Nucleic Acid Immunity

Abstract

Organisms throughout biology need to maintain the integrity of their genome. From bacteria to vertebrates, life has established sophisticated mechanisms to detect and eliminate foreign genetic material or to restrict its function and replication. Tremendous progress has been made in the understanding of these mechanisms which keep foreign or unwanted nucleic acids from viruses or phages in check. Mechanisms reach from restriction-modification systems and CRISPR/Cas in bacteria and archaea to RNA interference and immune sensing of nucleic acids, altogether integral parts of a system which is now appreciated as nucleic acid immunity. With inherited receptors and acquired sequence information, nucleic acid immunity comprises innate and adaptive components. Effector functions include diverse nuclease systems, intrinsic activities to directly restrict the function of foreign nucleic acids (e.g., PKR, ADAR1, IFIT1), and extrinsic pathways to alert the immune system and to elicit cytotoxic immune responses. These effects act in concert to restrict viral replication and to eliminate virus-infected cells. The principles of nucleic acid immunity are highly relevant for human disease. Besides its essential contribution to antiviral defense and restriction of endogenous retroelements, dysregulation of nucleic acid immunity can also lead to erroneous detection and response to self nucleic acids then causing sterile inflammation and autoimmunity. Even mechanisms of nucleic acid immunity which are not established in vertebrates are relevant for human disease when they are present in pathogens such as bacteria, parasites, or helminths or in pathogen-transmitting organisms such as insects. This review aims to provide an overview of the diverse mechanisms of nucleic acid immunity which mostly have been looked at separately in the past and to integrate them under the framework nucleic acid immunity as a basic principle of life, the understanding of which has great potential to advance medicine.

Keywords: ADAR1; Antiviral immunity; CRISPR/Cas; CpG-DNA; DNase; Immune sensing of nucleic acids; Innate immunity; OAS; Oligonucleotide; RIG-I; RNAi; RNase H; RNase L; SAMHD1; Sting; TLR3; TLR7; TLR8; TLR9; Trex1; cGAMP; cGAS.

Fig. 1

Mechanisms of nucleic acid immunity in species relevant for human disease. Biology has evolved a number of mechanisms to detect and eliminate foreign nucleic acids as introduced by viruses or bacteriophages. All species from bacteria to humans have established nucleases to directly degrade nucleic acids with structural characteristics or localizations which allow to distinguish them from regular cellular self nucleic acids. Other mechanisms are predominant in certain groups of species. Restriction-modification systems in bacteria and archaea apply sequence-specific modification of self nucleic acids which allows the specific detection and degradation of foreign nucleic acids (restriction endonucleases). Acquired sequence information is used by the CRISPR/Cas system in which new sequence information about pathogenic nucleic acids is integrated into the genome and thereby memorized in order to sequence-specifically degrade foreign nucleic acids. Sequence information is also used by RNA interference which serves antiviral nuclease functions (siRNA/DICER) as well as regulatory (microRNA) functions in higher multicellular organisms. In vertebrates, innate immune-sensing receptors including DICER-related helicases RIG-I and MDA5 dominate over RNAi as antiviral defense mechanism. While innate nucleic acid immune-sensing receptors elicit signaling pathways resulting in antiviral functions, a number of nucleic acid receptors (e.g., PKR, ADAR1, IFIT1) directly detect and restrict nucleic acid function and replication. Since the principles of nucleic acid immunity are either established in mammals themselves or in pathogens (bacteria, parasites, helminths) or pathogen-transmitting insects (e.g., mosquitoes), nucleic acid immunity as such is highly relevant for human health and disease.

Fig. 2

Overview of the time line of discoveries in nucleic acid immunity. This graph provides a noncomprehensive overview of the time lines when important principles, receptors, and ligands contributing to nucleic acid immunity have been described. Immune sensing of nucleic acids dates back to the early 1960s with the observation that nucleic acids such as long double-stranded RNA and specifically poly(I:C) can induce type I interferon. Later, it was appreciated that bacterial DNA is more active than vertebrate DNA. In 1995, the activity of bacterial DNA was attributed to a higher frequency of unmethylated CpG motifs in bacterial DNA. In 2000, TLR9 was identified as the immune receptor for the detection of unmethylated CpG motifs in DNA in the endosomal compartment. Sensing of cytoplasmic DNA remained unclear until in 2009 AIM2 and in 2012 cGAS were identified as the cytosolic receptors responsible for DNA-induced inflammasome activation and type I IFN induction, respectively. For immune sensing of RNA, the story of discoveries continued in 2001 with reports on TLR3-sensing long double-stranded RNA and was continued in 2004 with the appreciation of TLR7 and TLR8 as receptors sensing shorter forms of unmodified single and double-stranded RNA with great implications for the application of siRNA. Another milestone was reached with the immune sensing of cytoplasmic forms of RNA, specifically the detection of 5′-triphosphate short double-stranded forms of RNA by the cytosolic receptor RIG-I. The RIG-I-like receptor MDA5 added another cytosolic receptor which explained the induction of type I IFN by long double-stranded forms of RNA as observed early on in the 1960s. PKR identified in the late 1970s was the first of the receptors restricting nucleic acid function and replication without activating immunity and cytokines. SAMHD1 (depletion of dNTPs) and ADAR1 (A-to-I conversion in dsRNA) entered the field more recently in the context of genetic alterations in these genes identified in the context of inherited inflammatory syndromes (e.g., AGS). IFIT1 and IFIT5 are two other examples of more recently described receptors which inhibit the translation of mRNA. OAS1 was identified early on soon after PKR as a factor restricting viral replication by activating RNase L. Other nucleases contributing to nucleic acid immunity include RNase H structurally resolved in 2004, which degrades the RNA in DNA–RNA hybrids; furthermore, extracellular DNase I and endolysosomal DNase II are known since the mid-1950s. Knowledge around the function of the cytoplasmic DNase III which is also called Trex1 accumulated since 1999 and gained great impact on nucleic acid immunity like SAMHD1 and ADAR1 more recently in the context of inherited type I IFN-dependent inflammatory syndromes. Antiviral RNAi and the role of Dicer were first described in 2005, while the bacterial version of sequence-specific antiviral immunity, CRISPR/Cas, was identified in 2011. Restriction-modification systems are studied since the 1950s.

Fig. 3

Overview of functional components in nucleic acid immunity. The primary detection of specific forms of nucleic acids by highly specialized proteins is the central part of nucleic acid immunity. Upon binding of nucleic acids, the participating specialized proteins can either exert intrinsic direct effects on the nucleic acid which they have bound, or they can have indirect extrinsic effects which require the participation of additional signaling. Extrinsic effects that restrict viral replication and function can be located inside or outside cells, or both. Intrinsic direct effects include degradation or structural modification of the bound nucleic acids, or direct inhibition of translation. Extrinsic indirect effects via signaling pathways include mechanisms that restrict translation or replication, or that lead to degradation of nucleic acids. Extrinsic effects with activities beyond the infected cell include alarming neighboring cells, activating immune effector cells, and guiding immune cells to the site of infection. Together, these intrinsic and extrinsic activities represent the repertoire of nucleic acid immunity to restrict viral infection.

Fig. 4

Innate and adaptive components in nucleic acid immunity. In classical immunology, we distinguish innate and adaptive immunity. While innate immunity relies on receptors encoded in the germline, adaptive immunity acquires information about pathogens during the life span and memorizes such information for later usage. While adaptive immunity directed against proteins relies on the mechanism of genetic recombination to adapt to novel pathogen-derived proteins, in the adaptive part of nucleic acid immunity information about pathogen-derived nucleic acid sequences is acquired and memorized (CRISPR/Cas and RNA interference). Unlike the adaptive components, the innate components of nucleic acid immunity rely on germline-encoded receptors which detect certain structures indicating viral pathogens. Therefore, these receptors are identical throughout the life span, but regulation of receptor expression (e.g., by means of epigenetics) still allows adaptation to different environments (e.g., low or high burden of viral pathogens).

Fig. 5

Receptors and nucleases restricting function and replication of foreign nucleic acids. This graph provides an overview of the proteins which target foreign nucleic acids without involving the classical immune functions such as the induction of cytokines or the activation of immune cells. Such proteins can directly act on the foreign nucleic acid, or they can elicit pathways indirectly acting on the foreign nucleic acid. The endonuclease DNase I is the most abundant DNase in the extracellular space which degrades DNA down to tetramers. DNase II is the predominant endonuclease in the endolysosomal compartment of cells. The cytoplasmic DNase III (Trex1) is a 3′-to 5′ exonuclease which degrades both double- and single-stranded DNA. The cytoplasmic RNase H recognizes DNA–RNA hybrids and cleaves the RNA in such hybrids. In contrast, RNase L is indirectly activated by oligoadenylates which are formed by OAS1 upon binding to long double-stranded RNA. Furthermore, ADAR1 modifies long double-stranded RNA by A-to-I conversions destabilizing the double strand resulting in changes in the coding sequence of proteins. SAMHD1 depletes the pool of dNTPs which is the prerequisite for DNA formation. SAMHD1 hydrolyzes the triphosphate in dNTPs resulting in deoxynucleosides. At the same time, SAMHD1 has been proposed to be a 3′-exonuclease for single-stranded DNA and RNA. PKR and IFIT1/5 inhibit mRNA translation by phosphorylation of the elF2a and by replacing elF4 in the ribosomal complex, respectively. While PKR is activated by long double-stranded RNA, IFIT1 and IFIT5 bind 5′-triphosphate ends of single-stranded RNA.

Fig. 6

Immune-sensing receptors-detecting foreign nucleic acids and inducing indirect effector responses. This graph provides an overview of immune-sensing receptors of nucleic acids. TLR3 is the only one which besides its endosomal localization is also reported to be expressed on the cell membrane. TLR3 binds long double-stranded RNA which is not present in the cytosol of normal cells and is an indicator of foreign. TLR3 is expressed in myeloid immune cells and in a number of somatic cells including fibroblasts and endothelial cells. The other three TLRs expressed in the endolysosomal compartment of distinct immune cell subsets are TLR7, TLR8, and TLR9. TLR7 detects even short RNA, preferentially double-stranded and containing G and U. TLR8 detects single-stranded RNA. While TLR8 is expressed in human myeloid immune cells, TLR7 and TLR9 are predominantly expressed in human B cells and plasmacytoid dendritic cells. TLR9 detects single-stranded DNA containing unmethylated CpG dinucleotides. In the cytoplasm, RIG-I specifically detects RNA if it contains at least a short double strand with a blunt end and a 5′-triphosphate. The RIG-I-like receptor MDA5 detects long irregular forms of double-stranded RNA, but the exact definition of the ligand structure is unclear. Both RIG-I and MDA5 are widely expressed in immune cells and nonimmune cells, and induce a broad array of cell autonomous and extracellular antiviral responses including the production of type I interferon. MDA5 ligands also activate multiple other receptor pathways that depend on the detection of long double-stranded RNA, including PKR, ADAR1, and TLR3. The cytosolic receptor AIM2 detects long double-stranded DNA and activates the inflammasome. The other key receptor for the detection of DNA in the cytoplasm is cGAS. cGAS is activated by long double-stranded DNA and short forms of double-stranded DNA with single-stranded overhangs containing Gs, a structure which was termed Y-form DNA and which is presented during retroviral infection or by endogenous retroelements. Upon activation, cGAS catalyzes the formation of 2′–5′-cGAMP from GTP and ATP. 2′–5′-cGAMP acts as a second messenger which binds to the downstream signaling protein Sting which induces type I interferon via TBK1 and IRF3. 2′–5′-cGAMP can travel to and alarm neighboring cells via gap junctions. Sting also activates NF-κB activation and inflammatory cytokines.