategy to overcome host innate immunity by downregulating eIF4B protein. IMPORTANCE Influenza A virus (IAV) infection stimulates the host innate immune system, in part, by inducing interferons (IFNs). Secreted IFNs activate the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, leading to elevated transcription of a large group of IFN-stimulated genes that have antiviral function. To circumvent the host innate immune response, influenza virus has evolved multiple strategies for suppressing the production of IFNs. Here, we show that IAV infection induces lysosomal degradation of eIF4B protein; and eIF4B inhibits IAV replication by upregulating expression of interferon-induced transmembrane protein 3 (IFITM3), a key protein that protects the host from virus infection. Our finding illustrates a critical role of eIF4B in the host innate immune response and provides novel insights into the complex mechanisms by which influenza virus interacts with its host.

The Regulation of Autophagy by Influenza A Virus

Influenza A virus is a dreadful pathogen of animals and humans, causing widespread infection and severe morbidity and mortality. It is essential to characterize the influenza A virus-host interaction and develop efficient counter measures against the viral infection. Autophagy is known as a catabolic process for the recycling of the cytoplasmic macromolecules. Recently, it has been shown that autophagy is a critical mechanism underlying the interaction between influenza A virus and its host. Autophagy can be induced by the infection with influenza A virus, which is considered as a necessary process for the viral proliferation, including the accumulation of viral elements during the replication of influenza A virus. On the other hand, influenza A virus can inhibit the autophagic formation via interaction with the autophagy-related genes (Atg) and signaling pathways. In addition, autophagy is involved in the influenza virus-regulated cell deaths, leading to significant changes in host apoptosis. Interestingly, the high pathogenic strains of influenza A virus, such as H5N1, stimulate autophagic cell death and appear to interplay with the autophagy in distinct ways as compared with low pathogenic strains. This review discusses the regulation of autophagy, an influenza A virus driven process.

Avian Tembusu virus infection effectively triggers host innate immune response through MDA5 and TLR3-dependent signaling pathways

Avian Tembusu virus (ATMUV) is a newly emerged flavivirus that belongs to the Ntaya virus group. ATMUV is a highly pathogenic virus causing significant economic loss to the Chinese poultry industry. However, little is known about the role of host innate immune mechanism in defending against ATMUV infection. In this study, we found that ATMUV infection significantly up-regulated the expression of type I and type III interferons (IFN) and some critical IFN-stimulated genes (ISG) in vivo and in vitro. This innate immune response was induced by genomic RNA of ATMUV. Furthermore, we observed that ATMUV infection triggered IFN response mainly through MDA5 and TLR3-dependent signaling pathways. Strikingly, shRNA-based disruption of IPS-1, IRF3 or IRF7 expression significantly reduced the production of IFN in the 293T cell model. Moreover, NF-κB was shown to be activated in both chicken and human cells during the ATMUV infection. Inhibition of NF-κB signaling also resulted in a clear decrease in expression of IFN. Importantly, experiments revealed that treatment with IFN significantly impaired ATMUV replication in the chicken cell. Consistently, type I IFN also exhibited promising antiviral activity against ATMUV replication in the human cell. Together, these data indicate that ATMUV infection triggers host innate immune response through MDA5 and TLR3-dependent signaling that controls IFN production, and thereby induces an effective antiviral immunity.

Muscovy duck reovirus infection rapidly activates host innate immune signaling and induces an effective antiviral immune response involving critical interferons

Muscovy duck reovirus (MDRV) is a highly pathogenic virus in waterfowl and causes significant economic loss in the poultry industry worldwide. Because the host innate immunity plays a key role in defending against virus invasion, more and more attentions have been paid to the immune response triggered by viral infection. Here we found that the genomic RNA of MDRV was able to rapidly induce the production of interferons (IFNs) in host. Mechanistically, MDRV infection induced robust expression of IFNs in host mainly through RIG-I, MDA5 and TLR3-dependent signaling pathways. In addition, we observed that silencing VISA expression in 293T cells could significantly inhibit the secretion of IFNs. Remarkably, the production of IFNs was reduced by inhibiting the activation of NF-κB or knocking down the expression of IRF-7. Furthermore, our study showed that treatment of 293T cells and Muscovy duck embryo fibroblasts with IFNs markedly impaired MDRV replication, suggesting that these IFNs play an important role in antiviral response during the MDRV infection. Importantly, we also detected the induced expression of RIG-I, MDA5, TLR3 and type I IFN in Muscovy ducks infected with MDRV at different time points post infection. The results from in vivo studies were consistent with those in 293T cells infected with MDRV. Taken together, our findings reveal that the host can resist MDRV invasion by activating innate immune response involving RIG-I, MDA5 and TLR3-dependent signaling pathways that govern IFN production.

Targeted disruption of influenza A virus hemagglutinin in genetically modified mice reduces viral replication and improves disease outcome.

Influenza A virus can cause acute respiratory infection in animals and humans around the globe, and is still a major threat to animal husbandry and public health. Due to antigenic drift and antigenic shift of the virus, development of novel anti-influenza strategies has become an urgent task. Here we generated transgenic (TG) mice stably expressing a short-hairpin RNA specifically targeting hemagglutinin (HA) of influenza A virus, and investigated the susceptibility of the mice to influenza virus infection. We found that HA expression was dramatically disrupted in TG mice infected with WSN or PR8 virus. Importantly, the animals showed reduced virus production in lungs, slower weight loss, attenuated acute organ injury and consequently increased survival rates as compared to wild type (WT) mice after the viral infection. Moreover, TG mice exhibited a normal level of white blood cells following the virus infection, whereas the number of these cells was significantly decreased in WT mice with same challenge. Together, these experiments demonstrate that the TG mice are less permissive for influenza virus replication, and suggest that shRNA-based efficient disruption of viral gene expression in animals may be a useful strategy for prevention and control of a viral zoonosis.

Infection of goats with goatpox virus triggers host antiviral defense through activation of innate immune signaling

Goatpox, caused by goatpox virus (GTPV), is one of the most serious infectious diseases associated with high morbidity and mortality in goats. However, little is known about involvement of host innate immunity during the GTPV infection. For this, goats were experimentally infected with GTPV. The results showed that GTPV infection significantly induced mRNA expression of type I interferon (IFN)-α and IFN-β in peripheral blood lymphocytes, spleen and lung. In addition, GTPV infection enhanced expression of several inflammatory cytokines, including interleukin (IL)-1β, IL-6, IL-18; and tumor necrosis factor-α (TNF-α). Strikingly, infection with GTPV activated signal transducers and activators of transcription 3 (STAT3), a critical cytokine signaling molecule. Interestingly, the virus infection induced expression of suppressor of cytokine signaling (SOCS)-1. Importantly, the infection resulted in an increased expression of some critical interferon-stimulated genes, such as interferon-induced transmembrane protein (IFITM) 1, IFITM3, interferon stimulated gene (ISG) 15 and ISG20. Furthermore, we found that infection with GTPV up-regulated expression of Toll-like receptor (TLR) 2 and TLR9. These results revealed that GTPV infection activated host innate immune signaling and thereby triggered antiviral innate immunity. The findings provide novel insights into complex mechanisms underlying GTPV-host interaction and pathogenesis of GTPV.

3-Anhydro-6-hydroxy-ophiobolin A displays high in vitro and in vivo efficacy against influenza A virus infection

Influenza A virus (IAV) is an enveloped negative-strand RNA virus containing eight RNA segments that belongs to the family Orthomyxoviridae and can cause acute respiratory infection in humans and animals. Although vaccination is one of the major means for prophylaxis of influenza virus infection, a particular influenza vaccine only confers protection for no more than a few years. In addition, the outbreak of novel influenza infection cannot be predicted (Gao et al., 2013). Thus, vaccine is unable to provide immediate protection against sudden influenza outbreaks with unknown identity. Development of antiviral drugs for therapeutic treatment is an important strategy to reduce the duration and severity of influenza. However, more and more drug-resistant influenza virus strains are emerging due to its antigenic drift or antigenic shift, which increases the need for new antivirals. Over the past decades, progresses have been made in developing small molecule compounds for treatment of influenza viral infection. For example, previous experiments demonstrated that the novel NF-kappaB inhibitor SC75741 significantly protected mice against infection with highly pathogenic avian influenza A viruses (HPAIV) of the H5N1 and H7N7 subtypes (Haasbach et al., 2013). The MEK inhibitor U0126, targeting the intracellular Raf/MEK/ERK signaling pathway, is able to suppress propagation of both the 2009 pandemic IAV and HPAIV in vitro and in vivo (Droebner et al., 2011). Moreover, two identified novel antiIAV agents, obatoclax and gemcitabine possess broadspectrum antiviral activity. Obatoclax can inhibit IAV uptake and gemcitabine can suppress viral RNA transcription and replication (Denisova et al., 2012; Planz, 2013). Influenza virus utilizes cellular machinery for the replication and assembly of viral components and the release of progeny virions. Recent genome-wide RNAi screening has identified several host genes and molecular networks crucial for the viral replication (Karlas et al., 2010; Watanabe et al., 2010), providing potential ways for anti-influenza therapy. In our ongoing search for new bioactive compounds from fungi, we have recently reported 3-anhydro-6-hydroxyophiobolin A (L435-3), a new sesterterpene with antibacterial activities from the phytopathogenic fungus Bipolaris oryzae (Wang et al., 2013). In this study, we further examined the potential anti-influenza activity of L435-3 in vitro and in vivo. First, A549 cells were infected with influenza virus strain WSN virus and then treated with or without L435-3. We found that treatment with L435-3 could inhibit the IAV replication at very low concentration (Fig. 1A), indicating that L435-3 may be a potential therapeutic agent for treatment of influenza virus infections. To further confirm the inhibitory effect of L435-3 on IAV infection, we performed several experiments using A549 and MDCK cells infected with WSN or PR8 virus. As shown in Table S1, L435-3 displayed high activity against WSN or PR8 virus infection in MDCK cells, although its activity is lower than that of zanamivir. Then A549 cells were infected with WSN at a multiplicity of infection (MOI) of 0.2, and treated with 0.5 μM L435-3 at 1 h post-infection. Indeed, 0.5 μM L435-3 showed little cytotoxicity to A549 cells, and the influenza virus titers were markedly reduced by L435-3 treatment (Fig. 1B and 1C). Using Western blotting, we further confirmed that L435-3 treatment significantly inhibited influenza virus replication, since the levels of both viral HA and NP were markedly reduced in WSN-infected A549 cells treated with L435-3 (Fig. 1D). Together, these experiments demonstrate that L435-3 is a potent inhibitor of IAV replication in host cell. Next, we investigated the anti-influenza virus activity of L435-3 in vivo. To this end, Balb/c mice were separated into four groups (12 mice/each group). Two groups of mice were infected with WSN and then inoculated intranasally with L435-3 (0.3 mg/kg/mouse) or mock control. The other two groups of mice were only treated with L435-3 or mock control. All mice were monitored for body weight change and survival rate. As expected, WSN challenge resulted in obvious flu symptoms in all mice on day 2 post-infection. However, the symptoms were remarkably less severe in L435-3 treated group (Fig. 1E). The mice without L435-3 treatment showed a more weight loss as compared to L4353 treated group during the IAV infection (Fig. 1F). To further determine the efficacy of L435-3, we compared the mortality

Alpha/beta interferon receptor deficiency in mice significantly enhances susceptibility of the animals to pseudorabies virus infection

Pseudorabies virus, one of the neurotropic viruses, can infect numerous mammals. In particular, pseudorabies virus infection of swine occurs worldwide, and is a major threat to swine industry. However, the mechanism underlying the interaction between pseudorabies virus and host innate immune system is not fully understood. Here, we investigated the involvement of interferon α/β (IFN-α/β) receptor (IFNAR) in the pathogenesis of pseudorabies virus in a mouse model. The results showed that IFNAR-deficient (IFNAR-/-) mice were highly susceptible to the virus infection, as evidenced by markedly reduced survival rate of infected animals and increased viral replication. The expression of IFN-α/β and relevant interferon-stimulated genes in IFNAR-/- mice was significantly lower than that in wild-type (WT) littermates after the viral infection. Moreover, in response to the virus challenge, IFNAR-/- mice displayed elevated levels of inflammatory cytokines including interleukin 6 (IL-6) and IL-1β, and IFNAR-/- cells showed increased phosphorylation of STAT3. Collectively, these data reveal that the IFNAR-/- mice are more sensitive to pseudorabies virus infection than WT animals, and excessive IL-6/STAT3 response in IFNAR-/- mice may contribute to the pathogenesis. Our findings suggest that type I IFNs/IFNAR-dependent homeostatic control of the innate immunity is required for host defense against pseudorabies virus infection.