Plant RNA silencing machinery enlists four primary classes of proteins to achieve sequence-specific regulation of gene expression and mount an antiviral defense. These include Dicer-like ribonucleases (DCLs), Argonaute proteins (AGOs), dsRNA-binding proteins (DRBs), and RNA-dependent RNA polymerases (RDRs). Although at least four distinct endogenous RNA silencing pathways have been thoroughly characterized, a detailed understanding of the antiviral RNA silencing pathway is just emerging. In this report, we have examined the role of four DCLs, two AGOs, one DRB, and one RDR in controlling viral RNA accumulation in infected Arabidopsis plants by using a mutant virus lacking its silencing suppressor. Our results show that all four DCLs contribute to antiviral RNA silencing. We confirm previous reports implicating both DCL4 and DCL2 in this process and establish a minor role for DCL3. Surprisingly, we found that DCL1 represses antiviral RNA silencing through negatively regulating the expression of DCL4 and DCL3. We also implicate DRB4 in antiviral RNA silencing. Finally, we show that both AGO1 and AGO7 function to ensure efficient clearance of viral RNAs and establish that AGO1 is capable of targeting viral RNAs with more compact structures, whereas AGO7 and RDR6 favor less structured RNA targets. Our results resolve several key steps in the antiviral RNA silencing pathway and provide a basis for further in-depth analysis.interpathway regulation ͉ plant antiviral defense R NA silencing is a cellular mechanism that uses small RNA molecules (21-30 nt in length) as sequence-specific mediators to regulate the expression of a diverse array of genes at the transcriptional, posttranscriptional, or translational levels (1). In plants, these very small RNA species are termed small interfering RNAs (siRNAs) or micro RNAs (miRNAs) depending on the source of their precursors. They are generated by a family of double-stranded RNA (dsRNA)-specific RNases called Dicerlike ribonucleases (DCLs) (2). Once produced, the siRNAs and miRNAs are recruited by Argonaute proteins (AGOs) into RNA-induced silencing complexes (RISCs) to direct the cleavage or translational repression of homologous mRNAs or to remodel the homologous chromosomal DNA to achieve transcriptional silencing (3). Another family of dsRNA-binding proteins (DRBs) has been found to modulate the function of DCLs (4). Plants also encode RNA-dependent RNA polymerases (RDRs) to produce some of the dsRNA precursors that serve as templates for DCLs (2). In Arabidopsis, 4 DCLs, 10 AGOs, 5 DRBs, and up to 6 RDRs have been identified. They participate in at least four different endogenous RNA silencing pathways to achieve spatial and temporal regulation of gene expression throughout the plant life cycle and to condition the plant response to biotic and abiotic stresses (5).Although the plant RNA silencing mechanism was first revealed through studies aimed to unravel the complexity of plant antiviral defense strategies, the details of plant antiviral RNA silencing pathway(s) are far...

Dengue is an arthropod-borne infectious disease caused by dengue virus (DENV) infection and transmitted by Aedes mosquitoes. Approximately 50–100 million people are infected with DENV each year, resulting in a high economic burden on both governments and individuals. Here, we conducted a systematic review and meta-analysis to summarize information regarding the epidemiology, clinical characteristics, and serotype distribution and risk factors for global dengue outbreaks occurring from 1990 to 2015. We searched the PubMed, Embase and Web of Science databases through December 2016 using the term “dengue outbreak.” In total, 3,853 studies were identified, of which 243 studies describing 262 dengue outbreaks met our inclusion criteria. The majority of outbreak-associated dengue cases were reported in the Western Pacific Region, particularly after the year 2010; these cases were primarily identified in China, Singapore and Malaysia. The pooled mean age of dengue-infected individuals was 30.1 years; of the included patients, 54.5% were male, 23.2% had DHF, 62.0% had secondary infections, and 1.3% died. The mean age of dengue patients reported after 2010 was older than that of patients reported before 2010 (34.0 vs. 27.2 years); however, the proportions of patients who had DHF, had secondary infections and died significantly decreased after 2010. Fever, malaise, headache, and asthenia were the most frequently reported clinical symptoms and signs among dengue patients. In addition, among the identified clinical symptoms and signs, positive tourniquet test (OR = 4.86), ascites (OR = 13.91) and shock (OR = 308.09) were identified as the best predictors of dengue infection, DHF and mortality, respectively (both P < 0.05). The main risk factors for dengue infection, DHF and mortality were living with uncovered water container (OR = 1.65), suffering from hypotension (OR = 6.18) and suffering from diabetes mellitus (OR = 2.53), respectively (all P < 0.05). The serotype distribution varied with time and across WHO regions. Overall, co-infections were reported in 47.7% of the evaluated outbreaks, and the highest pooled mortality rate (2.0%) was identified in DENV-2 dominated outbreaks. Our study emphasizes the necessity of implementing programs focused on targeted prevention, early identification, and effective treatment.

SDE1/SGS2/RDR6, a putative RNA-dependent RNA polymerase (RdRP) from Arabidopsis thaliana, has previously been found to be indispensable for maintaining the posttranscriptional silencing of transgenes, but it is seemingly redundant for antiviral defense. To elucidate the antiviral role of this RdRP in a different host plant and to evaluate whether plant growth conditions affect its role, we down-regulated expression of the Nicotiana benthamiana homolog, NbRDR6, and examined the plants for altered susceptibility to various viruses at different growth temperatures. The results we describe here clearly show that plants with reduced expression of NbRDR6 were more susceptible to all viruses tested and that this effect was more pronounced at higher growth temperatures. Diminished expression of NbRDR6 also permitted efficient multiplication of tobacco mosaic virus in the shoot apices, leading to serious disruption with microRNA-mediated developmental regulation. Based on these results, we propose that NbRDR6 participates in the antiviral RNA silencing pathway that is stimulated by rising temperatures but suppressed by virus-encoded silencing suppressors. The relative strengths of these two factors, along with other plant defense components, critically influence the outcome of virus infections.RNA silencing is a surveillance system in eukaryotic organisms triggered by double-stranded RNA (dsRNA) that is subsequently digested by a dsRNA-specific RNase (Dicer or Dicer-like) into a small RNA species of 21 to 25 nucleotides (nt) long, called small interfering RNA (siRNA). The resultant siRNAs are then recruited into the RNA-induced silencing complex to direct the degradation of other RNAs with sequence complementarity to siRNAs (13). RNA silencing is thought to function primarily in defending eukaryotic cells against RNA molecular parasites, such as RNA viruses and transposon RNAs. Plant viruses, as well as some animal viruses, counteract this host defense mechanism by encoding suppressors of RNA silencing, which act at different steps of the pathway and with various strengths to ensure their successful systemic invasion of specific hosts (26,31).In addition to guarding the host against parasitic RNAs, recent studies have shown that processes highly related to RNA silencing are also involved in developmental regulation (22, 28), methylation of chromosomal DNA and histones, and chromatin maintenance (20,43). miRNA-mediated regulation of gene expression in both animal and plant systems is a particularly interesting discovery. Unlike siRNAs, miRNAs are encoded by genomes of eukaryotes in the form of partially double-stranded precursor molecules, which are processed by Dicer-like RNase(s) to release mature miRNAs. The miRNAs then mediate degradation or translational repression of the target RNAs (15). One well-studied example in plants is miR165/166. This miRNA targets the mRNA of three class III homeodomain leucine zipper (HD-ZIP III) transcription factors, PHABULOSA (PHB), PHAVOLUTA (PHV), and REVOLUTA (REV), for cleavage (10,...

SUMMARY Mitochondria play an integral role in cell death, autophagy, immunity, and inflammation. We previously showed that Nur77, an orphan nuclear receptor, induces apoptosis by targeting mitochondria. Here, we report that celastrol, a potent anti-inflammatory pentacyclic triterpene, binds Nur77 to inhibit inflammation and induce autophagy in a Nur77-dependent manner. Celastrol promotes Nur77 translocation from the nucleus to mitochondria, where it interacts with tumor necrosis factor receptor-associated factor 2 (TRAF2), a scaffold protein and E3 ubiquitin ligase important for inflammatory signaling. The interaction is mediated by an LxxLL motif in TRAF2 and results not only in the inhibition of TRAF2 ubiquitination but also in Lys63-linked Nur77 ubiquitination. Under inflammatory conditions, ubiquitinated Nur77 resides at mitochondria, rendering them sensitive to autophagy, an event involving Nur77 interaction with p62/SQSTM1. Together, our results identify Nur77 as a critical intracellular target for celastrol and unravel a mechanism of Nur77-dependent clearance of inflamed mitochondria to alleviate inflammation.

The capsid protein (CP) of Turnip crinkle virus (TCV) is a multifunctional protein needed for virus assembly, suppression of RNA silencing-based antiviral defense, and long-distance movement in infected plants. In this report, we have examined genetic requirements for the different functions of TCV CP and evaluated the interdependence of these functions. A series of TCV mutants containing alterations in the CP coding region were generated. These alterations range from single-amino-acid substitutions and domain truncations to knockouts of CP translation. The latter category also contained two constructs in which the CP coding region was replaced by either the cDNA of a silencing suppressor of a different virus or that of green fluorescent protein. These mutants were used to infect Arabidopsis plants with diminished antiviral silencing capability (dcl2 dcl3 dcl4 plants). There was a strong correlation between the ability of mutants to reach systemic leaves and the silencing suppressor activity of mutant CP. Virus particles were not essential for entry of the viral genome into vascular bundles in the inoculated leaves in the absence of antiviral silencing. However, virus particles were necessary for egress of the viral genome from the vasculature of systemic leaves. Our experiments demonstrate that TCV CP not only allows the viral genome to access the systemic movement channel through silencing suppression but also ensures its smooth egress by way of assembled virus particles. These results illustrate that efficient long-distance movement of TCV requires both functions afforded by the CP.