Sensing Infection by Adenovirus: Toll-Like Receptor-Independent Viral DNA Recognition Signals Activation of the Interferon Regulatory Factor 3 Master Regulator

Abstract

Infection with adenovirus vectors (AdV) results in rapid activation of innate immunity, which serves the dual purpose of stimulating inflammatory antiviral host defenses and the adaptive immune system. Viral recognition by macrophages, dendritic cells, and other cell types requires an ability to sense the presence of a foreign molecular pattern by "pattern recognition receptors." The nature of the adenoviral sensor, the target ligand of the sensor, and the downstream antiviral signaling response triggered by virus infection have not been defined for this nonenveloped double-stranded DNA (dsDNA) virus. We have identified four critical links involved in AdV recognition by murine antigen-presenting cells (APC) and primary lung fibroblasts: (i) viral recognition occurs chiefly via a Toll-like receptor (TLR)-independent nucleic acidsensing mechanism recognizing the viral dsDNA genome, (ii) the intact viral particle and capsid proteins are required for efficient intracellular delivery of the viral genome, (iii) delivery of the viral genome triggers interferon regulatory factor 3 (IRF3) phosphorylation, and (iv) IRF3 activation is the required dominant antiviral signaling pathway used by APC, whereas the "primary" involvement of NF-B, mitogen-activated protein kinase, or Akt pathways is less prominent. In this study we provide the first direct evidence that infection by a dsDNA virus stimulates an IRF3-mediated interferon and proinflammatory response through a TLR-independent DNA-sensing mechanism.Adenovirus (Ad) is a nonenveloped, double-stranded, linear DNA virus with a genome of approximately 35 kilobases and a particle size of 70 to 100 nm. The majority of Ad serotypes infect cells through a well-characterized, multistep process that includes high-affinity binding of the capsid protein fiber to the coxsackie-Ad receptor cell surface protein (1) and endocytic internalization stimulated by an RGD motif present in penton base binding to membrane integrins (61). Following internalization and subsequent endosome acidification, Ad escapes the endosomal compartment by directed membrane disruption (62) and transmigrates to the nuclear envelope by way of dynein/microtubule-mediated transport (31). Viral DNA passes through the nuclear pore complex (7) and establishes a noncovalent association with the nuclear matrix as a prerequisite to transcription by the host transcriptional machinery (51).The entire process of virus entry is extremely efficient, which in combination with a well-defined and easily manipulated viral genome prompted development of Ad vectors (AdV) for gene transfer applications. First-generation AdV are deficient for the E1 and E3 regions, rendering them replication deficient and able to carry up to 7 kb of foreign genomic material. Systemic administration of AdV results in high levels of transgene expression but also results in a robust inflammatory immune response (41, 66), which is followed by an equally vigorous adaptive immune response (65). The combination of the innate inflammatory response with the ...

The Role of Capsid Maturation on Adenovirus Priming for Sequential Uncoating

“…DNA accessibility in these conditions results from penton defects and core reorganization but not from core ejection, as all virions observed still retained electron dense contents, and only very rarely were short dsDNA stretches observed protruding from the virions. These properties correlate with the need for the partially disassembled virion to keep protecting its genome while trafficking in the cytosol until arrival to the nuclear pore, while at the same time allowing access to cellular sensors triggering inflammatory responses (15).…”

“…The partially disassembled virion does not proceed to later stages of the endocytic pathway but escapes the early endosome (14). At this point there must be a certain degree of dsDNA exposure to initiate inflammatory responses (15), but the virion is still stable enough to survive transport along microtubules to the nuclear pore (16,17). There, the final phase of uncoating takes place.…”

Interferon-α and interleukin-12 are induced, respectively, by double-stranded DNA and single-stranded RNA in human myeloid dendritic cells

“…As such, the dsDNA virus adenovirus stimulates type I IFN production through a TLR-independent sensing mechanism 35 and dsDNA is thought to be recognized by cytosolic DNA sensors. 15,16 As a result of recent intense research, LRRFIP1 has been proposed as a potent cytosolic sensor of dsDNA that enhances the induction of type I IFN genes via a b-catenin-dependent pathway, 17 but little is known about whether human myeloid DCs functionally express this sensor to provoke an IFN response.…”

NOD2 Signaling Contributes to the Innate Immune Response Against Helper-Dependent Adenovirus Vectors Independently of MyD88 In Vivo

“…TLR/MyD88 signaling is rapidly induced after stimulation with TLR ligands leading to the activation of pro-inflammatory genes and type I interferon (IFN) (Garcia-Sastre and Biron, 2006). Previously, we and others reported that TLR/MyD88 signaling is a major pathway for the induction of innate (Nociari et al, 2007;Yamaguchi et al, 2007) and acquired immune responses to Ad vector (Hartman et al, 2007;Suzuki et al, 2010b). However, our results showed that MyD88 deficiency leads to only a partial reduction in innate cytokine expression in response to HDAds, thereby underscoring the contribution of additional innate sensing pathways (Suzuki et al, 2010b).…”

“…The innate recognition of Ad by plasmacytoid dendritic cells mediated by TLRs depends on MyD88, whereas that by myeloid dendritic cells and macrophages is TLR-independent, possibly via cytosolic sensing of Ad (Nociari et al, 2007;Zhu et al, 2007). TLRs recognize microbes and/or microbial products at the cell surface and in the endosome, whereas nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) and retinoid acid-inducible gene I (RIG-I)-like receptors (RLRs) detect microbial components in the cytosol (Meylan and Tschopp, 2006;Kanneganti et al, 2007).…”