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Hit two birds with one stone: the multiple properties of (viral) RNA silencing suppressors Volume 23, issue 6, Novembre-Décembre 2019

Figure 1

Simplified representation of plant miRNA biogenesis and modes of action. Pri-miRNAs are transcribed by RNA polymerase II, capped (black sphere) and polyadenylated (A(n)). DCL1 and its cofactors, including DRB1 (double-stranded RNA binding 1, also referred to HYL1), ensure their processing into pre-miRNA. MiRNA duplexes are obtained following a second dicing step and methylated at their 3’ end by HEN1 (orange sphere). The duplex is exported into the cytoplasm and loaded into AGO1 to form the pre-RISC complex. The passenger strand is removed and the “active” RISC complex ensures cell transcripts silencing by their cleavage or by inhibiting their translation through their base-pairing complementarity to the guide strand.

Figure 2

Graphical representation of antiviral defense provided by RNA interference and counter-defenses mediated by VSR. Replication intermediates (double-stranded RNA) and/or double-stranded structures of RNA viruses as well as overlapping and/or locally structured transcripts of DNA viruses are recognized and cleaved (dicing) by Dicer-like enzymes (DCL). DCLs are associated with their DRB (double-stranded RNA binding) cofactors to generate primary small interfering RNAs duplexes (siRNAs). A methyl group (orange sphere) is added to each 3‘ hydroxyl end of the duplex by HEN1 (Hua enhancer 1). SiRNAs are loaded into an ARGONAUTE protein (AGO) to form the pre-RISC complex (RNA induced silencing complex). One of the duplex strand, then named passenger strand, is eliminated to form the active RISC complex. When infected with RNA viruses, viral transcripts are targeted by base complementarity with the guide strand loaded in AGO and will be mainly cleaved by AGO slicing activity. Cellular RNA dependent RNA polymerases (RDRs) make use of cleavage products to amplify the interfering signal. Secondary siRNAs are generated by DCL4 and associate with AGO to form RISC complexes. Regarding DNA viruses, cellular proteins involved in DNA methylation associate with RISC to initiate the methylation of the viral genome (red triangles). VSRs are represented in red boxes. VSRs expressed by RNA viruses for which an action has been demonstrated in TGS are framed by a double line. Geminiviridae express VSRs that inhibit the activity of cellular proteins involved in methylation [192, 193].

Figure 3

Transitivity mechanism representation. Following targeted RNA cleavage, primary siRNAs are used as primers by RNA-dependent RNA polymerase 6 (RDR6) to generate new dsRNA molecules (case for dependent primer spreading, left). RDR6 recruitment remains to be clarified in the independent primer spreading (right). Neo-synthetized dsRNA molecules are diced by DCLs to produce secondary siRNAs. This siRNA population is specific to the sequence targeted by primary siRNAs but also from its upstream and downstream regions, amplified by RDR6.

The black sphere represents the cap, the orange sphere the 3′ methyl group and A(n) the polyadenylated tail.

Figure 4

Biogenesis models of sfRNA flaviruses and ncRNA3 BNYVV. A) Top, schematic representation of West Nile virus (WNV) genome. XRN1 spreading on RNA is inhibited (full red stars) by the formation of pseudoknots (shown in blue) at SL-II and SL-IV positions leading to subsequent sfRNA1 and sfRNA2 accumulation. Almost all flaviviruses ensure sfRNA1 and sfRNA2 expression and additional species, sfRNA3 and sfRNA4 (empty red stars), may accumulate depending on flaviviruses and host studied (human, mosquito, cell cultures). SfRNAs seem to be a decoy for Dicer and Ago2 and viral siRNAs (vsiRNAs) accumulate. SL-II and SL-IV structures are also described as xrRNA1 and xrRNA2 (Xrn1-resistant RNA). SL, stem-loop; DB, dumbbell; ORF, open reading frame; UTR, untranslated region. The black sphere corresponds to the cap. For review see [211]. Image adapted from [212]. B) A population of BNYVV genomic RNA3 is degraded from 5‘ to 3’ by XRN4 exoribonuclease activity. About 1230 nucleotides are thus eliminated, including the open reading frame (ORF). The progression of XRN4 is blocked by the coremin sequence and non-coding RNA3 (ncRNA3) accumulate. A structural element containing the coremin sequence seems to induce XRN4 stalling, but this structure remains to be determined. A comparable degradation mechanism is described for BNYVV RNA5 [169].