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The innate immune system is the first line of the host defense program against pathogens and harmful substances. Antiviral innate immune responses can be triggered by multiple cellular receptors sensing viral components. The activated innate immune system produces interferons (IFNs) and cytokines that perform antiviral functions to eliminate invading viruses. Coronaviruses are single-stranded, positive-sense RNA viruses that have a broad range of animal hosts. Coronaviruses have evolved multiple means to evade host antiviral immune responses. Successful immune evasion by coronaviruses may enable the viruses to adapt to multiple species of host organisms. Coronavirus transmission from zoonotic hosts to humans has caused serious illnesses, such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease-2019 (COVID-19), resulting in global health and economic crises. In this review, we summarize the current knowledge of the mechanisms underlying host sensing of and innate immune responses against coronavirus invasion, as well as host immune evasion strategies of coronaviruses., Coronaviruses: Improved understanding of immune system responses needed Understanding how the innate immune system senses coronaviruses and how coronaviruses can escape detection could provide novel approaches to tackle infections. Coronaviruses, including SARS-CoV-2, constantly evolve to manipulate, obstruct and evade host immune responses. A team led by Ji-Seung Yoo, Hokkaido University, Sapporo, Japan, reviewed understanding of innate immune responses to coronaviruses and viral evasion strategies. Two major receptor families recognise RNA viruses upon infection, but how they respond to SARS-CoV-2 is unclear. One receptor, TLR7, plays a critical role in sensing coronavirus infections, and mutations in the TLR7 gene are associated with severe illness and mortality in young Covid-19 patients. Activating host TLR pathways may prove a useful therapeutic approach. Further in-depth investigations are needed into specific coronavirus proteins and viral mechanisms that suppress host immunity.

Background/aim Non-small cell lung cancer (NSCLC) is one among the most common cancers worldwide. Recently, dietary phytochemicals have been reported as an attractive approach to improve the symptoms of NSCLC patients. Tannic acid is a natural polyphenol, which is known to have anticancer effects on in vitro models of breast, gingival and colon cancer. However, the molecular mechanisms associated with the actions of tannic acid on A549 human lung cancer cells have not been elucidated. Materials and methods In this study, we analyzed the effect of tannic acid on A549 cells and their underlying mechanisms using western blotting, flow cytometry, invasion assay and tumorsphere formation assay. Results Tannic acid treatment suppressed the viability of A549 cells through cell cycle arrest and induction of the intrinsic pathways of apoptosis. In addition, the various malignant phenotypes of A549 cells including invasion, migration, and stemness were inhibited by tannic acid treatment. Conclusion Tannic acid could be used as an effective inhibitor of lung cancer progression.

Three-dimensional (3D) cell culture is well documented to regain intrinsic metabolic properties and to better mimic the in vivo situation than two-dimensional (2D) cell culture. Particularly, proline metabolism is critical for tumorigenesis since pyrroline-5-carboxylate (P5C) reductase (PYCR/P5CR) is highly expressed in various tumors and its enzymatic activity is essential for in vitro 3D tumor cell growth and in vivo tumorigenesis. PYCR converts the P5C intermediate to proline as a biosynthesis pathway, whereas proline dehydrogenase (PRODH) breaks down proline to P5C as a degradation pathway. Intriguingly, expressions of proline biosynthesis PYCR gene and proline degradation PRODH gene are up-regulated directly by c-Myc oncoprotein and p53 tumor suppressor, respectively, suggesting that the proline-P5C metabolic axis is a key checkpoint for tumor cell growth. Here, we report a metabolic reprogramming of 3D tumor cell growth by oncogenic Kaposi's sarcoma-associated herpesvirus (KSHV), an etiological agent of Kaposi's sarcoma and primary effusion lymphoma. Metabolomic analyses revealed that KSHV infection increased nonessential amino acid metabolites, specifically proline, in 3D culture, not in 2D culture. Strikingly, the KSHV K1 oncoprotein interacted with and activated PYCR enzyme, increasing intracellular proline concentration. Consequently, the K1-PYCR interaction promoted tumor cell growth in 3D spheroid culture and tumorigenesis in nude mice. In contrast, depletion of PYCR expression markedly abrogated K1-induced tumor cell growth in 3D culture, not in 2D culture. This study demonstrates that an increase of proline biosynthesis induced by K1-PYCR interaction is critical for KSHV-mediated transformation in in vitro 3D culture condition and in vivo tumorigenesis.

Non-small-cell lung cancer (NSCLC) is the most common lung cancer subtype and accounts for more than 80% of all lung cancer cases. Epidermal growth factor receptor (EGFR) phosphorylation by binding growth factors such as EGF activates downstream prooncogenic signaling pathways including KRAS-ERK, JAK-STAT, and PI3K-AKT. These pathways promote the tumor progression of NSCLC by inducing uncontrolled cell cycle, proliferation, migration, and programmed death-ligand 1 (PD-L1) expression. New cytotoxic drugs have facilitated considerable progress in NSCLC treatment, but side effects are still a significant cause of mortality. Gallic acid (3,4,5-trihydroxybenzoic acid, GA) is a phenolic natural compound, isolated from plant derivatives, that has been reported to show anticancer effects. We demonstrated the tumor-suppressive effect of GA, which induced the decrease of PD-L1 expression through binding to EGFR in NSCLC. This binding inhibited the phosphorylation of EGFR, subsequently inducing the inhibition of PI3K and AKT phosphorylation, which triggered the activation of p53. The p53-dependent upregulation of miR-34a induced PD-L1 downregulation. Further, we revealed the combination effect of GA and anti-PD-1 monoclonal antibody in an NSCLC-cell and peripheral blood mononuclear&ndash, cell coculture system. We propose a novel therapeutic application of GA for immunotherapy and chemotherapy in NSCLC.

Blood CD14+ monocytes are frontline immunomodulators categorized into classical, intermediate or non-classical subsets, and subsequently differentiated into M1 pro- or M2 anti-inflammatory macrophages on stimulation. Although the Zika virus (ZIKV) rapidly establishes viraemia, the target cells and immune responses, particularly during pregnancy, remain elusive. Furthermore, it is unknown whether African- and Asian-lineage ZIKV have different phenotypic impacts on host immune responses. Using human blood infection, we identified CD14+ monocytes as the primary target for African- or Asian-lineage ZIKV infection. When immunoprofiles of human blood infected with ZIKV were compared, a classical/intermediate monocyte-mediated M1-skewed inflammation by the African-lineage ZIKV infection was observed, in contrast to a non-classical monocyte-mediated M2-skewed immunosuppression by the Asian-lineage ZIKV infection. Importantly, infection of the blood of pregnant women revealed an enhanced susceptibility to ZIKV infection. Specifically, Asian-lineage ZIKV infection of pregnant women’s blood led to an exacerbated M2-skewed immunosuppression of non-classical monocytes in conjunction with a global suppression of type I interferon-signalling pathway and an aberrant expression of host genes associated with pregnancy complications. Also, 30 ZIKV+ sera from symptomatic pregnant patients showed elevated levels of M2-skewed immunosuppressive cytokines and pregnancy-complication-associated fibronectin-1. This study demonstrates the differential immunomodulatory responses of blood monocytes, particularly during pregnancy, on infection with different lineages of ZIKV. Both African and epidemic strains of Zika virus are shown to target CD14+ monocytes, which are more susceptible in pregnant women, but African strains are associated with inflammatory responses, and epidemic strains with immunotolerance.

HPV is a human oncogenic virus that causes a major public health problem worldwide, which is responsible for approximately 5% of total human cancers and almost all cases of cervical cancers. HPV capsid consists of two structure proteins, the major capsid L1 protein and the minor capsid L2 protein. While L2 plays critical roles during the viral life cycle, the molecular mechanism in viral entry remains elusive. Here, we performed fine mapping of the L2 N-terminal region and defined a short 36-amino-acid peptide, called L2N, which is critical for HPV infection. Specifically, L2N peptide with carboxyl-terminal lipidation acted as a potent and cross-type HPV inhibitor. Taken together, data from our study highlight the essential role of the L2N sequence at the early step of HPV entry and suggests the L2N lipopeptide as a new strategy to broadly prevent HPV infection., The amino (N)-terminal region of human papillomavirus (HPV) minor capsid protein (L2) is a highly conserved region which is essential for establishing viral infection. Despite its importance in viral infectivity, the role of the HPV N-terminal domain has yet to be fully characterized. Using fine mapping analysis, we identified a 36-amino-acid (aa) peptide sequence of the L2 N terminus, termed L2N, that is critical for HPV infection. Ectopic expression of L2N with the transmembrane sequence on the target cell surface conferred resistance to HPV infection. Additionally, L2N peptide with chemical or enzymatic lipidation at the carboxyl (C) terminus efficiently abrogated HPV infection in target cells. Among the synthetic L2N lipopeptides, a stearoylated lipopeptide spanning aa 13 to 46 (13-46st) exhibited the most potent anti-HPV activity, with a half-maximal inhibitory concentration (IC50) of ∼200 pM. Furthermore, we demonstrated that the 13-46st lipopeptide inhibited HPV entry by blocking trans-Golgi network retrograde trafficking of virion particles, leading to rapid degradation. Fundamentally, the inhibitory effect of L2N lipopeptides appeared to be evolutionarily conserved, as they showed cross-type inhibition among various papillomaviruses. In conclusion, our findings provide new insights into the critical role of the L2N sequence in the HPV entry mechanism and identify the therapeutic potential of L2N lipopeptide as an effective anti-HPV agent.

Severe fever with thrombocytopenia syndrome phlebovirus (SFTSV), listed in the World Health Organization Prioritized Pathogens, is an emerging phlebovirus with a high fatality(1–4). Owing to the lack of therapies and vaccines(5,6), there is a pressing need to understand SFTSV pathogenesis. SFSTV non-structural protein (NSs) has been shown to block type I interferon induction(7–11) and facilitate disease progression(12,13). Here, we report that SFTSV-NSs targets the tumour progression locus 2 (TPL2)-A20-binding inhibitor of NF-κB activation 2 (ABIN2)-p105 complex to induce the expression of interleukin-10 (lL-10) for viral pathogenesis. Using a combination of reverse genetics, a TPL2 kinase inhibitor and Tpl2(−/−) mice showed that NSs interacted with ABIN2 and promoted TPL2 complex formation and signalling activity, resulting in the marked upregulation of ll10 expression. Whereas SFTSV infection of wild-type mice led to rapid weight loss and death, Tpl2(−/−) mice or ll10(−/−) mice survived an infection. Furthermore, SFTSV-NSs P(102)A and SFTSV-NSs K(211)R that lost the ability to induce TPL2 signalling and IL-10 production showed drastically reduced pathogenesis. Remarkably, the exogenous administration of recombinant IL-10 effectively rescued the attenuated pathogenic activity of SFTSV-NSs P(102)A, resulting in a lethal infection. Our study demonstrates that SFTSV-NSs targets the TPL2 signalling pathway to induce immune-sup-pressive IL-10 cytokine production as a means to dampen the host defence and promote viral pathogenesis.

SUMMARY Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne virus with 12%−30% case mortality rates and is related to the Heartland virus (HRTV) identified in the United States. Together, SFTSV and HRTV are emerging segmented, negative-sense RNA viral (sNSV) pathogens with potential global health impact. Here, we characterize the amino-terminal cap-snatching endonuclease domain of SFTSV polymerase (L) and solve a 2.4-Å X-ray crystal structure. While the overall structure is similar to those of other cap-snatching sNSV endonucleases, differences near the C terminus of the SFTSV endonuclease suggest divergence in regulation. Influenza virus endonuclease inhibitors, including the US Food and Drug Administration (FDA) approved Baloxavir (BXA), inhibit the endonuclease activity in in vitro enzymatic assays and in cell-based studies. BXA displays potent activity with a half maximal inhibitory concentration (IC50) of ~100 nM in enzyme inhibition and an EC50 value of ~250 nM against SFTSV and HRTV in plaque assays. Together, our data support sNSV endonucleases as an antiviral target., In Brief Wang et al. solve the X-ray crystal structure of SFTSV L endonuclease domain and investigate the characteristics of SFTSV and HRTV endonuclease function. Resulting data support a mechanism for regulation. Baloxavir effectively inhibits the endonuclease activity of SFTSV and HRTV., Graphical Abstract

Cellular responses to pathogen invasion are crucial for maintaining cell homeostasis and survival. The interferon (IFN) system is one of the most effective cellular responses to viral intrusion in mammals. Viral recognition by innate immune sensors activates the antiviral IFN system. Retinoic acid-inducible gene I (RIG-I) like receptors (RLRs) are DExD/H box RNA helicases that sense viral invasion. RLRs recognize cytoplasmic viral RNAs and trigger antiviral responses, resulting in production of type I IFN and inflammatory cytokines. Unique and common sensing mechanisms among RLRs have been reported. In this review, recent progress in the understanding of antiviral responses by RLRs is summarized and discussed.

SUMMARY Excessive and unresolved neuroinflammation is a key component of the pathological cascade in brain injuries such as ischemic stroke. Here, we report that TRIM9, a brain-specific tripartite motif (TRIM) protein, was highly expressed in the peri-infarct areas shortly after ischemic insults in mice, but expression was decreased in aged mice, which are known to have increased neuroinflammation after stroke. Mechanistically, TRIM9 sequestered β-transducin repeat-containing protein (β-TrCP) from the Skp-Cullin-F-box ubiquitin ligase complex, blocking IκBα degradation and thereby dampening nuclear factor κB (NF-κB)-dependent proinflammatory mediator production and immune cell infiltration to limit neuroinflammation. Consequently, Trim9-deficient mice were highly vulnerable to ischemia, manifesting uncontrolled neuroinflammation and exacerbated neuropathological outcomes. Systemic administration of a recombinant TRIM9 adeno-associated virus that drove brain-wide TRIM9 expression effectively resolved neuroinflammation and alleviated neuronal death, especially in aged mice. These findings reveal that TRIM9 is essential for resolving NF-κB-dependent neuroinflammation to promote recovery and repair after brain injury and may represent an attractive therapeutic target., Graphical Abstract, In Brief Neuroinflammation drives pathology during brain injury. Zeng et al. show that TRIM9 is induced after ischemic insults in young mice, but not old mice, and promotes resolution of neuroinflammation. AAV-mediated TRIM9 therapy in aged mice restricts neuroinflammation and alleviates stroke damage, representing a potential therapeutic target for brain injury.

In response to stress, cells induce ribonucleoprotein aggregates, termed stress granules (SGs). SGs are transient loci containing translation-stalled mRNA, which is eventually degraded or recycled for translation. Infection of some viruses, including influenza A virus with a deletion of nonstructural protein 1 (IAVΔNS1), induces SG-like protein aggregates. Previously, we showed that IAVΔNS1-induced SGs are required for efficient induction of type I interferon (IFN). Here, we investigated SG formation by different viruses using green fluorescent protein (GFP)-tagged Ras-Gap SH3 domain binding protein 1 (GFP-G3BP1) as an SG probe. HeLa cells stably expressing GFP-G3BP1 were infected with different viruses, and GFP fluorescence was monitored live with time-lapse microscopy. SG formations by different viruses was classified into 4 different patterns: no SG formation, stable SG formation, transient SG formation, and alternate SG formation. We focused on encephalomyocarditis virus (EMCV) infection, which exhibited transient SG formation. We found that EMCV disrupts SGs by cleavage of G3BP1 at late stages of infection (>8 h) through a mechanism similar to that used by poliovirus. Expression of a G3BP1 mutant that is resistant to the cleavage conferred persistent formation of SGs as well as an enhanced induction of IFN and other cytokines at late stages of infection. Additionally, knockdown of endogenous G3BP1 blocked SG formation with an attenuated induction of IFN and potentiated viral replication. Taken together, our findings suggest a critical role of SGs as an antiviral platform and shed light on one of the mechanisms by which a virus interferes with host stress and subsequent antiviral responses.

Retinoic acid inducible gene I (RIG-I)-like receptors (RLRs) function as cytoplasmic sensors for viral RNA to initiate antiviral responses including type I interferon (IFN) production. It has been unclear how RIG-I encounters and senses viral RNA. To address this issue, we examined intracellular localization of RIG-I in response to viral infection using newly generated anti-RIG-I antibody. Immunohistochemical analysis revealed that RLRs localized in virus-induced granules containing stress granule (SG) markers together with viral RNA and antiviral proteins. Because of similarity in morphology and components, we termed these aggregates antiviral stress granules (avSGs). Influenza A virus (IAV) deficient in non-structural protein 1 (NS1) efficiently generated avSGs as well as IFN, however IAV encoding NS1 produced little. Inhibition of avSGs formation by removal of either the SG component or double-stranded RNA (dsRNA)-dependent protein kinase (PKR) resulted in diminished IFN production and concomitant enhancement of viral replication. Furthermore, we observed that transfection of dsRNA resulted in IFN production in an avSGs-dependent manner. These results strongly suggest that the avSG is the locus for non-self RNA sensing and the orchestration of multiple proteins is critical in the triggering of antiviral responses.

Background Japanese encephalitis virus (JEV) NS5 is a viral nonstructural protein that carries both methyltransferase and RNA-dependent RNA polymerase (RdRp) domains. It is a key component of the viral RNA replicase complex that presumably includes other viral nonstructural and cellular proteins. The biochemical properties of JEV NS5 have not been characterized due to the lack of a robust in vitro RdRp assay system, and the molecular mechanisms for the initiation of RNA synthesis by JEV NS5 remain to be elucidated. Results To characterize the biochemical properties of JEV RdRp, we expressed in Escherichia coli and purified an enzymatically active full-length recombinant JEV NS5 protein with a hexahistidine tag at the N-terminus. The purified NS5 protein, but not the mutant NS5 protein with an Ala substitution at the first Asp of the RdRp-conserved GDD motif, exhibited template- and primer-dependent RNA synthesis activity using a poly(A) RNA template. The NS5 protein was able to use both plus- and minus-strand 3'-untranslated regions of the JEV genome as templates in the absence of a primer, with the latter RNA being a better template. Analysis of the RNA synthesis initiation site using the 3'-end 83 nucleotides of the JEV genome as a minimal RNA template revealed that the NS5 protein specifically initiates RNA synthesis from an internal site, U81, at the two nucleotides upstream of the 3'-end of the template. Conclusion As a first step toward the understanding of the molecular mechanisms for JEV RNA replication and ultimately for the in vitro reconstitution of viral RNA replicase complex, we for the first time established an in vitro JEV RdRp assay system with a functional full-length recombinant JEV NS5 protein and characterized the mechanisms of RNA synthesis from nonviral and viral RNA templates. The full-length recombinant JEV NS5 will be useful for the elucidation of the structure-function relationship of this enzyme and for the development of anti-JEV agents.