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 ␣2 and ␣3, while helix ␣1 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.

A s a form of nucleic acid-based silencing, RNA interference (RNAi) plays essential roles in the cellular response to viral infection in plants and invertebrates (3,13,34,50). In virus-infected cells, aberrant accumulation of viral single-stranded RNA (ssRNA) triggers its conversion into viral replicative intermediate double-stranded RNA (vRI-dsRNA), which is processed by the dsRNA-specific endonuclease Dicer into 21-to 23-nucleotide (nt) small interfering RNAs (siRNAs). Next, the virus-derived siRNAs (viRNAs) are transferred from Dicer to Argonaute (AGO) proteins in the RNA-induced silencing complex (RISC), which then guides the specific degradation of homologous viral ssRNAs (1,8,11,50). In mammals, RNAi directed by viral and cellular microRNAs (miRNAs) also contributes to host innate immunity against viral infection (18,27,35).To combat RNAi-mediated immunity, many plant and animal viruses encode viral suppressors of RNA silencing (VSRs) that target different components in the RNAi machinery. By sequestering dsRNA and siRNA, plant VSRs like turnip crinkle virus (TCV) capsid protein and tombusvirus P19 protein inhibit the production of siRNAs and hinder the incorporation of siRNAs into RISC (25, 41, 52). Additionally, direct interaction with AGO protein is known as a common approach of many plant and insect VSRs, such as cucumber mosaic virus (CMV) 2b protein, TCV P38 protein, sweet potato mild mottle virus (SPMMV) P1 protein, and cricket paralysis virus (CrPV) 1A protein, for suppressing RISC-mediated mRNA cleavage (2,17,36,54).Although Dicer plays essential roles in RNAi immunity, the mechanism by which Dicer can be directly targeted by VSRs is still poorly understood. The core protein of hepatitis C virus (HCV) was previously reported to interact with Dicer; however, whether this interaction is required for the RNA silencing suppression activity of HCV core protein has not been determined (7, 51). Furthermore, several VSRs have been reported to be able to target both RNA and protein components in the RNAi machinery. For example, TCV P38 was shown to target both RNA duplexes and AGO1 (2, 33). A question that remains to be answered, however, is whether an interrelationship exists between diverse activities of VSRs that mediate RNA binding and interaction with RNAi protein components (51).The ideal model for studying viral pathogenesis and RNAi immunity is the persistent infection of Drosophila melanogaster cells with Flock House virus (FHV), the most extensively studied member of the Nodaviridae family, which encodes a well-defined VSR designated B2 (1,6,10,16,29). During the course of FHV infection, 5=-terminal vRI-dsRNA initiated by viral RNA-dependent RNA polymerase (RdRP) triggers RNAi immunity, which is suppressed by B2 protein because B2 associates with RdRP and binds to vRI-dsRNA, thereby leading to the inhibition of the production of siRNAs by Dicer-2 (Dcr-2) (1). Recently, an interaction of FHV B2 with the Piwi-Argonaut-Zwille (PAZ) domain of Dcr-2 was detected in vitro (45). Although whether this in...

We sequenced small (s) RNAs from field collected honeybees (Apis mellifera) and bumblebees ( Bombus pascuorum ) using the Illumina technology. The sRNA reads were assembled and resulting contigs were used to search for virus homologues in GenBank. Matches with Varroa destructor virus-1 (VDV1) and Deformed wing virus (DWV) genomic sequences were obtained for A. mellifera but not B . pascuorum . Further analyses suggested that the prevalent virus population was composed of VDV-1 and a chimera of 5’-DWV-VDV1-DWV-3’. The recombination junctions in the chimera genomes were confirmed by using RT-PCR, cDNA cloning and Sanger sequencing. We then focused on conserved short fragments (CSF, size > 25 nt) in the virus genomes by using GenBank sequences and the deep sequencing data obtained in this study. The majority of CSF sites confirmed conservation at both between-species (GenBank sequences) and within-population (dataset of this study) levels. However, conserved nucleotide positions in the GenBank sequences might be variable at the within-population level. High mutation rates (Pi>10%) were observed at a number of sites using the deep sequencing data, suggesting that sequence conservation might not always be maintained at the population level. Virus-host interactions and strategies for developing RNAi treatments against VDV1/DWV infections are discussed.

Retailers and manufacturers are increasingly selling extended warranties to obtain high profitability. In addition to the traditional extended warranty (EWR), that offers a free repair and replacement service, a new extended warranty (EWT) comes to the market, under which an additional trade-in service is provided during the warranty coverage. The service provider faces three important decisions: (1) Whether to offer EWR or EWT? (2) How to set the optimal selling prices of EWR and EWT? (3) When choosing to sell EWT, how to determine the optimal trade-in price? To address such challenging issues, we first develop two theoretical models regarding EWR and EWT for a retailer, and further consider the cases where a manufacturer sells the extended warranties and the upgraded product has different failure probabilities. The results show that EWT should never be offered at a higher price than EWR, and when the handling cost for used products is relatively low, EWT will outperform EWR. While the optimal EWR and EWT selling prices increase with the product failure probability, the optimal trade-in price decreases with it. Interestingly, the optimal trade-in discount is not always increasing or decreasing with the failure probability. Moreover, an earlier trade-in time is usually better for the service provider. Compared with the retailer, the manufacturer will always set a lower warranty selling price, but neither of them will always offer a lower trade-in price or discount. We also find that the upgraded product's failure probability will affect the retailer's optimal warranty strategy and profit.

No abstract