Intracellular detection of viral nucleic acids

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Highlights

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    Viral RNA and DNA are recognized by several intracellular innate immune receptors.

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    RIG-I-like helicases are key sensors of viral RNA in the cytoplasm.

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    Cytosolic viral DNA is recognized by cGAS, inducing STING activation via cGAMP.

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    The sensor IFI16 detects viral DNA both in the cytoplasm and nucleus.

Successful clearance of a microbial infection depends on the concerted action of both the innate and adaptive arms of the immune system. Accurate recognition of an invading pathogen is the first and most crucial step in eliciting effective antimicrobial defense mechanisms. In recent years, remarkable progress has been made towards understanding the molecular details of how the innate immune system recognizes microbial signatures, commonly called pathogen-associated molecular patterns (PAMPs). For viral pathogens, nucleic acids — both viral genomes and viral replication products — represent a major class of PAMPs that trigger antiviral host responses via activation of germline-encoded innate immune receptors. Here we summarize recent advances in intracellular innate sensing mechanisms of viral RNA and DNA.

Introduction

Virtually all cells of a mammalian host organism have the capacity to detect the presence of an invading pathogen by recognizing ‘non-self’ structural components through germline-encoded innate immune sensors, called pattern recognition receptors (PRRs). Over the past ten years, significant progress has been made in identifying the precise viral pathogen signatures (or PAMPs) recognized by PRRs, such as specific modifications (e.g. a 5′-triphosphate moiety) of viral RNA (vRNA), or mislocalized cytoplasmic viral DNA (vDNA) [1, 2]. Mammalian cells have evolved a large repertoire of PRRs, which can be grouped with respect to their subcellular localization. While Toll-like receptors (TLRs) and C-type lectin receptors (CLRs) recognize virion components in endosomes and on cell membranes (reviewed in [3]), the detection of incoming and actively replicating viruses is mediated by PRRs that are localized inside the cell. Most intracellular PRRs recognize viral nucleic acids and have the remarkable ability to distinguish ‘non-self’ RNA or DNA from the large pool of cellular RNAs and DNAs. At least three major classes of intracellular sensors of viral infection have been identified: (1) RIG-I-like receptors (RLRs) which sense vRNA species in the cytoplasm and play important roles in the detection of RNA viruses; (2) a structurally unrelated group of vDNA receptors (e.g. cGAS and IFI16) localized in the host cytoplasm and/or nucleus; and (3) members of the NOD-like receptor (NLR) family which, besides their established roles in sensing bacterial infections, have also been implicated in detecting viral pathogens. In addition, several other proteins have been implicated in vRNA or vDNA sensing, although their physiological roles have yet to be fully established (as discussed below).

Following ligand recognition, PRRs activate antiviral signaling cascades that converge on a group of well-characterized kinases, namely TANK-binding kinase 1 (TBK1), mitogen-activated protein kinases (MAPKs), and IκB kinase α (IKKα) and IKKβ. Through phosphorylation events, these kinases subsequently activate the interferon (IFN)-regulatory factors 3 and 7 (IRF3/7), AP-1, and NF-κB, respectively. These proteins transcriptionally induce the gene expression of type-I IFNs (mainly IFN-α subtypes and IFN-β), type-III IFN (IFN-λ), and other pro-inflammatory cytokines such as members of the interleukin (IL) protein family [1, 2]. Furthermore, some PRRs activate inflammasomes, which are caspase-1-activating multi-protein complexes that cleave pro-IL-1β and pro-IL-18 to generate their mature forms [4]. Secreted IFNs bind to their respective surface receptors on both infected and uninfected neighboring cells, inducing signal transduction that leads to the expression of numerous IFN-stimulated genes (ISGs) [5, 6]. ISGs encode for proteins that exert distinct antiviral effector functions such as cleavage of vRNA or induction of apoptosis. In addition, some ISGs encode for PRRs or for proteins involved in PRR signal transduction, leading to positive feedback amplification of the antiviral response in infected cells, and also sensitizing uninfected cells to fight off the viral attack. Induction of IFNs and other pro-inflammatory cytokines not only limits the spread of the viral pathogen to surrounding cells, but also facilitates viral clearance by recruiting and stimulating cells of the adaptive immune system.

In this opinion article, we summarize recent findings on the molecular mechanisms of how intracellular innate immune receptors detect vRNA and vDNA, and further outline unresolved questions in this rapidly progressing field.

Section snippets

Detection of vRNA by RLRs

Cytosolic vRNA is predominantly recognized by DExD/H-box RNA helicases of the RLR family (Figure 1). This family consists of retinoic acid-inducible gene-I (RIG-I), melanoma differentiation-associated gene 5 (MDA5), and laboratory of genetics and physiology 2 (LGP2), all of which are able to directly bind RNA through their helicase and C-terminal domains (CTD) [1, 2]. In addition, both RIG-I and MDA5 possess a pair of caspase activation and recruitment domains (CARDs), which mediate downstream

Intracellular sensing of vDNA

It has long been recognized that the presence of foreign DNA in the host cytoplasm — either arising from infection with DNA viruses or intracellular bacteria, or artificially introduced by transfection — can trigger innate immune activation. In addition, recent studies have indicated that host cells can also sense vDNA in the nucleus. Remarkable progress has been made in the past few years that has led to the identification of multiple intracellular vDNA receptors and a critical endoplasmic

Conclusions and perspectives

Although recent work has shed light on the molecules and pathways involved in vRNA and vDNA sensing, the exact nature of the physiological ligands for most receptors is still unknown. While several in vivo PAMPs have been identified for RIG-I using next-generation sequencing, physiological ligands of MDA5 and LGP2 during viral infection are largely unknown. Given that LGP2's function is still enigmatic, the identification of vRNA species recognized by LGP2 may clarify its role in antiviral

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

  • • of special interest

  • •• of outstanding interest

Acknowledgements

We apologize to all researchers whose critical contributions to the field could not be cited due to space constraints. Current research in the Gack laboratory is supported by National Institutes of Health Grants (AI087846, AI097699, and AI104415), a John and Virginia Kaneb Fellowship, and the Alexander and Margaret Stewart Trust Foundation.

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