The type I interferon system

Unlike the adaptive immune response, which has a broad repertoire of specific receptors (potentially 10⁷ to 10⁹ antigenic determinants can be discriminated) to recognize pathogens and acquire memory to a specific antigen, the innate immune system is equipped with a set of preset receptors specialized to detect non-self or danger signals - by-products of microbial replication (reviewed here [115, 116]).

These so called pattern recognition receptors (PRR) are encoded in the germline and

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are able to detect pathogen or danger associated molecular patterns (PAMPs or DAMPs) without prior encounter. The expression pattern of PRRs can be divers from just a few cell types to a wide range of cell types and tissues. Once the respective PAMP is encountered by the PRR, a signaling cascade induces the expression of interferons (IFNs), these in turn induce a broad range of interferon induced genes (ISGs) exerting diverse antiviral activities, in the producer cell itself, and in the surrounding cells. Since the receptors are already expressed in sentinel cells like dendritic cells and macrophages the response is very quick and an antiviral state can be established within minutes to hours [117]. The adaptive immune response on the other hand requires first activation signals from the innate immune system and takes days to weeks to establish a specific response towards a pathogen. The activity of IFN not only targets the replication of many pathogens but has also an effect on cellular function such as cell cycle arrest or apoptosis and can have a devastating effect on the organism (e.g. cytokine storm). Therefore the induction of the IFN system must be tightly regulated.

Interferons are grouped into three classes called type I, II and III IFNs according to their amino acid sequence. Type I IFNs consists of a large group of genes divided into IFNα (13 genes in humans and 14 genes in mice have been found) and one IFNβ gene, which are induced by viral infection. Other members of the type I IFN such as IFNε, IFNτ or IFNω have a less well defined role in other physiological pathways. IFNτ has been shown to be a multifunctional cytokine secreted by the trophoectoderm of the ruminant conceptus that manifests antiviral activity against HIV, FIV and ovine lentiviruses [118-120]. Type II IFN consists of one member called IFNγ, which is secreted by activated CD4⁺ T cells and natural killer cells (NK) rather than through viral infection. Type III IFNs called IFN-λ have been described more recently and are also induced by viral infection and activate the same pathways as IFN-α/β (reviewed here [121]).

Type I IFNs are induced by the activation of different PRRs, which are expressed in the cytoplasm, in the endosome or at the cell surface depending on their PAMP specificity (Figure 1.5). The PRRs use different downstream signaling pathways, which lead to the activation of type I IFN and proinflammatory cytokines. PRRs can be classified by their localization and the PAMP they recognize. Transmembrane PRRs of the Toll like receptor family (TLR) recognize PAMPs in the extracellular

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space (TLR2, 4, 5 and 6) and in the endosome (TLR3, 7, 8, 9). Extracellular PAMPs include mostly products from bacteria, parasites and fungi. TLR4 for example recognizes lipopolysaccharide (LPS) a cell wall component of gram-negative bacteria. TLRs of the endosome are able to detect nucleic acid: TLR 9 binds CpG DNA from viral or bacterial origin, TLR3 binds double stranded RNA (dsRNA) [122], while TLR7 and 8 bind ssRNA from viral origin [123-125].

Intracellular dsRNA is sensed by two helicases: retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene-5 (MDA5) [126]. The presence of DNA in the cytoplasm is usually a sign for viral infection or perceived as a danger signal and sensors for cytoplasmic DNA exist but the nature of these receptors and their ligand is still not well understood (reviewed here [127]). One way to sense cytoplasmic DNA is via transcription of dsDNA into dsRNA via cytoplasmic RNA polymerase III activating type I IFN using the RIG-I pathway [128, 129]. Candidates for dsDNA sensors are DNA-dependent activator of IRF (DAI) [130], DExD/H box protein DDX41 [131] and IFN inducible protein 16 (IFI16) [132]. Recently it has been shown that an enzyme called cyclic GMP-AMP synthase (cGAS) can produce a ribonucleotide moiety upon detection of cytoplasmic DNA activating type I IFNs [133, 134]. Reverse transcribed HIV-1, SIV or MLV cDNA is sensed by cGAS and induces IFNβ [135]. PRRs induce the expression of type I IFNs in a similar way using a variety of cellular signal transducer and adaptor proteins. Upon binding of dsRNA or its synthetic counterpart polyinosinic:polycytidylic acid (poly(I:C)) RIG-I and MDA5 recruit and activate a mitochondrion-associated adaptor called CARD adaptor inducing IFN-β (Cardif)/virus-induced signaling protein (VISA)/mitochondrial antiviral protein (MAVS)/IFN-β promoter stimulator protein 1 (IPS-1). MAVS interacts with tumor necrosis factor (TNF) receptor-associated factor 3 and 6 (TRAF3, 6). The E3 K63 ubiquitin ligase activity of TRAF6 will lead to autoubiquitination recruiting TAK1 binding proteins 2 and 3 (TAB2, 3), which serve as a platform to assemble the signaling cascade complex including transforming growth factor β-activated kinase 1 (TAK1). NF-κB is retained in the cytoplasm via inhibitor of NF-κB (IκB) and TAK1 phosphorylates the kinase of IκB (IKKα/β). IKKα/β then releases NF-κB from IκB leading to the translocation of NF-κB into the nucleus. On the other arm of the pathway TRAF3 binds directly to TRAF family member associated NF-κB activator (TANK), which in turn recruits TANK-binding kinase-1 (TBK-1). TBK-1

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phosphorylates the transcription factor IFN regulatory factor 3 (IRF3) and to some extent also IRF7. Phosphorylation of IRF3 leads to dimerization and exposes a nuclear localization signal (NLS). The IRF3 dimer then translocates into the nucleus where, together with IRF7, NF-κB, CBP/p300 and RNA polymerase, it activates expression of the IFNβ gene and other proinflammatory cytokines. The DNA sensors DDX41, IFI16 and cGAS activate TBK-1 via the ER associated adaptor called stimulator of IFN genes (STING) inducing IFNβ expression via IRF3 [136]. TLR3 and TLR4 signaling activates IRF3 and NF-κB directly via an adaptor called Toll-IL1 resistance (TIR) domain-containing adaptor inducing IFNβ (TRIF) and subsequent TBK-1 activation without the involvement of MAVS. Additionally, TLR3 and TLR4 activated AP-1 via the mitogen-activated protein kinase (MAP) pathway. NF-κB modulated by AP-1 induces the expression of proinflammatory cytokines like tumor necrosis factor α (TNFα), Interleukin 1 (IL1) and IL6 being the most important ones.

TLR7, 8 and 9 on the other hand recruit myeloid differentiation factor 88 (MyD88), which then recruits TRAF3 leading to IRF7 phosphorylation and TRAF6 leading to NF-κB activation via TAK1 inducing IFNβ without the involvement of IRF3.

Type I IFNs act on the secreting cell itself (autocrine) as well as on neighboring cells (paracrine). IFNα/β bind to a heterodimeric receptor composed of IFNAR1 and IFNAR2 gene products (IFNαβ receptors 1 and 2). The cytoplasmic tail of IFNAR1 is associated with tyrosine kinase 2 (Tyk2) and the cytoplasmic tail of IFNAR2 is associated with the tyrosine kinase Janus kinase 1 (JAK1). Upon ligand induced dimerization, Tyk2 phosphorylates IFNAR1, creating a docking site for signal transducer and activation of transcription 2 (STAT2) and a weak association with STAT1. STAT2 is then phosphorylated by Tyk2, while JAK1 phosphorylates STAT1.

That permits the dimerization of STAT1 and STAT2 which are then transported into the nucleus, where this heterodimer recruits IRF-9, forming the heterotrimeric IFN stimulated gene factor 3 (ISGF3). ISGF3 binds to the IFN-stimulated response element (ISRE), which is present in the promoter of most ISGs and initiates their transcription. Several hundred genes are known to be induced by IFN signaling, such as the protein kinase R (PKR), the 2’5’-oligoadenlyte synthetase (OAS)/RNaseL system, Mx proteins and proteins of the APOBEC family and TRIM family (discussed below). PKR uses dsRNA as cofactor and phosphorylates the α subunit of the eukaryotic translation initiation factor 2 (eIF2α), which interrupts mRNA translation.

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The OAS/RNaseL system degrades cellular and viral RNAs. Mx proteins are small dynamin-like GTPases, which exhibit antiviral activities against a wide range of RNA viruses including Influenza [137, 138].

29 1.6.1.2 Innate immune sensing of HIV-1

Although HIV-1 is very sensitive to the action of type I IFNs and its replication can be inhibited in cells in an antiviral state 100 to 1000-fold [139], in patients type I IFNs are not able to control the virus since using it as a treatment failed to give positive results. In fact it may actually be detrimental for the patients and might contribute to progression to AIDS (reviewed here [140]). Our knowledge of how HIV-1 is sensed by the innate immune system and induces IFN or avoids detection is still limited but in recent years the picture became clearer. As most viruses HIV-1 has evolved strategies to avoid detection by the innate immune system and evade antiviral activities (reviewed here [121, 141, 142]). The main cell type producing type I IFNs are plasmacytoid dendritic cells (pDCs). They are the only cell type next to B cells to express TLR7. Via endocytosis pDCs take up HIV-1 particles and the genomic ssRNA is sensed by TLR7 in the endosome [143]. Cell free virus is able to stimulate type I IFN induction but the stimulation is greatly enhanced when pDCs make cell to cell contact with infected CD4⁺ T cells [144]. In the same study it was shown that fusion was not required for TLR7 stimulation with cell free virus in the case of cell to cell contact fusion lead to a greater stimulation of type I IFN. The exact HIV-1 ligand for TLR7 is not known but HIV-1 ssRNA must be delivered to the endosome after fusion but before reverse transcription takes place. Probably cytosolic ssRNA is taken up by the early endosome via autophagy since knockdown of Atg7 in pDCs led to a decrease in type I IFN production after HIV-1 infection [145]. Conventional DCs but not pDCs express TLR8 and HIV-1 infected DCs showed increased replication mediated by both TRL8 signaling and DC-SIGN signaling [146]. One report shows that newly produced capsid in DCs is able to induce type I IFN via IRF3 and cycophilin A (CypA) [147]. CypA has been shown to bind very strongly to HIV-1 CA [148] and is able to promote an early step in the HIV-1 replication in specific cell types [149]. It is not clear how CypA is stimulating type I IFN production but the involvement of an unknown sensor has been postulated [147].

1.6.2 Intrinsic HIV-1 restriction factors