Hepatitis E virus infection activates NOD-like receptor family pyrin domain-containing 3 inflammasome antagonizing interferon response but therapeutically targetable.

BACKGROUND AND AIMS: HEV infection is the most common cause of liver inflammation, but the pathogenic mechanisms remain largely unclear. We aim to explore whether HEV infection activates inflammasomes, crosstalk with antiviral interferon response, and the potential of therapeutic targeting. APPROACH AND RESULTS: We measured IL-1ß secretion, the hallmark of inflammasome activation, in serum of HEV-infected patients and rabbits, and in cultured macrophage cell lines and primary monocyte-derived macrophages. We found that genotypes 3 and 4 HEV infection in rabbits elevated IL-1ß production. A profound increase of IL-1ß secretion was further observed in HEV-infected patients (1,733 ± 1,234 pg/mL; n = 70) compared to healthy persons (731 ± 701 pg/mL; n = 70). Given that macrophages are the drivers of inflammatory response, we found that inoculation with infectious HEV particles robustly triggered NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation in primary macrophages and macrophage cell lines. We further revealed that the ORF2 capsid protein and the formed integral viral particles are responsible for activating inflammasome response. We also identified NF-κB signaling activation as a key upstream event of HEV-induced NLRP3 inflammasome response. Interestingly, inflammasome activation antagonizes interferon response to facilitate viral replication in macrophages. Pharmacological inhibitors and clinically used steroids can effectively target inflammasome activation. Combining steroids with ribavirin simultaneously inhibits HEV and inflammasome response without cross-interference. CONCLUSIONS: HEV infection strongly activates NLRP3 inflammasome activation in macrophages, which regulates host innate defense and pathogenesis. Therapeutic targeting of NLRP3, in particular when combined with antiviral agents, represents a viable option for treating severe HEV infection.

Comparative assessment of favipiravir and remdesivir against human coronavirus NL63 in molecular docking and cell culture models.

Human coronavirus NL63 (HCoV-NL63) mainly affects young children and immunocompromised patients, causing morbidity and mortality in a subset of patients. Since no specific treatment is available, this study aims to explore the anti-SARS-CoV-2 agents including favipiravir and remdesivir for treating HCoV-NL63 infection. We first successfully modelled the 3D structure of HCoV-NL63 RNA-dependent RNA polymerase (RdRp) based on the experimentally solved SARS-CoV-2 RdRp structure. Molecular docking indicated that favipiravir has similar binding affinities to SARS-CoV-2 and HCoV-NL63 RdRp with LibDock scores of 75 and 74, respectively. The LibDock scores of remdesivir to SARS-CoV-2 and HCoV-NL63 were 135 and 151, suggesting that remdesivir may have a higher affinity to HCoV-NL63 compared to SARS-CoV-2 RdRp. In cell culture models infected with HCoV-NL63, both favipiravir and remdesivir significantly inhibited viral replication and production of infectious viruses. Overall, remdesivir compared to favipiravir is more potent in inhibiting HCoV-NL63 in cell culture. Importantly, there is no evidence of resistance development upon long-term exposure to remdesivir. Furthermore, combining favipiravir or remdesivir with the clinically used antiviral cytokine interferon-alpha resulted in synergistic effects. These findings provided a proof-of-concept that anti-SARS-CoV-2 drugs, in particular remdesivir, have the potential to be repurposed for treating HCoV-NL63 infection.

cGAS-STING effectively restricts murine norovirus infection but antagonizes the antiviral action of N-terminus of RIG-I in mouse macrophages.

Although cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling has been well recognized in defending DNA viruses, the role of cGAS-STING signaling in regulating infection of RNA viruses remains largely elusive. Noroviruses, as single-stranded RNA viruses, are the main causative agents of acute viral gastroenteritis worldwide. This study comprehensively investigated the role of cGAS-STING in response to murine norovirus (MNV) infection. We found that STING agonists potently inhibited MNV replication in mouse macrophages partially requiring the JAK/STAT pathway that induced transcription of interferon (IFN)-stimulated genes (ISGs). Loss- and gain-function assays revealed that both cGAS and STING were necessary for host defense against MNV propagation. Knocking out cGAS or STING in mouse macrophages led to defects in induction of antiviral ISGs upon MNV infection. Overexpression of cGAS and STING moderately increased ISG transcription but potently inhibited MNV replication in human HEK293T cells ectopically expressing the viral receptor CD300lf. This inhibitory effect was not affected by JAK inhibitor treatment or expression of different MNV viral proteins. Interestingly, STING but not cGAS interacted with mouse RIG-I, and attenuated its N-terminus-mediated anti-MNV effects. Our results implicate an essential role for mouse cGAS and STING in regulating innate immune response and defending MNV infection. This further strengthens the evidence of cGAS-STING signaling in response to RNA virus infection.

Drug screening identified gemcitabine inhibiting hepatitis E virus by inducing interferon-like response via activation of STAT1 phosphorylation.

Exposure to hepatitis E virus (HEV) bears a high risk of developing chronic infection in immunocompromised patients, including organ transplant recipients and cancer patients. We aim to identify effective anti-HEV therapies through screening and repurposing safe-in-human broad-spectrum antiviral agents. In this study, a safe-in-human broad-spectrum antiviral drug library comprising of 94 agents was used. Upon screening, we identified gemcitabine, a widely used anti-cancer drug, as a potent inhibitor of HEV replication. The antiviral effect was confirmed in a range of cell culture models with genotype 1 and 3 HEV strains. As a cytidine analog, exogenous supplementation of pyrimidine nucleosides effectively reversed the antiviral activity of gemcitabine, but the level of pyrimidine nucleosides per se does not affect HEV replication. Surprisingly, similar to interferon-alpha (IFNα) treatment, gemcitabine activates STAT1 phosphorylation. This subsequently triggers activation of interferon-sensitive response element (ISRE) and transcription of interferon-stimulated genes (ISGs). Cytidine or uridine effectively inhibits gemcitabine-induced activation of ISRE and ISGs. As expected, JAK inhibitor 1 blocked IFNα, but not gemcitabine-induced STAT1 phosphorylation, ISRE/ISG activation, and anti-HEV activity. These effects of gemcitabine were completely lost in STAT1 knockout cells. In summary, gemcitabine potently inhibits HEV replication by triggering interferon-like response through STAT1 phosphorylation but independent of Janus kinases. This represents a non-canonical antiviral mechanism, which utilizes the innate defense machinery that is distinct from the classical interferon response. These results support repurposing gemcitabine for treating hepatitis E, especially for HEV-infected cancer patients, leading to dual anti-cancer and antiviral effects.

Murine norovirus replicase augments RIG-I-like receptors-mediated antiviral interferon response.

Noroviruses are the main causative agents for acute viral gastroenteritis worldwide. RIG-I-like receptors (RLRs) triggered interferon (IFN) activation is essential for host defense against viral infections. In turn, viruses have developed sophisticated strategies to counteract host antiviral response. This study aims to investigate how murine norovirus (MNV) replicase interacts with RLRs-mediated antiviral IFN response. Counterintuitively, we found that the MNV replicase NS7 enhances the activation of poly (I:C)-induced IFN response and the transcription of downstream interferon-stimulated genes (ISGs). Interestingly, NS7 protein augments RIG-I and MDA5-triggered antiviral IFN response, which conceivably involves direct interactions with the caspase activation and recruitment domains (CARDs) of RIG-I and MDA5. Consistently, RIG-I and MDA5 exert anti-MNV activity in human HEK293T cells with ectopic expression of viral receptor CD300lf. This effect requires the activation of JAK/STAT pathway, and is further enhanced by NS7 overexpression. These findings revealed an unconventional role of MNV NS7 as augmenting RLRs-mediated IFN response to inhibit viral replication.

Guanylate-binding protein 2 orchestrates innate immune responses against murine norovirus and is antagonized by the viral protein NS7.

Noroviruses are the main causative agents of acute viral gastroenteritis, but the host factors that restrict their replication remain poorly identified. Guanylate-binding proteins (GBPs) are interferon (IFN)-inducible GTPases that exert broad antiviral activity and are important mediators of host defenses against viral infections. Here, we show that both IFN-γ stimulation and murine norovirus (MNV) infection induce GBP2 expression in murine macrophages. Results from loss- and gain-of-function assays indicated that GBP2 is important for IFN-γ-dependent anti-MNV activity in murine macrophages. Ectopic expression of MNV receptor (CD300lf) in human HEK293T epithelial cells conferred susceptibility to MNV infection. Importantly, GBP2 potently inhibited MNV in these human epithelial cells. Results from mechanistic dissection experiments revealed that the N-terminal G domain of GBP2 mediates these anti-MNV effects. R48A and K51A substitutions in GBP2, associated with loss of GBP2 GTPase activity, attenuated the anti-MNV effects of GBP2. Finally, we found that nonstructural protein 7 (NS7) of MNV co-localizes with GBP2 and antagonizes the anti-MNV activity of GBP2. These findings reveal that GBP2 is an important mediator of host defenses against murine norovirus.

Lipopolysaccharide restricts murine norovirus infection in macrophages mainly through NF-kB and JAK-STAT signaling pathway.

The inflammasome machinery has recently been recognized as an emerging pillar of innate immunity. However, little is known regarding the interaction between the classical interferon (IFN) response and inflammasome activation in response to norovirus infection. We found that murine norovirus (MNV-1) infection induces the transcription of IL-1ß, a hallmark of inflammasome activation, which is further increased by inhibition of IFN response, but fails to trigger the release of mature IL-1ß. Interestingly, pharmacological inflammasome inhibitors do not affect viral replication, but slightly reverse the inflammasome activator lipopolysaccharide (LPS)-mediated inhibition of MNV replication. LPS efficiently stimulates the transcription of IFN-ß through NF-ĸB, which requires the transcription factors IRF3 and IRF7. This activates downstream antiviral IFN-stimulated genes (ISGs) via the JAK-STAT pathway. Moreover, inhibition of NF-ĸB and JAK-STAT signaling partially reverse LPS-mediated anti-MNV activity, suggesting additional antiviral mechanisms activated by NF-ĸB. This study reveals additional insight in host defense against MNV infection.

MDA5 against enteric viruses through induction of interferon-like response partially via the JAK-STAT cascade.

Enteric viruses including hepatitis E virus (HEV), human norovirus (HuNV), and rotavirus are causing global health issues. The host interferon (IFN) response constitutes the first-line defense against viral infections. Melanoma Differentiation-Associated protein 5 (MDA5) is an important cytoplasmic receptor sensing viral infection to trigger IFN production, and on the other hand it is also an IFN-stimulated gene (ISG). In this study, we investigated the effects and mode-of-action of MDA5 on the infection of enteric viruses. We found that MDA5 potently inhibited HEV, HuNV and rotavirus replication in multiple cell models. Overexpression of MDA5 induced transcription of important antiviral ISGs through IFN-like response, without triggering of functional IFN production. Interestingly, MDA5 activates the expression and phosphorylation of STAT1, which is a central component of the JAK-STAT cascade and a hallmark of antiviral IFN response. However, genetic silencing of STAT1 or pharmacological inhibition of the JAK-STAT cascade only partially attenuated the induction of ISG transcription and the antiviral function of MDA5. Thus, we have demonstrated that MDA5 effectively inhibits HEV, HuNV and rotavirus replication through provoking a non-canonical IFN-like response, which is partially dependent on JAK-STAT cascade.

FDA-drug screening identifies deptropine inhibiting hepatitis E virus involving the NF-κB-RIPK1-caspase axis.

Hepatitis E virus (HEV) infection is the leading cause of acute hepatitis worldwide and can develop into chronic infection in immunocompromised patients, promoting the development of effective antiviral therapies. In this study, we performed a screening of a library containing over 1000 FDA-approved drugs. We have identified deptropine, a classical histamine H1 receptor antagonist used to treat asthmatic symptoms, as a potent inhibitor of HEV replication. The anti-HEV activity of deptropine appears dispensable of the histamine pathway, but requires the inhibition on nuclear factor-κB (NF-κB) activity. This further activates caspase mediated by receptor-interacting protein kinase 1 (RIPK1) to restrict HEV replication. Given deptropine being widely used in the clinic, our results warrant further evaluation of its anti-HEV efficacy in future clinical studies. Importantly, the discovery that NF-κB-RIPK1-caspase pathway interferes with HEV infection reveals new insight of HEV-host interactions.

αß T-cell receptor bias in disease and therapy (Review).

The diversity and specificity of T cell receptors (TCR), the characteristics of T-cell surface marker, are central to the adaptive immunity. TCR variability is required for successful immunization coverage because this structural foundation is indispensable for the valid identification of short antigen peptides (derived from degraded antigens) that are presented by major histocompatibility molecules on the surfaces of antigen-presenting cells. Despite the vast T-cell repertoire, biased αß TCR has become a common theme in immunology. To date, numerous examples of TCR bias have been observed in various diseases. Immunotherapy strategies that are based on αß T cell responses are also emerged as a prominent component of clinical treatment. In the present review, we briefly summarize the current knowledge regarding basic structural information and the molecular mechanisms underlying TCR diversity. Moreover, we outline the role of TCR repertoire bias in some diseases, and its application for therapeutic interventions, as these play significant roles in disease progression, even with patients with a good prognosis.