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Orthomyxo- and bunyaviruses steal the 5' cap portion of host RNAs to prime their own transcription in a process called "cap snatching." We report that RNA modification of the cap portion by host 2'-O-ribose methyltransferase 1 (MTr1) is essential for the initiation of influenza A and B virus replication, but not for other cap-snatching viruses. We identified with in silico compound screening and functional analysis a derivative of a natural product from Streptomyces , called trifluoromethyl-tubercidin (TFMT), that inhibits MTr1 through interaction at its S -adenosyl-l-methionine binding pocket to restrict influenza virus replication. Mechanistically, TFMT impairs the association of host cap RNAs with the viral polymerase basic protein 2 subunit in human lung explants and in vivo in mice. TFMT acts synergistically with approved anti-influenza drugs.

Binding of dsDNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) triggers formation of the metazoan second messenger c[G(2',5')pA(3',5')p], which binds the signaling protein STING with subsequent activation of the interferon (IFN) pathway. We show that human hSTING(H232) adopts a "closed" conformation upon binding c[G(2',5')pA(3',5')p] and its linkage isomer c[G(2',5')pA(2',5')p], as does mouse mSting(R231) on binding c[G(2',5')pA(3',5')p], c[G(3',5')pA(3',5')p] and the antiviral agent DMXAA, leading to similar "closed" conformations. Comparing hSTING to mSting, 2',5'-linkage-containing cGAMP isomers were more specific triggers of the IFN pathway compared to the all-3',5'-linkage isomer. Guided by structural information, we identified a unique point mutation (S162A) placed within the cyclic-dinucleotide-binding site of hSTING that rendered it sensitive to the otherwise mouse-specific drug DMXAA, a conclusion validated by binding studies. Our structural and functional analysis highlights the unexpected versatility of STING in the recognition of natural and synthetic ligands within a small-molecule pocket created by the dimerization of STING., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Background: Upper respiratory tract infection is a frequent cause of morbidity worldwide. Although the course of infection is usually mild, it is responsible for enormous social and economic costs. Immunostimulation with bacterial extracts consisting of ribosomal RNA and proteoglycans, such as Ribomunyl, was introduced into the clinic in the 1980s as a new treatment concept, but did not achieve widespread application. Ribomunyl has been proposed to activate innate immunity, but the contribution of its RNA content as well as its antiviral potential has not been studied., Methods: Peripheral blood mononuclear cells from healthy donors and immune cells from adenoids were incubated with Ribomunyl either by itself or formulated in a complex with cationic polypeptides such as poly-l-arginine or protamine, and induction of cytokines was quantified by ELISA., Results: Ribomunyl in complex with either poly-l-arginine or protamine, but not on its own, was able to strongly induce interferon-α (P<0.01) and interleukin-12 (P<0.01) in peripheral blood mononuclear cells, whereas induced tumour necrosis factor-α and interleukin-6 levels were independent of the formulation. Comparable results were obtained in immune cells from adenoids, suggesting efficacy also in virus-affected tissue. Cell sorting, RNase digests and selective receptor expression show that the RNA in Ribomunyl acts as an agonist of Toll-like receptor (TLR)7 and TLR8., Conclusions: Ribomunyl is, in principle, able to potently induce antiviral interferon-α and interleukin-12 via TLR7 and TLR8, respectively, but only when formulated in a complex with cationic polypeptides.

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. [ABSTRACT FROM AUTHOR]

Intracellular RIG-I receptors represent key innate sensors of RNA virus infection, and RIG-I activation results in the induction of hundreds of host effector genes, including interferon-stimulated genes (ISGs). Synthetic RNA agonists targeting RIG-I have shown promise as antivirals against a broad spectrum of viruses, including influenza A virus (IAV), in both in vitro and mouse models of infection. Herein, we demonstrate that treatment of a ferret airway epithelial (FRL) cell line with a RIG-I agonist rapidly and potently induced expression of a broad range of ISGs and resulted in potent inhibition of growth of different IAV strains. In ferrets, a single intravenous injection of RIG-I agonist was associated with upregulated ISG expression in peripheral blood mononuclear cells and lung tissue, but not in nasal tissues. In a ferret model of viral contact transmission, a single treatment of recipient animals 24 h prior to cohousing with IAV-infected donors did not reduce virus transmission and shedding but did result in reduced lung virus titers 6 days after treatment. A single treatment of the IAV-infected donor animals also resulted in reduced virus titers in the lungs 2 days later. Thus, a single intravenous treatment with RIG-I agonist prior to infection or to ferrets with an established IAV infection can reduce virus growth in the lungs. These findings support further development of RIG-I agonists as effective antiviral treatments to limit the impact of IAV infections, particularly in reducing virus replication in the lower airways. [ABSTRACT FROM AUTHOR]

Summary: Immune sensing of DNA is critical for antiviral immunity but can also trigger autoimmune diseases such as lupus erythematosus (LE). Here we have provided evidence for the involvement of a damage-associated DNA modification in the detection of cytosolic DNA. The oxidized base 8-hydroxyguanosine (8-OHG), a marker of oxidative damage in DNA, potentiated cytosolic immune recognition by decreasing its susceptibility to 3′ repair exonuclease 1 (TREX1)-mediated degradation. Oxidizative modifications arose physiologically in pathogen DNA during lysosomal reactive oxygen species (ROS) exposure, as well as in neutrophil extracellular trap (NET) DNA during the oxidative burst. 8-OHG was also abundant in UV-exposed skin lesions of LE patients and colocalized with type I interferon (IFN). Injection of oxidized DNA in the skin of lupus-prone mice induced lesions that closely matched respective lesions in patients. Thus, oxidized DNA represents a prototypic damage-associated molecular pattern (DAMP) with important implications for infection, sterile inflammation, and autoimmunity. [ABSTRACT FROM AUTHOR]