Sensors of RNA virus infection

Many signaling pathways leading to the IFNα/β induction upon virus infection, have been recently discovered. These involve specific cellular receptors that can detect the presence of virus by recognising viral molecular signatures. These viral molecular signatures are part of the PAMPs (Pathogen-Associated Molecular Patterns) that contain many other potential patterns from other pathogen, including bacteria and funghi. The PAMPs are recognised by a wide range of receptors, called PRRs (Pattern Recognition Receptors) that comprise the Pathogen-Resistance Protein (R-protein) in plants, the Toll-Like Receptors (TLRs), the Nod-Like Receptors (NLRs) and the recently discovered Rig-like receptors (RLRs) in animals (Fig.11).

The R proteins were the first PRR to be discovered. These are crucial for immune defence against all the possible pathogen-derived molecules invading plants. Considering the fact that these R genes are of real importance for the survival of plants, it is not surprising to find that kind of pathogen-resistance protein in other organisms (Nimchuk et al., 2003). The next PRR to be discovered was the Toll receptor that was identified in the fruit fly, Drosophila melanogaster, as an essential receptor for the establishment of the dorso-ventral pattern in developing embryos. In the mid-90s, toll-mutant flies were shown to be highly susceptible to fungal infection (Lemaitre et al., 1996; Yamamoto and Akirz, 2005). The receptor Toll was identified as a key mediator of innate immune defences in Drosophila melanogaster. A year later, the identification of a Toll-like receptor (TLR) in the human genome was reported and it was later called TLR4 (Medzhitov et al., 1997). Sequencing of human and murine genomes further allowed the identification of 11 TLRs in mice and 10 TLRs in human. The ability of the TLRs to recognise microbes and directly initiate specific signal transduction cascades that alert the host defences, can also be observed in the two additional families of innate receptors:

the NLRs and the RLRs. Unlike the TLRs that are essentially found at the plasma membrane, these families consist of soluble proteins that survey the cytoplasm for signs that broadcast the presence of intracellular invaders. Until now, the NLRs have been shown to detect bacteria and many other pathogens, whereas the RLRs only recognise viruses (Creagh and O'Neill, 2006). NLRs share high structural and functional homology with plant R genes and search of the human genome database revealed 22 R-gene homologous classified in two main subclasses: the NODs (Nucleotides Oligomerization Domain leucine-rich repeat protein) counting 5 members and the NALPs (NACHT Leucine rich domain and Pyrin-containing

protein) containing 14 members. Some of the NLRs activation is proposed to occur via a mechanism similar to the mechanism described for the apoptosome (Reith and Mach, 2001).

NACHT

TLRs RLRs

Pattern recognition receptors (PRRs)

A B Helicase

TIR

NLRs (NOD1)

LRR R protein

Figure 11: Domain organization of the PRRs. All of the proteins have the NACHT-LRR configuration, with the exception of the RLRs, which contain a helicase domain. The A and B represent the Walker A and Walker B motifs. Not all of the possible N-terminal domains are represented; the caspase-recruitement domains (CARD) are specific to the RLRs and are also present in the NLRs when there are no BIR (baculoviral inhibitory repeat) or acidic domains. The Toll-IL-1 receptor or coiled-coil domains (TIR/CC) are N-terminal domains specific to the R protein or the TLRs (Adapted from (Ting and Davis, 2005).

It is likely that the initiation of innate immunity involves an important cooperation between the TLRs, the NLRs and the RLRs depending on the pathogen, providing a tightly controlled combinatorial repertoire for triggering host defences. TLRs seem to be specifically active in immune cells such as dendritic cells whereas NLRs and especially RLRs are expressed in more various cell lines. The RLR family comprises three DExD/H-box-containing RNA helicases: retinoic-acid-inducible gene 1 (RIG-1), melanoma differentiation-associated gene 5 (Mda-5) and laboratory of genetics and physiology 2 (LGP2). RIG-I and Mda-5 are both ubiquitously expressed in most tissues and are part of the ISGs, which allows autocrine and paracrine amplification of the sensing system. RIG-I and Mda-5 encode two caspase recruitment domains (CARD) at the N-terminus, followed by an RNA helicase domain. The helicase domain recognises viral RNA and regulates signal transduction in an ATPase-dependent manner, whereas the CARD domain is involved in the signal transduction downstream (Kang et al., 2004). LGP2 lacks completely the CARD domains and functions as a dominant-negative regulator of RIG-I/Mda-5 mediated signaling (Rothenfusser et al., 2005;

Yoneyama et al., 2005). The recognition of the molecular viral signature via the helicase domain of RIG-I and Mda-5 likely induces a conformationnal change enabling their CARD

domains to interact with the CARD-like domain of a protein anchored to the outer mitochondrial membrane called Cardif (MAVS/IPS-1/VISA) (Kawai et al., 2005; Meylan et al., 2005; Seth et al., 2005; Xu et al., 2005). This interaction leads indirectly to the activation of several different kinases such as TBK-1/IKKε and IKKγ and activate IRF-3 and NF-κB respectively, inducing type I IFNs and the antiviral state of the cell (Fig. 12) (Hornung et al., 2006; Pichlmair et al., 2006).

Figure 12: RIG-I and Mda-5 are cellular RNA helicases that, upon activation, stimulate IFN gene expression. RIG-I recognises RNAs with 5’triphosphates, whereas Mda-5 recognises dsRNA

(From (Basler and Garcia-Sastre, 2007).

Studies of RIG-I and Mda-5-deficient mice have revealed that RIG-I is essential for the recognition of a set of specific ssRNA viruses, including Paramyxoviruses, Flaviviruses, Orthomyxoviruses and Rhabdoviruses, whereas Mda-5 is important for the recognition of a different set of RNA virus that includes Picornaviruses and alphaviruses (Basler and Garcia-Sastre, 2007; Kato et al., 2006). In vitro studies have also shown that both RIG-I and Mda-5 can bind to polyI/C and respond to polyI/C and RNA viruses (Yoneyama et al., 2005). In addition to this, it has been shown that RIG-I is more likely to recognise RNAs with 5’triphosphates than dsRNA, whereas Mda-5 specifically recognises dsRNA (Basler and Garcia-Sastre, 2007). The role of these helicases is to distinguish between self RNA and non-self RNA coming from the viruses. At the end of the year 2006, Veit Hornung et al. provided evidence that uncapped unmodifided 5’-triphosphate RNA present in viruses known to be recognised by RIG-I, but absent in viruses known to be detected by Mda-5, serves as PAMP for the detection of viral infection by RIG-I in the cytosol of eukaryotic cells and that this

property is not confined to immune cells. They further observed that RIG-I does not activate the IFNβ activation with RNA containing 5’di- or 5’-monophosphate (Hornung et al., 2006).

Many of the RNA species in the cytosol are known to lack free 5’-triphosphate group although all RNA transcripts generated in the nucleus of a eukaryotic cell initially contain a 5’tri-phophate. Indeed cellular self RNA escapes detection by RIG-I because they are known to undergo several modifications before being transported to the cytoplasm: messenger RNA acquires a 7-methyl-guanosine cap structure at its 5′-end; transfer RNA undergoes 5′ cleavage and a series of nucleotide modifications; and ribosomal RNA undergoes several cleavages and finally associates with ribosomal proteins (Bowie and Fitzgerald, 2007) (Fig. 13).

Figure 13: Discrimination of self and non-self RNA by RIG-I. Viral infection leads to the accumulation of non-self RNAs in the cytoplasm, such as dsRNA and 5-triphosphate RNA. Cellular RNA synthesis takes place in the nucleus by 3 different RNA polymerases. The 5’ ppp of these RNA are eventually removed or masked, thus self RNA species do not activate RIG-I (From (Yoneyama and Fujita, 2007).