Fig 1.
Domains organization and functions of Dicer proteins in metazoans.
(A) Dicer carries a DExD/H-box helicase, which is divided into 3 subdomains: HEL1, HEL2i, and HEL2. The domain of unknown function (DUF283) is regulating pre-miRNA binding and the PAZ domain is involved in the recognition of the 3′-overhanged extremities. The catalytic core is composed of 2 RNase III domains that each processes one strand of the duplex. Finally, the terminal dsRBD is involved in the binding of the dsRNA minor groove. (B) Position of the different domains in the tridimensional structure of human Dicer in complex with TRBP determined by cryo-EM [22]. The color code used for the domains is the same as in A. TRBP is in orange (adapted from PDB structure n°5ZAK). cryo-EM, cryo-electron microscopy; dsRBD, dsRNA-binding domain; miRNA, microRNA; PAZ, Piwi–Argonaute–Zwille; siRNA, small interfering RNA; TRBP, TAR RNA-binding protein.
Fig 2.
Antiviral RNAi pathways in Caenorhabditis elegans and Drosophila melanogaster.
Upon viral infection, dsRNA triggers RNAi after its recognition by Dicer. In C. elegans, Dicer helped by the dsRBP RDE-4 and another DExD/H-box helicase, DRH-1, recognizes the dsRNA and processes it into primary 22 nt siRNA duplexes. These duplexes serve as a template for the generation of secondary 22G-siRNA by the polymerase RRF-1 and a third helicase called DRH-3. They are finally loaded into an Argonaute protein, WAGO. In D. melanogaster, Dicer-2 recognizes and cleaves the viral dsRNA into 21nt-siRNA duplexes. Strand selection occurs with the help of the dsRBP R2D2, and the passenger strand is sliced by Ago2 before being degraded by the nuclease C3PO. The guide strand is then 2′-O-methylated at the 3′ end by HEN1 to be stabilized. For both worms and flies, the loaded Argonaute protein can then cleave viral complementary sequences, resulting in the antiviral state. dsRNA, double-stranded RNA; RNAi, RNA interference; siRNA, small interfering RNA; vsiRNA, virus-derived small interfering RNA.
Fig 3.
RLRs are directly involved in the IFN-I response upon viral infection. RIG-I and MDA5 can activate the mitochondrial adaptor protein MAVS via their N-terminal CARD domains. Their central DExD/H-box helicase domain shares the same organization as the Dicer one and, together with the CTD, is involved in dsRNA recognition. LGP2 lacks the signaling CARD domains but possesses the whole helicase domain and the CTD. CARD, caspase activation and recruitment domain; CTD, carboxyl-terminal domain; IFN, interferon; MAVS, mitochondrial antiviral signaling adaptator; MDA5, melanoma differentiation–associated gene 5; RIG-I, retinoic acid–inducible gene I; RLR, RIG-I-like receptor; LGP2, laboratory of genetics and physiology 2.
Fig 4.
Mode of action of RLRs upon viral infection.
Both dsRNA replication intermediates and 5′ terminal motifs can be recognized by cytosolic RLRs. RIG-I detects 5′ di- or tri-phosphorylated dsRNA via its CTD and helicase domains whereas MDA5 recognizes long dsRNA structures. Upon dsRNA binding, RIG-I opens and homodimerizes to mediate interactions with MAVS CARD domains at the surface of mitochondria. By contrast, MDA5 constantly shifts between open and close conformations and when it is activated, it polymerizes along the dsRNA forming helicoidal filament to expose its CARD domains and activate MAVS. Once activated, MAVS aggregates and triggers a signaling cascade through the TRAF protein that activates the TBK1 kinase. The latter then phosphorylates the cytosolic transcription factors, IRF3 and IRF7, thereby allowing their dimerization and their translocation into the nucleus where they activate the transcription of IFN-I genes. CARD, caspase activation and recruitment domain; CTD, carboxyl-terminal domain; dsRNA, double-stranded RNA; IFN, interferon; MAVS, mitochondrial antiviral signaling adaptator; MDA5, melanoma differentiation–associated gene 5; RIG-I, retinoic acid–inducible gene I; RLR, RIG-I-like receptor.
Fig 5.
Noncanonical functions of helicases during viral infection.
(A) In insects, Dicer-2 is involved in 2 antiviral pathways in addition to its canonical role in RNAi. First, its helicase domain is necessary to induce the transcription of Vago, a Drosophila cytokine. Vago inhibits viral replication through a yet to be defined mechanism. Second, in Drosophila and mosquitoes, Dicer-2-generated siRNAs can be used by a transposon-encoded retrotranscriptase to generate both linear and circular vDNA molecules. These vDNA can be a source of new vsiRNA duplexes, thereby amplifying the antiviral signal in the cell. (B) In the case of mammalian Dicer, its involvement in direct catalytic antiviral pathways remains debated. During viral infection of undifferentiated ESCs, Dicer is responsible for the production of vsiRNAs functionally involved in the blocking of viral replication. Artificial generation of a helicase-truncated form of Dicer (N1) allows to uncover antiviral RNAi functions in differentiated cells as well. The recent discovery in human ES cells of a naturally occurring isoform of Dicer called Avid, which lacks the HEL2i domain, provides some support to the existence of antiviral RNAi in humans. (C) In human cells, Dicer is also at the center of many interactions, which could modulate either its own or the interacting protein functions. For instance, human Dicer interacts with PKR via its helicase domain and modulates its function during SINV infection. LGP2 is also interacting with the Dicer helicase domain and inhibits Dicer catalytic activity. (D) In mammalian cells, RLRs can be involved in steric obstruction of viral dsRNA. Thus, RIG-I can block the binding and catalytic action of viral proteins (polymerase or capsid) or displace them (NS1), thereby allowing recognition of the dsRNA by other antiviral proteins. dsRNA, double-stranded RNA; ESC, embryonic stem cell; HBV, hepatitis B virus; IAV, influenza A virus; PKR, protein kinase R; RLR, RIG-I-like receptor; RNAi, RNA interference; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; SINV, Sindbis virus; siRNA, small interfering RNA; vDNA, viral DNA.