In vitro biological evaluation and in silico insights into the antiviral activity of standardized olive leaves extract against SARS-CoV-2.

There is still a great global need for efficient treatments for the management of SARS-CoV-2 illness notwithstanding the availability and efficacy of COVID-19 vaccinations. Olive leaf is an herbal remedy with a potential antiviral activity that could improve the recovery of COVID-19 patients. In this work, the olive leaves major metabolites were screened in silico for their activity against SARS-CoV-2 by molecular docking on several viral targets such as methyl transferase, helicase, Plpro, Mpro, and RdRp. The results of in silico docking study showed that olive leaves phytoconstituents exhibited strong potential antiviral activity against SARS-CoV-2 selected targets. Verbacoside demonstrated a strong inhibition against methyl transferase, helicase, Plpro, Mpro, and RdRp (docking scores = -17.2, -20, -18.2, -19.8, and -21.7 kcal/mol.) respectively. Oleuropein inhibited 5rmm, Mpro, and RdRp (docking scores = -15, -16.6 and -18.6 kcal/mol., respectively) respectively. Apigenin-7-O-glucoside exhibited activity against methyl transferase and RdRp (docking score = -16.1 and -19.4 kcal/mol., respectively) while Luteolin-7-O-glucoside inhibited Plpro and RdRp (docking score = -15.2 and -20 kcal/mol., respectively). The in vitro antiviral assay was carried out on standardized olive leaf extract (SOLE) containing 20% oleuropein and IC50 was calculated. The results revealed that 20% SOLE demonstrated a moderate antiviral activity against SARS-CoV-2 with IC50 of 118.3 µg /mL. Accordingly, olive leaf could be a potential herbal therapy against SARS-CoV-2 but more in vivo and clinical investigations are recommended.

Ailanthone inhibits non-small cell lung cancer growth and metastasis through targeting UPF1/GAS5/ULK1 signaling pathway.

BACKGROUND: Targeting long non-coding RNAs (LncRNAs) is a novel and promising approach in cancer therapy. In our previous study, we investigated the effects of ailanthone (aila), the main active compound derived from the stem barks of Ailanthus altissima (Mill.) Swingle, on the growth of non-small cell lung cancer (NSCLC) cells. Although we observed significant inhibition of NSCLC cell growth of aila, the underlying mechanisms involving LncRNAs, specifically LncRNA growth arrest specific 5 (GAS5), remain largely unknown. METHODS: To further explore the impact of aila on NSCLC, we performed a series of experiments. Firstly, we confirmed the inhibitory effect of aila on NSCLC cell growth using multiple assays, including MTT, wound healing, transwell assay, as well as subcutaneous and metastasis tumor mice models in vivo. Next, we utilized cDNA microarray and RT-QPCR to identify GAS5 as the primary target of aila. To verify the importance of GAS5 in aila-induced tumor inhibition, we manipulated GAS5 expression levels by constructing GAS5 over-expression and knockdown NSCLC cell lines. Furthermore, we investigated the upstream and downstream signaling pathways of GAS5 through western blot and RT-QPCR analysis. RESULTS: Our results showed that aila effectively increased GAS5 expression, as determined by microarray analysis. We also observed that aila significantly enhanced GAS5 expression in a dose- and time-dependent manner across various NSCLC cell lines. Notably, over-expression of GAS5 led to a significant suppression of NSCLC cell tumor growth; while aila had minimal inhibitory effect on GAS5-knockdown NSCLC cells. Additionally, we discovered that aila inhibited ULK1 and autophagy, and this inhibition was reversed by GAS5 knockdown. Moreover, we found that aila up-regulated GAS5 expression by suppressing UPF1-mediated nonsense-mediated mRNA decay (NMD). CONCLUSION: In summary, our findings suggest that aila promotes GAS5 expression by inhibiting UPF1-mediated NMD, leading to the repression of ULK1-mediated autophagy and subsequent inhibitory effects on NSCLC cells. These results indicate that aila is a potent enhancer of GAS5 and holds promising potential for application in NSCLC therapy. However, our research is currently focused only on NSCLC. It remains to be determined whether aila can also inhibit the growth of other types of tumors through the UPF1/GAS5/ULK1 signaling pathway. In future studies, we can further investigate the mechanisms by which aila suppresses other types of tumors and potentially broaden the scope of its application in cancer therapy.

In Silico Exploration of Metabolically Active Peptides as Potential Therapeutic Agents against Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is regarded as a fatal neurodegenerative disease that is featured by progressive damage of the upper and lower motor neurons. To date, over 45 genes have been found to be connected with ALS pathology. The aim of this work was to computationally identify unique sets of protein hydrolysate peptides that could serve as therapeutic agents against ALS. Computational methods which include target prediction, protein-protein interaction, and peptide-protein molecular docking were used. The results showed that the network of critical ALS-associated genes consists of ATG16L2, SCFD1, VAC15, VEGFA, KEAP1, KIF5A, FIG4, TUBA4A, SIGMAR1, SETX, ANXA11, HNRNPL, NEK1, C9orf72, VCP, RPSA, ATP5B, and SOD1 together with predicted kinases such as AKT1, CDK4, DNAPK, MAPK14, and ERK2 in addition to transcription factors such as MYC, RELA, ZMIZ1, EGR1, TRIM28, and FOXA2. The identified molecular targets of the peptides that support multi-metabolic components in ALS pathogenesis include cyclooxygenase-2, angiotensin I-converting enzyme, dipeptidyl peptidase IV, X-linked inhibitor of apoptosis protein 3, and endothelin receptor ET-A. Overall, the results showed that AGL, APL, AVK, IIW, PVI, and VAY peptides are promising candidates for further study. Future work would be needed to validate the therapeutic properties of these hydrolysate peptides by in vitro and in vivo approaches.

Yin and yang regulation of stress granules by Caprin-1.

Stress granules (SGs) are cytoplasmic biomolecular condensates containing proteins and RNAs in response to stress. Ras-GTPase-activating protein binding protein 1 (G3BP1) is a core SG protein. Caprin-1 and ubiquitin specific peptidase 10 (USP10) interact with G3BP1, facilitating and suppressing SG formation, respectively. The crystal structures of the nuclear transport factor 2-like (NTF2L) domain of G3BP1 in complex with the G3BP1-interacting motif (GIM) of Caprin-1 and USP10 show that both GIMs bind to the same hydrophobic pocket of G3BP1. Moreover, both GIMs suppressed the liquid-liquid phase separation (LLPS) of G3BP1, suggesting that Caprin-1 likely facilitates SG formation via other mechanisms. Thus, we dissected various domains of Caprin-1 and investigated their role in LLPS in vitro and SG formation in cells. The C-terminal domain of Caprin-1 underwent spontaneous LLPS, whereas the N-terminal domain and GIM of Caprin-1 suppressed LLPS of G3BP1. The opposing effect of the N- and C-terminal domains of Caprin-1 on SG formation were demonstrated in cells with or without the endogenous Caprin-1. We propose that the N- and C-terminal domains of Caprin-1 regulate SG formation in a "yin and yang" fashion, mediating the dynamic and reversible assembly of SGs.

Phillygenin activates PKR/eIF2α pathway and induces stress granule to exert anti-avian infectious bronchitis virus.

The prevalence of avian infectious bronchitis virus (IBV) is still one of causes inducing severe losses of production in the poultry industry worldwide. Vaccination does not completely prevent IBV infection and spread due to immune failure and viral mutations. ForsythiaeFructus and its compounds have been widely used in a lot of prescriptions of the traditional Chinese medicine for a long history, and it is well-known as safety and efficiency in heat-clearing and detoxifying. This study aims to investigate the anti-IBV activity and mechanism of phillygenin. The results showed that phillygenin inhibited IBV replication by disturbing multiple stages of the virus life cycle, including viral adsorption, invasion, internalization, and release in Vero cells. After being treated with 100, 125 and 150 µg/mL phillygenin, the expression of G3BP1 was significantly increased and the phosphorylation of PKR/eIF2α was activated, which increased stress granule, thereby triggering the antiviral response in Vero cells. The anti-virus activity of PHI was decreased when G3BP1 was interfered by si-RNA, and G3BP1 was down-regulated when PKR/eIF2α was interfered by si-RNA. In conclusion, our findings indicate that phillygenin activates PKR/eIF2α pathway and induces stress granule formation to exert anti-IBV, which holds promise to develop into a novel anti-IBV drug. Further study in vivo is needed to explore phillygenin as a potential and effective drug to prevent IB in poultry.

High-Throughput Fluorescent Assay for Inhibitor Screening of Proteases from RNA Viruses.

Spanish flu, polio epidemics, and the ongoing COVID-19 pandemic are the most profound examples of severe widespread diseases caused by RNA viruses. The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) demands affordable and reliable assays for testing antivirals. To test inhibitors of viral proteases, we have developed an inexpensive high-throughput assay based on fluorescent energy transfer (FRET). We assayed an array of inhibitors for papain-like protease from SARS-CoV-2 and validated it on protease from the tick-borne encephalitis virus to emphasize its versatility. The reaction progress is monitored as loss of FRET signal of the substrate. This robust and reproducible assay can be used for testing the inhibitors in 96- or 384-well plates.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp13 helicase.

The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global public health challenge. While the efficacy of vaccines against emerging and future virus variants remains unclear, there is a need for therapeutics. Repurposing existing drugs represents a promising and potentially rapid opportunity to find novel antivirals against SARS-CoV-2. The virus encodes at least nine enzymatic activities that are potential drug targets. Here, we have expressed, purified and developed enzymatic assays for SARS-CoV-2 nsp13 helicase, a viral replication protein that is essential for the coronavirus life cycle. We screened a custom chemical library of over 5000 previously characterized pharmaceuticals for nsp13 inhibitors using a fluorescence resonance energy transfer-based high-throughput screening approach. From this, we have identified FPA-124 and several suramin-related compounds as novel inhibitors of nsp13 helicase activity in vitro. We describe the efficacy of these drugs using assays we developed to monitor SARS-CoV-2 growth in Vero E6 cells.

Structure-Based Virtual Screening Identifies Multiple Stable Binding Sites at the RecA Domains of SARS-CoV-2 Helicase Enzyme.

With the emergence and global spread of the COVID-19 pandemic, the scientific community worldwide has focused on search for new therapeutic strategies against this disease. One such critical approach is targeting proteins such as helicases that regulate most of the SARS-CoV-2 RNA metabolism. The purpose of the current study was to predict a library of phytochemicals derived from diverse plant families with high binding affinity to SARS-CoV-2 helicase (Nsp13) enzyme. High throughput virtual screening of the Medicinal Plant Database for Drug Design (MPD3) database was performed on SARS-CoV-2 helicase using AutoDock Vina. Nilotinib, with a docking value of -9.6 kcal/mol, was chosen as a reference molecule. A compound (PubChem CID: 110143421, ZINC database ID: ZINC257223845, eMolecules: 43290531) was screened as the best binder (binding energy of -10.2 kcal/mol on average) to the enzyme by using repeated docking runs in the screening process. On inspection, the compound was disclosed to show different binding sites of the triangular pockets collectively formed by Rec1A, Rec2A, and 1B domains and a stalk domain at the base. The molecule is often bound to the ATP binding site (referred to as binding site 2) of the helicase enzyme. The compound was further discovered to fulfill drug-likeness and lead-likeness criteria, have good physicochemical and pharmacokinetics properties, and to be non-toxic. Molecular dynamic simulation analysis of the control/lead compound complexes demonstrated the formation of stable complexes with good intermolecular binding affinity. Lastly, affirmation of the docking simulation studies was accomplished by estimating the binding free energy by MMPB/GBSA technique. Taken together, these findings present further in silco investigation of plant-derived lead compounds to effectively address COVID-19.

Identification of dibucaine derivatives as novel potent enterovirus 2C helicase inhibitors: In vitro, in vivo, and combination therapy study.

Enterovirus A71 (EV-A71) is a human pathogen causing hand, foot and mouth disease (HFMD) which seriously threatened the safety and lives of infants and young children. However, there are no licensed direct antiviral agents to cure the HFMD. In this study, a series of quinoline formamide analogues as effective enterovirus inhibitors were developed, subsequent systematic structure-activity relationship (SAR) studies demonstrated that these quinoline formamide analogues exhibited good potency to treat EV-A71 infection. As described, the most efficient EV-A71 inhibitor 6i showed good anti-EV-A71 activity (EC50 = 1.238 µM) in RD cells. Furthermore, compound 6i could effectively prevent death of virus infected mice at dose of 6 mg/kg. When combined with emetine (0.1 mg/kg), this treatment could completely prevent the clinical symptoms and death of virus infected mice. Mechanism study indicated that compound 6i inhibited EV-A71 via targeting 2C helicase, thus impeding RNA remodeling and metabolism. Taken together, these data indicated that 6i is a promising EV-A71 inhibitor and worth extensive preclinical investigation as a lead compound.

Zucchini consensus motifs determine the mechanism of pre-piRNA production.

PIWI-interacting RNAs (piRNAs) of between approximately 24 and 31 nucleotides in length guide PIWI proteins to silence transposons in animal gonads, thereby ensuring fertility1. In the biogenesis of piRNAs, PIWI proteins are first loaded with 5'-monophosphorylated RNA fragments called pre-pre-piRNAs, which then undergo endonucleolytic cleavage to produce pre-piRNAs1,2. Subsequently, the 3'-ends of pre-piRNAs are trimmed by the exonuclease Trimmer (PNLDC1 in mouse)3-6 and 2'-O-methylated by the methyltransferase Hen1 (HENMT1 in mouse)7-9, generating mature piRNAs. It is assumed that the endonuclease Zucchini (MitoPLD in mouse) is a major enzyme catalysing the cleavage of pre-pre-piRNAs into pre-piRNAs10-13. However, direct evidence for this model is lacking, and how pre-piRNAs are generated remains unclear. Here, to analyse pre-piRNA production, we established a Trimmer-knockout silkworm cell line and derived a cell-free system that faithfully recapitulates Zucchini-mediated cleavage of PIWI-loaded pre-pre-piRNAs. We found that pre-piRNAs are generated by parallel Zucchini-dependent and -independent mechanisms. Cleavage by Zucchini occurs at previously unrecognized consensus motifs on pre-pre-piRNAs, requires the RNA helicase Armitage, and is accompanied by 2'-O-methylation of pre-piRNAs. By contrast, slicing of pre-pre-piRNAs with weak Zucchini motifs is achieved by downstream complementary piRNAs, producing pre-piRNAs without 2'-O-methylation. Regardless of the endonucleolytic mechanism, pre-piRNAs are matured by Trimmer and Hen1. Our findings highlight multiplexed processing of piRNA precursors that supports robust and flexible piRNA biogenesis.

Disruption of galectin-3 and galectin-3 binding protein (G3BP) interaction by dietary pectic polysaccharides (DPP) - Arrest of metastasis, inhibition of proliferation and induction of apoptosis.

Galectin-3 and galectin-3 binding proteins (G3BP) are implicated as key players in metastasis. In the current study, we evaluated the effect of pectic polysaccharides on galectin-3 and G3BP mediated metastasis in vitro (cells) and in vivo (tissues). In vitro study (double immunostaining) confirms the presence of galectin-3 on the cell surface and G3BP in the interlinking region of the cells confirming the role of G3BP in bridging galectin-3 molecules. Dietary carrot (Daucus carota) pectic polysaccharide (CRPP) blocked the expression of galectin-3 and G3BP more effectively (80%), whereas the expressions were reduced to 60% upon treatment with swallow root (Decalepis hamiltonii) pectic polysaccharide (SRPP), ß­carotene and deferoxamine (antiproliferative drug). Ginger (Gingiber officinale) pectic polysaccharide (GRPP) showed only 20% reduction. CRPP reduced 80% of tumor incidence followed by cyclophosphamide - a chemotherapeutic drug (77%), SRPP (67%) and GRPP (45%). Further 3-5 folds reduction in galectin-3/G3BP expression followed by infiltration of macrophages into the deeper layer of the skin by CRPP and SRPP suggested the anticancer property via immunomodulation. Surface Plasmon Resonance (SPR) studies confirm galectin-3 and G3BP interaction, which are disrupted during the treatment with dietary pectic polysaccharides (DPP) (Supplementary Scheme-1). Overall data demonstrate the role of DPPs as potential anticancer alternatives.

Computational screening of medicinal plant phytochemicals to discover potent pan-serotype inhibitors against dengue virus.

Emergence of Dengue as one of the deadliest viral diseases prompts the need for development of effective therapeutic agents. Dengue virus (DV) exists in four different serotypes and infection caused by one serotype predisposes its host to another DV serotype heterotypic re-infection. We undertook virtual ligand screening (VLS) to filter compounds against DV that may inhibit inclusively all of its serotypes. Conserved non-structural DV protein targets such as NS1, NS3/NS2B and NS5, which play crucial role in viral replication, infection cycle and host interaction, were selected for screening of vital antiviral drug leads. A dataset of plant based natural antiviral derivatives was developed. Molecular docking was performed to estimate the spatial affinity of target compounds for the active sites of DV's NS1, NS3/NS2B and NS5 proteins. The drug likeliness of the screened compounds was followed by ADMET analysis whereas the binding behaviors were further elucidated through molecular dynamics (MD) simulation experiments. VLS screened three potential compounds including Canthin-6-one 9-O-beta-glucopyranoside, Kushenol W and Kushenol K which exhibited optimal binding with all the three conserved DV proteins. This study brings forth novel scaffolds against DV serotypes to serve as lead molecules for further optimization and drug development against all DV serotypes with equal effect against multiple disease causing DV proteins. We therefore anticipate that the insights given in the current study could be regarded valuable towards exploration and development of a broad-spectrum natural anti-dengue therapy.

Breathing new life into West Nile virus therapeutics; discovery and study of zafirlukast as an NS2B-NS3 protease inhibitor.

The West Nile virus (WNV) has spread throughout the world causing neuroinvasive diseases with no treatments available. The viral NS2B-NS3 protease is essential for WNV survival and replication in host cells and is a promising drug target. Through an enzymatic screen of the National Institute of Health clinical compound library, we report the discovery of zafirlukast, an FDA approved treatment for asthma, as an inhibitor for the WNV NS2B-NS3 protease. Zafirlukast was determined to inhibit the protease through a mixed mode mechanism with an IC50 value of 32 µM. A structure activity relationship study of zafirlukast revealed the cyclopentyl carbamate and N-aryl sulfonamide as structural elements crucial for NS2B-NS3 protease inhibition. Replacing the cyclopentyl with a phenyl improved inhibition, resulting in an IC50 of 22 µM. Experimental and computational docking analysis support the inhibition model of zafirlukast and analogs binding at an allosteric site on the NS3 protein, thereby disrupting the NS2B cofactor from binding, resulting in protease inhibition.

A conformational switch high-throughput screening assay and allosteric inhibition of the flavivirus NS2B-NS3 protease.

The flavivirus genome encodes a single polyprotein precursor requiring multiple cleavages by host and viral proteases in order to produce the individual proteins that constitute an infectious virion. Previous studies have revealed that the NS2B cofactor of the viral NS2B-NS3 heterocomplex protease displays a conformational dynamic between active and inactive states. Here, we developed a conformational switch assay based on split luciferase complementation (SLC) to monitor the conformational change of NS2B and to characterize candidate allosteric inhibitors. Binding of an active-site inhibitor to the protease resulted in a conformational change of NS2B and led to significant SLC enhancement. Mutagenesis of key residues at an allosteric site abolished this induced conformational change and SLC enhancement. We also performed a virtual screen of NCI library compounds to identify allosteric inhibitors, followed by in vitro biochemical screening of the resultant candidates. Only three of these compounds, NSC135618, 260594, and 146771, significantly inhibited the protease of Dengue virus 2 (DENV2) in vitro, with IC50 values of 1.8 µM, 11.4 µM, and 4.8 µM, respectively. Among the three compounds, only NSC135618 significantly suppressed the SLC enhancement triggered by binding of active-site inhibitor in a dose-dependent manner, indicating that it inhibits the conformational change of NS2B. Results from virus titer reduction assays revealed that NSC135618 is a broad spectrum flavivirus protease inhibitor, and can significantly reduce titers of DENV2, Zika virus (ZIKV), West Nile virus (WNV), and Yellow fever virus (YFV) on A549 cells in vivo, with EC50 values in low micromolar range. In contrast, the cytotoxicity of NSC135618 is only moderate with CC50 of 48.8 µM on A549 cells. Moreover, NSC135618 inhibited ZIKV in human placental and neural progenitor cells relevant to ZIKV pathogenesis. Results from binding, kinetics, Western blot, mass spectrometry and mutagenesis experiments unambiguously demonstrated an allosteric mechanism for inhibition of the viral protease by NSC135618.

Identification, ontogeny and expression analysis of a novel laboratory of genetics and physiology 2 (LGP2) transcript in Asian seabass, Lates calcarifer.

LGP2 (laboratory of genetics and physiology 2) is an important member of the retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), which plays a significant role in antiviral innate immunity. In this study, we have cloned the full-length cDNA sequence of LGP2 from Asian seabass, Lates calcarifer (AsLGP2). The complete AsLGP2 cDNA sequence consisted of 2586 nucleotides encoding a putative protein of 681 amino acids with a molecular mass of 77.6 kDa. From the AsLGP2 protein, four different conserved domains were predicted: a DExDc (DEAD/DEAH box helicase domain), a bacterial type III restriction enzyme domain (RES III), a HELICc (Helicase superfamily c-terminal domain and a RIG-I_C-RD (RIG-I C-terminal regulatory domain). The transcript of AsLGP2 could be detected in all the 11 tissues tested in healthy animals with high expression noticed in tissues facing external environment such as gill, hindgut and skin. The ontogenic expression profile of AsLGP2 implies a possible maternal transfer of this gene as it has been detected in all early embryonic developmental stages along with unfertilized eggs. Viral analogue, poly I:C, injection resulted in rapid up-regulated expression in different tissues with the highest modulation of expression observed in kidney followed by liver and gill. A rapid response of AsLGP2 expression was also observed in the different tissues of Vibrio alginolyticus-injected L. calcarifer, while significant change in expression was noticed following Staphylococcus aureus infection. Similarly, exposure to different pathogen-mimicking microbial analogues such as poly I:C, LPS and PGN resulted in enhanced expression of AsLGP2 in SISK cell-line. Taking together, these observations suggest that AsLGP2 can act as both antiviral and antibacterial cytosolic receptor and may play a significant role in embryonic and larval development in marine euryhaline teleosts like Asian seabass.

Sen1, the homolog of human Senataxin, is critical for cell survival through regulation of redox homeostasis, mitochondrial function, and the TOR pathway in Saccharomyces cerevisiae.

Mutations in the Senataxin gene, SETX are known to cause the neurodegenerative disorders, ataxia with oculomotor apraxia type 2 (AOA2), and amyotrophic lateral sclerosis 4 (ALS4). However, the mechanism underlying disease pathogenesis is still unclear. The Senataxin N-terminal protein-interaction and C-terminal RNA/DNA helicase domains are conserved in the Saccharomyces cerevisiae homolog, Sen1p. Using genome-wide expression analysis, we first show alterations in key cellular pathways such as: redox, unfolded protein response, and TOR in the yeast sen1 ΔN mutant (N-terminal truncation). This mutant exhibited growth defects on nonfermentable carbon sources, was sensitive to oxidative stress, and showed severe loss of mitochondrial DNA. The growth defect could be partially rescued upon supplementation with reducing agents and antioxidants. Furthermore, the mutant showed higher levels of reactive oxygen species, lower UPR activity, and alterations in mitochondrial membrane potential, increase in vacuole acidity, free calcium ions in the cytosol, and resistance to rapamycin treatment. Notably, the sen1 ∆N mutant showed increased cell death and shortened chronological life span. Given the strong similarity of the yeast and human Sen1 proteins, our study thus provides a mechanism for the progressive neurological disorders associated with mutations in human senataxin.

The evolution and functional characterization of miiuy croaker cytosolic gene LGP2 involved in immune response.

The laboratory of genetics and physiology 2 (LGP2) is a member of retinoic acid-inducible gene I (RIG-I)-like receptors (RLR receptors), which may participate in the immune regulation process. The role of LGP2 on modulating signaling was ambiguous, some researchers suggested that the regulation mechanism of LGP2 to melanoma differentiation-associated gene 5 (MDA5) and retinoic acid inducible gene-I (RIG-I) were different. In this study, the bioinformatics and functions of LGP2 from miiuy croaker (mmLGP2) were characterized. Comparative genomic analysis showed that the evolution of LGP2 in mammals was more conserved than it in fish. LGP2 contains three structural domains: ResIII, HelicaseC and RD, and ResIII structural domain of LGP2 was extremely conservative. The mmLGP2 was ubiquitously expressed in the tested miiuy croaker tissues and the expressions were significantly upregulated after stimulation with poly(I:C), indicating that LGP2 might participate in the immune response, especially antiviral immunity. Furthermore, immunofluorescence of miiuy croaker LGP2 presents in the cytoplasm in Hela cells. The overexpression of mmLGP2 can activate ISRE, but cannot activate NF-κB luciferase reporter, implying that mmLGP2 might act as a positive regulator in immune responses through activating ISRE to induce the expression of IFN. The research of mmLGP2 will enrich the information of fish LGP2, and the functional experiments will be helpful for the future research about fish immune systems.

Cloning, characterization, and expression analysis of LGP2 cDNA from goose, Anser cygnoides.

Laboratory of genetics and physiology 2 ( LGP2: ) is a homologue of the retinoic acid inducible gene-I and melanoma differentiation associated gene 5 that lacks the caspase activation and recruitment domain required for signaling. It plays a pivotal role in host immune response. In this study, we cloned and characterized the full-length open reading frame ( ORF: ) sequence of LGP2 in the Qingyuan goose (Anser cygnoides) and evaluated the mRNA expression of this gene post infection with an H5N1 highly pathogenic avian influenza virus ( HPAIV: ). The full-length goose LGP2 ORF (2,028 bp) encoded a polypeptide of 675 amino acids. The deduced amino acid sequence contained 5 main overlapping structural domains-2 DEAD/DEAH box helicase domains, one conserved restriction domain of bacterial type III restriction enzyme, one helicase superfamily C-terminal domain and one C-terminal regulatory domain. Quantitative real-time PCR analysis indicated that goose LGP2 was constitutively expressed in all 19 investigated tissues, but the expression level was different among them. It was high expressed in the trachea, jejunum, bursa, kidney and heart, but low in the glandular stomach, lung, liver, spleen, crop and muscular stomach. A significant increase in the transcription of LGP2 was detected in the brain, spleen and lungs of geese post infection with H5N1 HPAIV versus uninfected tissues. These findings indicated that goose LGP2 was an important receptor that is involved in the host antiviral innate immune defense to H5N1 HPAIV in geese.

Isolation and Characterization of Pepper Genes Interacting with the CMV-P1 Helicase Domain.

Cucumber mosaic virus (CMV) is a destructive pathogen affecting Capsicum annuum (pepper) production. The pepper Cmr1 gene confers resistance to most CMV strains, but is overcome by CMV-P1 in a process dependent on the CMV-P1 RNA1 helicase domain (P1 helicase). Here, to identify host factors involved in CMV-P1 infection in pepper, a yeast two-hybrid library derived from a C. annuum 'Bukang' cDNA library was screened, producing a total of 76 potential clones interacting with the P1 helicase. Beta-galactosidase filter lift assay, PCR screening, and sequencing analysis narrowed the candidates to 10 genes putatively involved in virus infection. The candidate host genes were silenced in Nicotiana benthamiana plants that were then inoculated with CMV-P1 tagged with the green fluorescent protein (GFP). Plants silenced for seven of the genes showed development comparable to N. benthamiana wild type, whereas plants silenced for the other three genes showed developmental defects including stunting and severe distortion. Silencing formate dehydrogenase and calreticulin-3 precursor led to reduced virus accumulation. Formate dehydrogenase-silenced plants showed local infection in inoculated leaves, but not in upper (systemic) leaves. In the calreticulin-3 precursor-silenced plants, infection was not observed in either the inoculated or the upper leaves. Our results demonstrate that formate dehydrogenase and calreticulin-3 precursor are required for CMV-P1 infection.

Paramyxovirus V protein interaction with the antiviral sensor LGP2 disrupts MDA5 signaling enhancement but is not relevant to LGP2-mediated RLR signaling inhibition.

The interferon antiviral system is a primary barrier to virus replication triggered upon recognition of nonself RNAs by the cytoplasmic sensors encoded by retinoic acid-inducible gene I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology gene 2 (LGP2). Paramyxovirus V proteins are interferon antagonists that can selectively interact with MDA5 and LGP2 through contact with a discrete helicase domain region. Interaction with MDA5, an activator of antiviral signaling, disrupts interferon gene expression and antiviral responses. LGP2 has more diverse reported roles as both a coactivator of MDA5 and a negative regulator of both RIG-I and MDA5. This functional dichotomy, along with the concurrent interference with both cellular targets, has made it difficult to assess the unique consequences of V protein interaction with LGP2. To directly evaluate the impact of LGP2 interference, MDA5 and LGP2 variants unable to be recognized by measles virus and parainfluenza virus 5 (PIV5) V proteins were tested in signaling assays. Results indicate that interaction with LGP2 specifically prevents coactivation of MDA5 signaling and that LGP2's negative regulatory capacity was not affected. V proteins only partially antagonize RIG-I at high concentrations, and their expression had no additive effects on LGP2-mediated negative regulation. However, conversion of RIG-I to a direct V protein target was accomplished by only two amino acid substitutions that allowed both V protein interaction and efficient interference. These results clarify the unique consequences of MDA5 and LGP2 interference by paramyxovirus V proteins and help resolve the distinct roles of LGP2 in both activation and inhibition of antiviral signal transduction. Importance: Paramyxovirus V proteins interact with two innate immune receptors, MDA5 and LGP2, but not RIG-I. V proteins prevent MDA5 from signaling to the beta interferon promoter, but the consequences of LGP2 targeting are poorly understood. As the V protein targets MDA5 and LGP2 simultaneously, and LGP2 is both a positive and negative regulator of both MDA5 and RIG-I, it has been difficult to evaluate the specific advantages conferred by LGP2 targeting. Experiments with V-insensitive proteins revealed that the primary outcome of LGP2 interference is suppression of its ability to synergize with MDA5. LGP2's negative regulation of MDA5 and RIG-I remains intact irrespective of V protein interaction. Complementary experiments demonstrate that RIG-I can be converted to V protein sensitivity by two amino acid substitutions. These findings clarify the functions of LGP2 as a positive regulator of MDA5 signaling, demonstrate the basis for V-mediated LGP2 targeting, and broaden our understanding of paramyxovirus-host interactions.