Plants and invertebrates can protect themselves from viral infection through RNA silencing. This antiviral immunity involves production of virus-derived small interfering RNAs (viRNAs) and results in specific silencing of viruses by viRNA-guided effector complexes. The proteins required for viRNA production as well as several key downstream components of the antiviral immunity pathway have been identified in plants, flies, and worms. Meanwhile, viral mechanisms to suppress this small RNA-directed immunity by viruses are being elucidated, thereby illuminating an ongoing molecular arms race that likely impacts the evolution of both viral and host genomes.

BackgroundAdapter trimming is a prerequisite step for analyzing next-generation sequencing (NGS) data when the reads are longer than the target DNA/RNA fragments. Although typically used in small RNA sequencing, adapter trimming is also used widely in other applications, such as genome DNA sequencing and transcriptome RNA/cDNA sequencing, where fragments shorter than a read are sometimes obtained because of the limitations of NGS protocols. For the newly emerged Nextera long mate-pair (LMP) protocol, junction adapters are located in the middle of all properly constructed fragments; hence, adapter trimming is essential to gain the correct paired reads. However, our investigations have shown that few adapter trimming tools meet both efficiency and accuracy requirements simultaneously. The performances of these tools can be even worse for paired-end and/or mate-pair sequencing.ResultsTo improve the efficiency of adapter trimming, we devised a novel algorithm, the bit-masked k-difference matching algorithm, which has O(kn) expected time with O(m) space, where k is the maximum number of differences allowed, n is the read length, and m is the adapter length. This algorithm makes it possible to fully enumerate all candidates that meet a specified threshold, e.g. error ratio, within a short period of time. To improve the accuracy of this algorithm, we designed a simple and easy-to-explain statistical scoring scheme to evaluate candidates in the pattern matching step. We also devised scoring schemes to fully exploit the paired-end/mate-pair information when it is applicable. All these features have been implemented in an industry-standard tool named Skewer (https://sourceforge.net/projects/skewer). Experiments on simulated data, real data of small RNA sequencing, paired-end RNA sequencing, and Nextera LMP sequencing showed that Skewer outperforms all other similar tools that have the same utility. Further, Skewer is considerably faster than other tools that have comparative accuracies; namely, one times faster for single-end sequencing, more than 12 times faster for paired-end sequencing, and 49% faster for LMP sequencing.ConclusionsSkewer achieved as yet unmatched accuracies for adapter trimming with low time bound.

In eukaryotic RNA-based antiviral immunity, viral double-stranded RNA is recognized as a pathogen-associated molecular pattern and processed into small interfering RNAs (siRNAs) by the host ribonuclease Dicer. After amplification by host RNA-dependent RNA polymerases in some cases, these virus-derived siRNAs guide specific antiviral immunity through RNA interference and related RNA silencing effector mechanisms. Here, I review recent studies on the features of viral siRNAs and other virus-derived small RNAs from virus-infected fungi, plants, insects, nematodes and vertebrates and discuss the innate and adaptive properties of RNA-based antiviral immunity.

RNA silencing is a sequence-specific RNA degradation mechanism that is operational in plants and animals. Here, we show that flock house virus (FHV) is both an initiator and a target of RNA silencing in Drosophila host cells and that FHV infection requires suppression of RNA silencing by an FHV-encoded protein, B2. These findings establish RNA silencing as an adaptive antiviral defense in animal cells. B2 also inhibits RNA silencing in transgenic plants, providing evidence for a conserved RNA silencing pathway in the plant and animal kingdoms.

Innate immunity against bacterial and fungal pathogens is mediated by Toll and immune deficiency (Imd) pathways, but little is known about the antiviral response in Drosophila. Here, we demonstrate that an RNA interference pathway protects adult flies from infection by two evolutionarily diverse viruses. Our work also describes a molecular framework for the viral immunity, in which viral doublestranded RNA produced during infection acts as the pathogen trigger whereas Drosophila Dicer-2 and Argonaute-2 act as host sensor and effector, respectively. These findings establish a Drosophila model for studying the innate immunity against viruses in animals.

BackgroundCRISPR/Cas systems confer immunity against invading nucleic acids and phages in bacteria and archaea. CRISPR/Cas13a (known previously as C2c2) is a class 2 type VI-A ribonuclease capable of targeting and cleaving single-stranded RNA (ssRNA) molecules of the phage genome. Here, we employ CRISPR/Cas13a to engineer interference with an RNA virus, Turnip Mosaic Virus (TuMV), in plants.ResultsCRISPR/Cas13a produces interference against green fluorescent protein (GFP)-expressing TuMV in transient assays and stable overexpression lines of Nicotiana benthamiana. CRISPR RNA (crRNAs) targeting the HC-Pro and GFP sequences exhibit better interference than those targeting other regions such as coat protein (CP) sequence. Cas13a can also process pre-crRNAs into functional crRNAs.ConclusionsOur data indicate that CRISPR/Cas13a can be used for engineering interference against RNA viruses, providing a potential novel mechanism for RNA-guided immunity against RNA viruses and for other RNA manipulations in plants.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-017-1381-1) contains supplementary material, which is available to authorized users.

Diverse eukaryotic hosts produce virus-derived small interfering RNAs (siRNAs) to direct antiviral immunity by RNA interference (RNAi). However, it remains unknown whether the mammalian RNAi pathway has a natural antiviral function. Here, we show that infection of hamster cells and suckling mice by Nodamura virus (NoV), a mosquito-transmissible RNA virus, requires RNAi suppression by its B2 protein. Loss of B2 expression or its suppressor activity leads to abundant production of viral siRNAs and rapid clearance of the mutant viruses in mice. However, viral small RNAs detected during virulent infection by NoV do not have the properties of canonical siRNAs. These findings have parallels with the induction and suppression of antiviral RNAi by the related Flock house virus in fruit flies and nematodes and reveal a mammalian antiviral immunity mechanism mediated by RNAi.

In diverse eukaryotic organisms, Dicer-processed, virus-derived small interfering RNAs direct antiviral immunity by RNA silencing or RNA interference. Here we show that in addition to core dicing and slicing components of RNAi, the RNAi-mediated viral immunity in Arabidopsis thaliana requires host RNA-directed RNA polymerase (RDR) 1 or RDR6 to produce viral secondary siRNAs following viral RNA replication-triggered biogenesis of primary siRNAs. We found that the two antiviral RDRs exhibited specificity in targeting the tripartite positive-strand RNA genome of cucumber mosaic virus (CMV). RDR1 preferentially amplified the 5′-terminal siRNAs of each of the three viral genomic RNAs, whereas an increased production of siRNAs targeting the 3′ half of RNA3 detected in rdr1 mutant plants appeared to be RDR6-dependent. However, siRNAs derived from a single-stranded 336-nucleotide satellite RNA of CMV were not amplified by either antiviral RDR, suggesting avoidance of the potent RDR-dependent silencing as a strategy for the molecular parasite of CMV to achieve preferential replication. Our work thus identifies a distinct mechanism for the amplification of immunity effectors, which together with the requirement for the biogenesis of endogenous siRNAs, may play a role in the emergence and expansion of eukaryotic RDRs.antiviral immunity | cucumber mosaic virus | RNA silencing | RNAdependent RNA polymerase | secondary small interfering RNA R NA silencing or RNA interference in fungi, nematodes and plants requires amplification of small interfering RNAs by eukaryotic RNA-directed RNA polymerases (RDRs) (1-3). Plant and fungal RDRs convert transcripts of target genes into dsRNA that is subsequently processed into secondary siRNAs by a Dicer or Dicer-like (DCL) nuclease. In contrast, Caenorhabditis elegans RDRs, such as RRF-1, may directly manufacture secondary siRNAs without dicing a dsRNA precursor (1-3). The genome of Arabidopsis thaliana encodes six RDRs that are grouped into four clusters (1, 2, 4), among which little is known about cluster III, consisting of RDRs 3a, 3b, and 3c. RDR2 and RDR6 are both required for the short-distance spread of transgene silencing and for the perception, but not the production, of the long-distance mobile silencing signal (1, 5-9). RDR2 is also essential for the biogenesis of the DCL3-dependent 24-nucleotide (nt) repeat-associated siRNAs (rasiRNAs) derived from transposons, retroelements, and other elements, which are the most abundant endogenous small RNAs in A. thaliana (3). Similarly, RDR6 coupled with DCL4 or DCL1 is responsible for the biogenesis of transacting siRNAs (tasiRNAs) and natural antisense siRNAs (nat-siRNAs), which silence expression of their target genes like microRNAs (miRNAs) (1-3).RNA silencing controls antiviral immunity in fungi, plants, and invertebrates by producing virus-derived siRNAs to be loaded in an Argonaute protein for antiviral silencing (10-12). In A. thaliana, DCL4 and DCL2 produce viral siRNAs against distinct positive (+)-strand RNA viruses in a hierarc...