Adenovirus Detection by the cGAS/STING/TBK1 DNA Sensing Cascade

Abstract

T he human serotype 5 adenovirus (Ad5) is a nonenveloped linear double-stranded DNA virus associated with upper respiratory tract disease in humans. It has been extensively studied as a model for virus and host cell interactions. Replication-defective recombinant Ad5 vectors (rAdV) deleted in E1 and E3 coding domains have been characterized in gene therapy, vaccine, and oncolytic vector strategies in the murine model. Although nonpermissive for Ad5 replication, the murine model of rAdV infection provides a valuable resource for characterizing how the innate and adaptive immune response orchestrates an antiviral response to nonenveloped DNA viruses.Virus uptake by immune sentinel cells such as macrophage and dendritic cells is vital to initiating the antiviral immune response. In addition to antigen-presenting cells (APCs), other cell types, including endothelial cells or tissue-specific cells such as hepatocytes, when exposed to virus, also contribute to the host antiviral response. In vitro studies of isolated bone marrow-derived APCs or representative cell lines have revealed a cell-specific antiviral innate response, where activation of the type I interferon (IFN) cascade is a dominant feature (1-4). A valuable marker for early events in the antiviral recognition response is activation of the transcription factor interferon response factor 3 (IRF3). Following infection, cytosolic IRF3 undergoes phosphorylation as a primary response to adenovirus uptake. Activation occurs in a MyD88/TRIF-independent manner; it requires integrin-dependent endosomal entry, escape, and presentation of viral DNA to the cytosolic compartment (3).In rAdV-responsive murine cell lines, the STING/TBK1 cascade is required for IRF3 phosphorylation (5, 6). STING (7,8) functions as an adaptor linking DNA recognition signaling to activation of the TBK1 kinase. TBK1 activation (9) leads to C-terminal IRF3 phosphorylation, dimerization, and translocation to the nucleus (10, 11). In the nucleus, IRF3, in collaboration with additional transcription factors (NF-B and AP1), results in transcriptional activation of IRF3-responsive genes (including IFN-␤) (12). This sequence of events contributes to the primary antiviral response to adenovirus infection. The translation of primary response transcripts such as IFN-␤ leads to autocrine/paracrine secondary signaling. The combination of primary and secondary response functions leads to expression of a complete antiviral response, which is distinct for different cell types.Using various screening protocols, cell lines, and output assays, an extensive list of cytosolic DNA sensors, including DAI, RNA polymerase (Pol) III, IFI16, DDX41, and Aim 2, has been established (reviewed in reference 13). However, the DNA sensor involved in recognizing infection by adenovirus leading to early IRF3 activation has not been convincingly established. The recent identification of cyclic dinucleotide activation of STING (14-18) and the elegant discovery of cyclic-GMP-AMP synthase (cGAS) as a DNA sensor (19,20) provide an...

Membrane Perturbation-Associated Ca 2+ Signaling and Incoming Genome Sensing Are Required for the Host Response to Low-Level Enveloped Virus Particle Entry

“…The cGAS/STING/TBK-1 pathway is capable of sensing cytoplasmic AdV DNA, leading to IRF3 activation (50,51). In our studies, however, infection with 1,000 PFU/ cell of AdV particles failed to elicit a response (data not shown), while infection with 0.02 PFU/cell of HCMV particles efficiently induced a response.…”

“…For example, VLPs and fusogenic liposomes efficiently induce type I IFN and ISGs in human immune cells (22). Similarly, AdV activates the cGAS/STING pathway and elicits IRF3 activation in murine macrophages (50,51), while AdV infection of primary mouse lung fibroblasts leads to type I IFN induction (55). Our studies also highlight the exquisite sensitivity of cells to incoming lowlevel virus particle entry.…”

Antigen expression determines adenoviral vaccine potency independent of IFN and STING signaling

“…Indeed for rAd5 and chAd63, the IFN-driven gene expression profile was abrogated entirely in the absence of STING, leading us to hypothesize that STING acts as a proximal and dominant PRR after vaccination with certain rAds. STING likely senses rAd-derived DNA exposed as the viral particles uncoat in transfected cells (37,38,(41)(42)(43). We speculate that accessibility of rAd-derived DNA to DNA sensors may differ due to different uncoating processes across adenoviral serotypes, leading to differential ISG induction observed in our study.…”

“…This suggests that high and persistent Ag expression by rAd5 and chAd3 may not only result from attenuated IFN induction by these vectors but also from specific and positive regulation vated by cytosolic DNA via the action of DNA sensors, such as cyclic GMP-AMP synthase (37-39), or directly activated by bacterially generated cyclic dinucleotides (39,40) and may detect viral fusion events (41). Recent in vitro studies have shown that STING activates innate pathways after adenoviral infection (42,43) and mediates a key innate signaling pathway for sensing a variety of viruses with dsDNA or RNA genomes (44). Mice were vaccinated with rAd5 or chAd63 at a low dose (3 × 10 7 PU) to focus on innate regulation of CD8 T cell responses in the context of more limiting Ag, as high doses of rAds can mask the influence of innate pathways in mice (18,19).…”

Quantitative Analysis of Virus-associated RNAI Expression following Transduction with a Replication-incompetent Adenovirus Vector In Vitro and In Vivo

“…Although adaptive immune responses following administration with Ad vectors has been relatively well studied [3], the mechanisms underlying Ad-induced innate immune responses have remained to be clarified. Several groups, including ours, have reported that Ad components, including capsid proteins, Ad genome DNA, and transcripts, are recognized by cellular receptors and sensors, leading to the induction of innate immune responses [4][5][6][7][8][9].…”