Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA

Abstract

Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon–inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.

Cytosolic sensing of immuno-stimulatory DNA, the enemy within

“…DNA binding induces a conformational change in the active site of the enzyme, which relieves auto-inhibition and allows for an atypical phosphodiester bond to be formed between the 2′ hydroxyl group of GMP and the 5′ phosphate of AMP, which is followed by cyclization to form an additional canonical 3′–5′ linkage. Short DNA fragments of 15–20 base pairs bind efficiently to cGAS and induce its activity in vitro [8]; however, longer DNA elements are required to fully activate cGAS, particularly in cells [9]. A cooperative mechanism by which longer DNA elements are recognized by a network of cGAS dimers has been recently described [10].…”

Cellular and molecular regulation of innate inflammatory responses

“…cGAS, which was previously thought to recognize cytoplasmic dsDNA over 40 bp in a sequence-independent manner, is recently shown to recognize unpaired guanosines flanking short (12-20 bp) dsDNA (Y-form DNA) found in human immunodeficiency virus type 1 to induce type I IFN production. 25,26 In neutrophils, transcription factor Sox2 could directly recognize microbial DNA through its high-mobility-group domain to activate innate immunity against microbial infection. [27][28][29] NLRs consist of a large group of intracellular PRRs that includes NODs (nucleotide-binding oligomerization domains), NLRPs (LRR-and pyrin-domain (PYD)-containing protein), CIITA (Class II, major histocompatibility complex, transactivator), IPAF (ICE protease-activating factor) and NAIPs (neuronal apoptosis inhibitory protein), which vary in the effector domain they use to transduce downstream signals.…”

Advanced Design of Dumbbell-shaped Genetic Minimal Vectors Improves Non-coding and Coding RNA Expression

“…This pathway is also activated by unpaired guanosines in Y-shaped or hairpin loop structures but all non-transcribed standard loops of our dumbbells were chosen to be (T) 4 tetra loops. 51 Notably, most of the DNA sensing pathways are cell-type dependent and we would not expect strong immune sensing in activated human T cells or the tissue culture cell lines we used for our …”