RNAi as a nucleicacidbased immune defence

RNAi is a form of highly specific defence reaction, based on the specific base-pairing between small RNAs and invading nucleic acids. Different types of small RNAs, called microRNAs (miRNAs), small interfering RNAs (siRNAs), or Piwi-associated RNAs (piRNAs), have been described in insects and other multicellular organisms.

4.3.1.1 RNAi pathways miRNAs are produced from nuclear genes, which are transcribed in pri-miRNAs. These precursors, which contain stem loops, are processed in the nucleus by the RNaseIII Drosha, to generate pre-miRNAs. Pre-miRNAs exit the nucleus through the exportin-5 system, to access the cytosol, where they are processed by another RNaseIII enzyme, Dicer-1, to generate miRNAs (Lee et al., 2004). miRNA duplexes are dissociated with the help of the dsRNA-binding protein R3D1, and incorporated into the miRNA-dependent RNAi-silencing complex (miRISC) (Jiang et al., 2005). The miRISC contains the RNaseH-like enzyme Argonaute-1 (AGO-1) that will be guided by the miRNA towards complementary RNA sequences (Okamura et al., 2004). Binding of the miRISC to an RNA molecule can result either in translation inhibition if the complementarity between the miRNA and the RNA molecule is not perfect, or in RNA cleavage if the complementarity is complete (Brodersen et al., 2008). miRNAs can also affect the chromatin structure, and affect transcription of the gene encoding the corresponding mRNA. miRNAs, which also exist in vertebrates and in plants, play important roles in development. As a result, Dicer-1 and AGO-1 mutant flies are embryonic lethal (Lee et al., 2004; Okamura et al, 2004).

Small interfering RNAs (siRNAs) are produced from dsRNA molecules, which are recognized by the second Dicer enzyme encoded by the Drosophila genome, Dicer-2, and processed into 22 bp fragments (Lee et al., 2004). The dsRNA molecules can have an endogenous origin, resulting from annealing of RNAs generated by bidirectional transcription of overlapping genes, from transcription of palindromic sequences generating hairpin structures, or from inverted-repeat pseudogenes. In this case, Dicer-2 generates endo-siRNAs, which are then incorporated in an siRNA-dependent RNAi-silencing complex (siRISC) containing the AGO-2 enzyme. R3D1 is also required for this step (reviewed in Obbard and Finnegan, 2008). Importantly, dsRNA can also have an exogenous origin, and betray the presence of foreign nucleic acids in the Drosophila cells. Indeed, many viruses have dsRNA genomes (e.g. Reoviridae, Birnaviridae), or generate dsRNA replicative intermediates in the cytosol of infected cells. These exo-siRNAs are generated by Dicer-2, and incorporated into an AGO-2-containing RISC complex. However, this incorporation requires R2D2 instead of R3D1, revealing differences in the processing of siRNAs of endogenous or exogenous origin (Figure 4.2).

Finally, Piwi-associated RNAs (piRNAs) are substantially longer (24-30 nt) than miRNAs and siRNAs; they are involved in heterochromatin maintenance. As their name indicates, piRNAs are

Entry

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Entry

Replication 5

dsRNA

Pirnas Name Images

Dicing siRNAs

Dicing

Replication 5

RISC

Slicing

RISC

siRNAs

RISC

RISC

Figure 4.2 siRNA-mediated antiviral defence in Drosophila. Double-stranded RNA (dsRNA) molecules produced during replication of a (+) single-stranded RNA virus are recognized by the RNaselll enzyme Dicer-2 in the cytosol of infected cells and are processed into 21-22 nt short interfering RNA (siRNA) duplexes (Dicing). R2D2 separates the two siRNA strands and loads the guide strand on to the Argonaute protein AGO-2 in the RNAi-silencing complex (RISC), which can then target single-stranded RNA molecules of complementary sequence. Viral RNA molecules are then cleaved by AGO-2 (Slicing).

associated with Piwi proteins, which form a distinct subfamily in the AGO superfamily. In total, the Drosophila genome encodes five AGO proteins, two of which (AGO-1 and AGO-2) belong to the Argonaute subfamily and have been mentioned above, whereas the three remaining members belong to the Piwi subfamily (Piwi, Aubergine, and AGO-3) and are involved in the production and function of piRNAs. Unlike miRNAs and siR-NAs, piRNAs are not generated from a dsRNA precursor, and do not require Dicer enzymes. Rather, these small RNAs are produced by an original amplification mechanism involving Piwi,

Aubergine, and AGO-3 (Girard and Hannon, 2007) (Figure 4.3).

4.3.1.2 RNAi and virus control in Drosophila RNAi was first shown to be an important antiviral defence mechanisms in plants. Plants have expanded the Dicer family to four members, with Dicer-like (DCL)1 primarily dedicated to the production of miRNAs. DCL2, 3, and 4 process long dsRNA molecules and participate in the control of RNA (DCL2, 4) or DNA (DCL3) virus infections (reviewed in Ding and Voinnet, 2007). In insects, Dicer-2 plays an important role in the resistance to viral infections, as shown by the increased susceptibility of Dicer-2 mutant flies to infection by the RNA viruses DCV, CrPV, FHV, and SINV (Galiana-Arnoux et al, 2006; van Rij et al, 2006; Wang et al, 2006). Curiously, however, Dicer-2 mutant flies are as resistant as wild-type controls to infection with the dsRNA virus DXV (Zambon et al, 2006). AGO-2 and r2d2 mutant flies have also been shown to be more susceptible to DCV, CrPV, FHV, and DXV infections. Increased lethality of virus towards mutant flies correlates with increased viral load and high levels of viral RNAs in infected flies, in good agreement with the mode of action of RNA interference. West Nile virus (WNV), an arbovirus of the Flavoviridae family that can readily infect Drosophila, also replicates to higher titres in AGO-2 mutant flies than in wild-type controls (Chotkowski et al., 2008). However, as in the case of DXV, a similar increase in viral titres is not observed in Dicer-2 mutant flies. Curiously, piwi appears to be involved in the control of the viral load in both DXV- and WNV-infected flies, even though this gene is reportedly essentially expressed in the germ line (Zambon et al., 2006; Chotkowski et al., 2008).

In addition to genetic evidence, the role of RNA interference as an antiviral defence in Drosophila is confirmed by the detection of siRNAs corresponding to viral sequences in infected flies. Altogether, these data point to a mechanism whereby dSRNA corresponding either to the viral genome or to replication intermediate forms is detected by Dicer-2 and cleaved into siRNAs. The guide strand of the siRNAs is then incorporated in a R2D2-dependent manner into AGO-2-containing RISC complexes, which will degrade viral RNAs in the cytosol of

Sense gypsy

Antisense gypsy

Sense gypsy

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Secondary antisense piRNA 5'

5' Secondary sense piRNA

Secondary antisense piRNA 5'

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AGO-3

Accumulation of antisense piRNA

Sequence-specific Piwi-dependent formation of heterochromatin: silencing of gypsy

Accumulation of antisense piRNA

Sequence-specific Piwi-dependent formation of heterochromatin: silencing of gypsy

Figure 4.3 Model for synthesis of piRNAs and silencing of gypsy expression in Drosophila. The flamenco locus, which consists of defective integrated copies of gypsy, produces primary Piwi-associated RNA molecules (piRNAs). Antisense primary piRNAs guide Piwi (or Aubergine) to complementary sequences, which are cleaved by the slicer protein Piwi to generate the 5' end of secondary piRNAs. The 3' end of these piRNAs is generated by an unidentified mechanism. Secondary piRNAs of sense polarity then associate with AGO-3, and guide it to flamenco transcripts, which are cleaved to generate more antisense piRNAs.

infected cells (Figure 4.2). Consistent with this idea, transgenic flies expressing FHV dsRNA are protected against a challenge with FHV, but not with the unrelated DCV (Galiana-Arnoux et al., 2006). Thus, the siRNA pathway provides flies with a highly specific antiviral defence system, based on the pairing of complementary nucleic acids.

Another RNAi pathway, the piRNA pathway, plays an important role in the control of nucleic acid parasites like transposons or endogenous ret-roviruses in Drosophila. Genetic studies have shown that two loci located in heterochromatic regions of the X chromosome and known as flamenco and

X-TAS restrict the ability of endogenous retroviruses of the Gypsy, Idefix, or ZAM family (flamenco) or transposons of the P element family (X-TAS) to translocate within the genome. Flamenco and X-TAS correspond to insertion hotspots for different types of mobile genetic elements, which will lead to the generation of large quantities of distinct piRNAs. These piRNAs associate with Piwi proteins and guide them to silence transposons or endogenous retroviruses dispersed all over the genome (Figure 4.3). Interestingly, a similar mechanism is present in mammalian genomes to control transposon mobility in the germ line (reviewed in Girard and Hannon, 2007). Expression of transposons is also controlled in somatic tissues, by endo-siRNAs (Obbard and Finnegan, 2008).

4.3.1.3 Viral suppressors of RNAi RNAi is an efficient antiviral defence mechanism in plants, and as a result viruses that successfully replicate in plant cells express suppressors of RNAi or viral suppressors of RNAi (VSRs). A few dozen VSRs have so far been identified from plant viruses. Interestingly, these molecules are extremely diverse both structurally and in terms of mode of action, suggesting that they have evolved independently to provide a variety of solutions to counter the host's RNAi-based defence (Ding and Voinnet, 2007). While some plant VSRs encode dsRNA-binding proteins that prevent interaction of dsRNA with Dicer enzymes (e.g. turnip crinkle virus p38), others prevent assembly of the RISC complex (e.g. gemini virus AC4), interfere with slicing (e.g. cauliflower mosaic virus 2b), or even promote ubiquitin-dependent proteolysis of key molecules of the RNAi machinery such as AGO-1 (e.g. Polerovirus P0).

In agreement with the proposed critical role of RNAi in antiviral defence in insects, insect viruses have also been shown to express VSRs. Further, as would be expected if RNAi were a crucial antiviral defence, Dicer-2, AGO-2, and r2d2 are among the fastest-evolving genes in the Drosophila genome (Obbard et al., 2006). The best-characterized VSR is the B2 protein from FHV. B2 is a 106 amino acid molecule, which dimerizes and forms a four-helix bundle. It is synthesized at high levels in infected cells, and binds dsRNA with nanomolar affinity. By contrast, it does not bind DNA, single-stranded RNA, or DNA-RNA hybrids. Interaction with dsRNA is sequence-independent, and B2 binds with affinity to dsRNAs as short as 17 bp. Hence, B2 interacts with both long dsRNA and siRNA, and can interfere with RNAi both before and after cleavage by Dicer enzymes (Chao et al., 2005). Multiple B2 proteins may associate with a dsRNA molecule, thus coating the FHV replication intermediates, and preventing interaction with Dicer or other molecules of the RNAi machinery. In agreement with the proposed function of B2, B2-deficient viruses are not virulent, and are barely detected in injected wildtype flies. These viruses can, however, replicate in

Dicer-2 or AGO-2 mutant flies, proving that B2 acts in vivo as a suppressor of RNAi (Galiana-Arnoux et al, 2006; Wang et al, 2006).

The Dicistroviruses DCV and CrPV have also been shown to encode VSRs. Interestingly, even though the two viruses are closely related and the two VSRs map to the N-terminus of the ORF1, the two suppressor proteins do not show any sequence similarity, and appear to function differently (Figure 4.1). Indeed, sequence analysis of the first 100 amino acids of DCV ORF1 reveals the presence of a canonical dsRNA-binding domain, whereas the N-terminus of CrPV ORF1 does not contain any known structural motifs. In vitro experiments with recombinant protein confirmed that the DCV VSR, known as DCV-1A, binds long dsRNAs with high affinity in a sequence-independent manner, and prevents processing by Dicer-2 (van Rij et al, 2006). Unlike B2, DCV-1A does not bind to siRNAs, and seems to act only upstream of Dicer. Globally however, both FHV B2 and DCV-1A act by sequestering dsRNA, even though they do not share any sequence similarity, providing a nice example of convergent evolution in two insect viruses to counteract RNAi. The mode of action of the VSR of CrPV remains mysterious (Wang et al., 2006). It is fascinating that DCV and CrPV, which are two closely related members of Dicistroviridae family, sharing high sequence similarity throughout their genomes, are completely divergent in the N-terminus of their ORF1. Examination of the N-terminus of ORF1 from the other sequenced Dicistroviruses reveals that they are not related, suggesting that they may all have evolved original strategies to evade host defences. Interestingly, two of these viruses have motifs at the N-terminus of ORF1 that may hint to the function of the suppressors (Figure 4.1). In the case of aphid lethal paralysis virus (ALPV), a discrete Pox protein repeats of ankyrin-C-terminal (PRANC) motif is observed. This motif is present at the C-terminus of a variety of poxvirus proteins, and is related to F-box proteins. Thus, this protein may function in a manner similar to P0 from plant poleroviruses and target one component of the RNAi pathway for degradation. The second case is the shrimp virus Taura syndrome virus (TSV), which encodes a baculo-virus inhibitor of apoptosis repeat (BIR) domain.

As indicated by its name, this domain is present in some viral proteins that interfere with the apop-totic pathway. Thus, for some Dicistroviruses, the N-terminus of ORF1 may be used to interfere with antiviral defences other than RNAi.

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