Human Virus-Derived Small RNAs Can Confer Antiviral Immunity in Mammals.

RNA interference (RNAi) functions as a potent antiviral immunity in plants and invertebrates; however, whether RNAi plays antiviral roles in mammals remains unclear. Here, using human enterovirus 71 (HEV71) as a model, we showed HEV71 3A protein as an authentic viral suppressor of RNAi during viral infection. When the 3A-mediated RNAi suppression was impaired, the mutant HEV71 readily triggered the production of abundant HEV71-derived small RNAs with canonical siRNA properties in cells and mice. These virus-derived siRNAs were produced from viral dsRNA replicative intermediates in a Dicer-dependent manner and loaded into AGO, and they were fully active in degrading cognate viral RNAs. Recombinant HEV71 deficient in 3A-mediated RNAi suppression was significantly restricted in human somatic cells and mice, whereas Dicer deficiency rescued HEV71 infection independently of type I interferon response. Thus, RNAi can function as an antiviral immunity, which is induced and suppressed by a human virus, in mammals.

Diversity synthesis of indole derivatives via catalyst control cyclization reaction of 2-indolylmethanols and azonaphthalene.

A series of indole-fused scaffolds and derivatives was synthesized via the cyclization reaction of 2-indolylmethanols with azonaphthalene. These reactions were realized under mild reaction conditions through catalyst control, providing structurally diverse indole derivatives with moderate to excellent yields. This protocol also shows good substrate adaptability, especially in six-membered ring products.

Discovery of a Novel Macrocyclic ATP Citrate Lyase Inhibitor.

ATP citrate lyase (ACLY) is an important metabolic enzyme involved in the synthesis of fatty acid and cholesterol. The inhibition of ACLY is considered as a promising therapeutic strategy for various metabolic diseases and numerous malignancies. In this study, a novel macrocyclic compound 2 has been identified as a potent ACLY inhibitor with the "ring closing" strategy for conformational restriction based on NDI-091143. It showed potent ACLY inhibitory activity and binding affinity comparable to the positive control. Furthermore, compared with the positive control (T1/2 = 3.36 min), the metabolic stability of 2 in HLMs (T1/2 = 531.22 min) was significantly improved. All of these results characterized 2 as a promising lead compound worthy of further study.

Opposite effects of Drosophila C3PO on gene silencing mediated by esi-2.1 and miRNA-bantam.

Translin/TRAX complex, also named as C3PO, is evolutionarily conserved and participates in diverse cellular processes in different organisms from yeast to human. C3PO plays a critical role in the activation of RNA-induced silencing complexes by promoting the unwinding and degradation of passenger strand of exogenous siRNAs (exo-siRNAs) in Drosophila and human. Moreover, human C3PO (hC3PO) has been found to broadly repress miRNAs by degrading miRNA precursors. However, the effect of Drosophila melanogaster C3PO (dmC3PO) on endogenous siRNA (endo-siRNA) and miRNA pathways remains unknown. Here, we found that the loss of dmC3PO promoted the accumulation of the passenger strand of esi-2.1 (hp-CG4068B), and resulted in the de-repression of the DNA-damage-response gene mutagensensitive 308 (mus308), which is an endogenous slicer target of esi-2.1 in Drosophila. Moreover, we also found that depletion of dmC3PO increased the accumulation of miR-bantam. Taken together, our findings indicated that dmC3PO not only involves in siRNA pathway triggered by dsRNA, but also regulates the abundance of certain endogenous small RNAs in Drosophila.

Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone.

RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2CATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3'-to-5' unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2CATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2CATPase facilitated EV71 RNA synthesis in vitro; when 2CATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2CATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2CATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2CATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our understanding of enteroviruses and the two types of RNA remodeling activities.

A cypovirus VP5 displays the RNA chaperone-like activity that destabilizes RNA helices and accelerates strand annealing.

For double-stranded RNA (dsRNA) viruses in the family Reoviridae, their inner capsids function as the machinery for viral RNA (vRNA) replication. Unlike other multishelled reoviruses, cypovirus has a single-layered capsid, thereby representing a simplified model for studying vRNA replication of reoviruses. VP5 is one of the three major cypovirus capsid proteins and functions as a clamp protein to stabilize cypovirus capsid. Here, we expressed VP5 from type 5 Helicoverpa armigera cypovirus (HaCPV-5) in a eukaryotic system and determined that this VP5 possesses RNA chaperone-like activity, which destabilizes RNA helices and accelerates strand annealing independent of ATP. Our further characterization of VP5 revealed that its helix-destabilizing activity is RNA specific, lacks directionality and could be inhibited by divalent ions, such as Mg(2+), Mn(2+), Ca(2+) or Zn(2+), to varying degrees. Furthermore, we found that HaCPV-5 VP5 facilitates the replication initiation of an alternative polymerase (i.e. reverse transcriptase) through a panhandle-structured RNA template, which mimics the 5'-3' cyclization of cypoviral positive-stranded RNA. Given that the replication of negative-stranded vRNA on the positive-stranded vRNA template necessitates the dissociation of the 5'-3' panhandle, the RNA chaperone activity of VP5 may play a direct role in the initiation of reoviral dsRNA synthesis.

Characterization of a nodavirus replicase revealed a de novo initiation mechanism of RNA synthesis and terminal nucleotidyltransferase activity.

Nodaviruses are a family of positive-stranded RNA viruses with a bipartite genome of RNAs. In nodaviruses, genomic RNA1 encodes protein A, which is recognized as an RNA-dependent RNA polymerase (RdRP) and functions as the sole viral replicase protein responsible for its RNA replication. Although nodaviral RNA replication has been studied in considerable detail, and nodaviruses are well recognized models for investigating viral RNA replication, the mechanism(s) governing the initiation of nodaviral RNA synthesis have not been determined. In this study, we characterized the RdRP activity of Wuhan nodavirus (WhNV) protein A in detail and determined that this nodaviral protein A initiates RNA synthesis via a de novo mechanism, and this RNA synthesis initiation could be independent of other viral or cellular factors. Moreover, we uncovered that WhNV protein A contains a terminal nucleotidyltransferase (TNTase) activity, which is the first time such an activity has been identified in nodaviruses. We subsequently found that the TNTase activity could function in vitro to repair the 3' initiation site, which may be digested by cellular exonucleases, to ensure the efficiency and accuracy of viral RNA synthesis initiation. Furthermore, we determined the cis-acting elements for RdRP or TNTase activity at the 3'-end of positive or negative strand RNA1. Taken together, our data establish the de novo synthesis initiation mechanism and the TNTase activity of WhNV protein A, and this work represents an important advance toward understanding the mechanism(s) of nodaviral RNA replication.

The nonstructural protein 2C of a Picorna-like virus displays nucleic acid helix destabilizing activity that can be functionally separated from its ATPase activity.

Picorna-like viruses in the Picornavirales order are a large group of positive-strand RNA viruses that include numerous important pathogens for plants, insects, and humans. In these viruses, nonstructural protein 2C is one of the most conserved proteins and contains ATPase activity and putative RNA helicase activity. Here we expressed 2C protein of Ectropis obliqua picorna-like virus (EoV; genus Iflavirus, family Iflaviridae, order Picornavirales) in a eukaryotic expression system and determined that EoV 2C displays ATP-independent nucleic acid helix destabilizing and strand annealing acceleration activity in a concentration-dependent manner, indicating that this picornaviral 2C is more like an RNA chaperone than like the previously predicted RNA helicase. Our further characterization of EoV 2C revealed that divalent metal ions, such as Mg(2+) and Zn(2+), inhibit 2C-mediated helix destabilization to different extents. Moreover, we determined that EoV 2C also contains ATPase activity like that of other picornaviral 2C proteins and further assessed the functional relevance between its RNA chaperone-like and ATPase activities using mutational analysis as well as their responses to Mg(2+). Our data show that, when one of the two 2C activities was dramatically inhibited or almost abolished, the other activity could remain intact, showing that the RNA chaperone-like and ATPase activities of EoV 2C can be functionally separated. This report reveals that a picorna-like virus 2C protein displays RNA helix destabilizing and strand annealing acceleration activity, which may be critical for picornaviral replication and pathogenesis, and should foster our understanding of picorna-like viruses and viral RNA chaperones.

Targeting of dicer-2 and RNA by a viral RNA silencing suppressor in Drosophila cells.

RNA interference (RNAi) is a eukaryotic gene-silencing mechanism that functions in antiviral immunity in diverse organisms. To combat RNAi-mediated immunity, viruses encode viral suppressors of RNA silencing (VSRs) that target RNA and protein components in the RNAi machinery. Although the endonuclease Dicer plays key roles in RNAi immunity, little is known about how VSRs target Dicer. Here, we show that the B2 protein from Wuhan nodavirus (WhNV), the counterpart of Flock House virus (FHV), suppresses Drosophila melanogaster RNAi by directly interacting with Dicer-2 (Dcr-2) and sequestering double-stranded RNA (dsRNA) and small interfering RNA (siRNA). Further investigations reveal that WhNV B2 binds to the RNase III and Piwi-Argonaut-Zwille (PAZ) domains of Dcr-2 via its C-terminal region, thereby blocking the activities of Dcr-2 in processing dsRNA and incorporating siRNA into the RNA-induced silencing complex (RISC). Moreover, we uncover an interrelationship among diverse activities of WhNV B2, showing that RNA binding enhances the B2-Dcr-2 interaction by promoting B2 homodimerization. Taken together, our findings establish a model of suppression of Drosophila RNAi by WhNV B2 targeting both Dcr-2 and RNA and provide evidence that an interrelationship exists among diverse activities of VSRs to antagonize RNAi.

Internal initiation is responsible for synthesis of Wuhan nodavirus subgenomic RNA.

Nodaviruses are small nonenveloped spherical viruses with a bipartite genome of RNAs. In nodaviruses, subgenomic RNA3 (sgRNA3) plays a critical role in viral replication and survival, as it coordinates the replication of two viral genomic RNAs (RNA1 and RNA2) and encodes protein B2, which is a potent RNA-silencing inhibitor. Despite its importance, the molecular mechanism of nodaviral sgRNA3 synthesis is still poorly understood. Here, we propose that sgRNA3 of Wuhan nodavirus (WhNV) is internally initiated from a promoter on the negative template of genomic RNA1. Serial deletion and mutation analyses further revealed that the core promoter of WhNV sgRNA3 is between nucleotide positions -22 and +6 of its transcription start site. Besides, a stem-loop structure of WhNV sgRNA3 was computationally predicted upstream of sgRNA3's transcription start site. Both the secondary structure and the primary sequence were determined to be required for promoter activity. Furthermore, our results show that the synthesis of WhNV sgRNA3 is counterregulated by the replication of WhNV genomic RNA2, which encodes a viral capsid precursor protein. And this sgRNA3 synthesis is also able to trans-activate the replication of RNA2. Altogether, findings in this study indicate that there is a newly discovered internal initiation model for the synthesis of nodaviral sgRNA.

RNA binding by a novel helical fold of b2 protein from wuhan nodavirus mediates the suppression of RNA interference and promotes b2 dimerization.

Wuhan nodavirus (WhNV) is a newly identified member of the Nodaviridae family with a bipartite genome of positive-sense RNAs. A nonstructural protein encoded by subgenomic RNA3 of nodaviruses, B2, serves as a potent RNA silencing suppressor (RSS) by sequestering RNA duplexes. We have previously demonstrated that WhNV B2 blocks RNA silencing in cultured Drosophila cells. However, the molecular mechanism by which WhNV B2 functions remains unknown. Here, we successfully established an RNA silencing system in cells derived from Pieris rapae, a natural host of WhNV, by introducing into these cells double-stranded RNA (dsRNA)-expressing plasmids or chemically synthesized small interfering RNAs (siRNAs). Using this system, we revealed that the WhNV B2 protein inhibited Dicer-mediated dsRNA cleavage and the incorporation of siRNA into the RNA-induced silencing complex (RISC) by sequestering dsRNA and siRNA. Based on the modeled B2 3-dimensional structure, serial single alanine replacement mutations and N-terminal deletion analyses showed that the RNA-binding domain of B2 is formed by its helices α2 and α3, while helix α1 mediates B2 dimerization. Furthermore, positive feedback between RNA binding and B2 dimerization was uncovered by gel shift assay and far-Western blotting, revealing that B2 dimerization is required for its binding to RNA, whereas RNA binding to B2 in turn promotes its dimerization. All together, our findings uncovered a novel RNA-binding mode of WhNV B2 and provided evidence that the promotion effect of RNA binding on dimerization exists in a viral RSS protein.

Deciphering the Pharmacological Mechanisms of Taohe-Chengqi Decoction Extract Against Renal Fibrosis Through Integrating Network Pharmacology and Experimental Validation In Vitro and In Vivo.

Taohe-Chengqi decoction (THCQ), a classical traditional Chinese medicinal (TCM) formula, has been extensively used for treating chronic kidney disease (CKD). However, the biological activity and mechanisms of action of its constituents against renal fibrosis have not yet been investigated thoroughly. This study was aimed at devising an integrated strategy for investigating the bioactivity constituents and possible pharmacological mechanisms of the n-butanol extract of THCQ (NE-THCQ) against renal fibrosis. The n-butanol extract of THCQ was prepared by the solvent extraction method. The components of NE-THCQ were analyzed using UPLC-Q/TOF-MS/MS techniques and applied for screening the active components of NE-THCQ according to their oral bioavailability and drug-likeness index. Then, we speculated the potential molecular mechanisms of NE-THCQ against renal fibrosis through pharmacological network analysis. Based on data mining techniques and topological parameters, gene ontology, and pathway enrichment, we established compound-target (C-T), protein-protein interaction (PPI) and compound-target-pathway (C-T-P) networks by Cytoscape to identify the hub targets and pathways. Finally, the potential molecular mechanisms of NE-THCQ against renal fibrosis, as predicted by the network pharmacology analyses, were validated experimentally in renal tubular epithelial cells (HK-2) in vitro and against unilateral ureteral obstruction models in the rat in vivo. We identified 26 components in NE-THCQ and screened seven bioactive ingredients. A total of 118 consensus potential targets associated with renal fibrosis were identified by the network pharmacology approach. The experimental validation results demonstrated that NE-THCQ might inhibit the inflammatory processes, reduce ECM deposition and reverse EMT via PI3K/AKT/mTOR and HIF-1α/VEGF signaling pathways to exert its effect against renal fibrosis. This study identified the potential ingredients of the NE-THCQ by UPLC-Q/TOF-MS/MS and explained the possible mechanisms of NE-THCQ against renal fibrosis by integrating network pharmacology and experimental validation.

Non-structural proteins of Periplaneta fuliginosa densovirus inhibit cellular gene expression and induce necrosis in Sf9 cell cultures.

The non-structural protein NS1 of Periplaneta fuliginosa densovirus (PfDNV) is a multifunctional protein that has previously been shown to possess ATP-binding, ATPase, site-specific DNA-binding, helicase, and transcription activation activities. We report here an investigation of the cytopathogenicity of this viral non-structural (NS) protein, as well as other two NSs, NS2, and NS3, in cultured insect cells. The expression of NS1 alone potently inhibited cellular gene expression, whereas NS2 and NS3 did not produce a similar effect. The inhibition of gene expression by NS1 was confirmed to be specific and not a simple manifestation of toxicity. For example, NS1 inhibited expression of several reporter genes under the control of different RNA polymerase II promoters, whereas it did not inhibit expression from a T7 RNA polymerase promoter construct. Mapping analysis identified the carboxy-terminal peptide of this protein as the region important for the inhibition of cellular gene expression, suggesting that this inhibition is independent of its DNA-binding activity. Next, the mutagenesis assay showed that ATP-binding was essential for the unique function of this protein. Furthermore, we found that NS2 and NS3 cooperatively enhanced the NS1-induced transcription inhibition. Co-expression of all the three NS proteins in Sf9 cells also led to necrotic cell death by ATP depletion.

Pathogenicity of a new China variety of Metarhizium anisopliae (M. Anisopliae var. Dcjhyium) to subterranean termite Odontotermes formosanus.

The pathogenicity of a new China variety of Metarhizium anisopliae (M. anisopliae var. dcjhyium) against the subterranean termite Odontotermes formosanus and the effect of the fungal fermented solution on hemolymph intracellular calcium were studied in laboratory. Conidia from the M. anisopliae var. dcjhyium were highly virulent for O. formosanus causing approximately 100% mortality 3 days post-inoculation in the concentration of 3x10(8) conidia/ml. The conidial clumps with conidial chains distributed on the cadavers of termite. When the termite was treated with 3x10(5) conidia/ml for 2 days, two constitutive proteins (91 and 105kDa) disappeared and a new specific protein (40kDa) appeared in the hemolymph of survivors relative to the controls. Hemolymph cells treated by the fungal fermented solution had a significantly higher level of intracellular calcium than controls 30min after treatment (x1.7). When the termite O. formosanus was infected by the entomopathogenic fungus M. anisopliae var. dcjhyium, hyphae invaded the integument and body cavity of the termite; well-developed muscles and fat tissue in the thorax of termite were decomposed and absorbed by hyphae, and formed the net structure; Hyphae seriously destroyed hemolymph, various tissues, pipelines and produced large number of conidia in the body of termite.

RNA-binding properties of Dendrolimus punctatus tetravirus p17 protein.

The RNA-binding properties of the p17 protein of Dendrolimus punctatus tetravirus were analysed. We have demonstrated that p17 protein, a nonstructural protein with a potentially important role in viral replication, is a RNA-binding protein, which has not been previously described for any member of the family Tetraviridae. P17 protein was expressed in Escherichia coli and was used in UV cross-linking analysis, using a digoxigenin-UTP-labeled RNA probe and chemical cross-linking analysis. The analyses demonstrated that p17 protein could bind to RNA. When analysed for capacity of p17 to form oligomers, the protein could form dimers and tetramers. Furthermore, the circular dichroism spectrums of viral RNA 3'-UTR proved that their secondary structures were consistent with yeast tRNA.

Characterization of the promoter elements and transcription profile of Periplaneta fuliginosa densovirus nonstructural genes.

Periplaneta fuliginosa Densovirus (PfDNV), an autonomous invertebrate parvovirus that infects the cockroach, is unusual in that alternative splicing is involved in the structural gene expression. The expression strategy for nonstructural (NS) genes has yet not been reported. Northern blot analysis of cockroach larvae infected with PfDNV revealed two transcripts for the NS genes, one of 2.6 kb, and the other of 1.9 kb. The two transcripts were shown to begin at a common initiator consensus sequence, CAGT, located in the terminus of ITR. The 1.9 kb transcript was produced by splicing out the ns3 gene from the 2.6 kb transcript. To understand the mechanism of transcriptional regulation of NS genes, the 5'-flanking sequence of ns3 gene (325 bp), which encompasses the region from the 5'-terminus of the viral genome to the initiator ATG codon of the ns3 gene, was cloned and fused to a luciferase reporter gene. The luciferase reporter assay showed that this sequence possessed promoter activity in Sf9, Ld652, Tn368, and S2 cell lines. Subsequent promoter deletion analysis showed that the promoter exhibited TATA-dependent and TATA-independent transcriptional activities. Moreover, we found that the promoter activity of the 325-bp fragment in S2 cells could be enhanced significantly by co-transfection of the nonstructural protein NS1 and that the NS1 binding element, (CAC)(4) repeat, mediated the promoter activity activated by NS1 protein.

Characterization of the RNA-binding regions in protein p36 of Heliothis armigera cypovirus 14.

Some proteins of cypovirus (CPV) bind to RNA, probably contributing to the replication of viral genome. However, little is known about whether any protein from Heliothis armigera cypovirus (HaCPV) could bind to RNA. In this study, we cloned the ORF of segment 9 (S9) of HaCPV, serotype 14, into pMAL-c2X for the generation and purification of maltose binding protein (MBP) fused protein p36 (MBP-p36). The analysis of the RNA-binding properties of MBP-p36 revealed that p36, but not MBP alone, bound to ssRNA of CPV. Furthermore, the ssRNA-binding activities of p36 were significantly inhibited or completely eliminated by protein denaturants or unsuitable concentrations of NaCl. Importantly, the formation of ssRNA/p36 was only competitively inhibited by a heavy dose of competitive non-viral ssRNA or dsRNA, but not by ssDNA and dsDNA, suggesting that p36 bound to both ssRNA and dsRNA, but not DNA. Moreover, the characterization of different mutants of p36 revealed that the regions 1-26aa, 154-170aa, and 229-238aa, but not region 291-320aa, may be crucial for the ssRNA-binding ability of p36. Conceivably, the sensitivity of p36 to denaturants and the synergetic effect of different regions suggest that the RNA-binding ability of p36 may be conformation-dependent. Thus, our findings provide new insights into understanding the genomic function of HaCPV-14.

Characterization of a newly discovered China variety of Metarhizium anisopliae (M. anisopliae var. dcjhyium) for virulence to termites, isoenzyme, and phylogenic analysis.

The efficacy of a new virulent Metarhizium anisopliae variety (M. anisopliae var. dcjhyium, DQ288247) obtained from Odontotermes formosanus in China was evaluated against the subterranean termite, O. formosanus, in the laboratory. The new variety was compared with four other virulent M. anisopliae isolates and was found to be highly infectious and virulent against termites. M. anisopliae var. dcjhyium could cause approximately 100% mortality of termites 3 days post-inoculation in the concentration of 3x10(8) conidia/ml. There were also differences in relative hyhal growth and isoenzymes. M. anisopliae var. dcjhyium showed a different isoenzyme band pattern from the four isolates of M. anisopliae (AB027337, AB099510, AB099941 and AF280631). The phylogenetic tree of the 18S rDNA sequences revealed the taxonomic and evolutionary position of M. anisopliae var. dcjhyium. M. anisopliae var. dcjhyium and four isolates of M. anisopliae formed a monophyletic group, supported by a 99% bootstrap value. M. anisopliae var. dcjhyium formed a distinct variety, which had a special characterization of unique bands of isoenzyme, high virulence and low repellency against termites when compared with four other isolates of M. anisopliae.

Isolation and RNA1 nucleotide sequence determination of a new insect nodavirus from Pieris rapae larvae in Wuhan city, China.

A new insect nodavirus is isolated from Pieris rapae larvae in Wuhan city, China and tentatively designated Wuhan nodavirus (WhNV). We here report the physicochemical characterization of WhNV and determine the nucleotide sequences of its larger segment of genome, RNA1. The results show that WhNV particles are isometric, non-enveloped, with a diameter of about 29nm. The virus has a major capsid protein and a minor capsid protein with estimated molecular mass of 40 and 44kDa, respectively. WhNV RNA1 is determined to be 3149nt long, containing a 1014-amino-acid open reading frame (ORF) encoding protein A with a calculated molecular mass of 114,608Da. The protein A shows 39 and 27% identity to its homologues in Pariacoto virus (PaV) and Striped jack necrosis nervous virus (SJNNV), respectively, but shows only 24% or less identity to its homologues in other insect Nodaviruses such as Nodamura virus (NoV), Black beetle virus (BBV), Boolarra virus (BoV) and Flock house virus (FHV). Predicted domains for six RNA-dependent RNA polymerase motifs and putative ORFs (protein B) are confirmed by sequence analysis of WhNV RNA1.