Cutting Edge: Antibody-mediated TLR7-dependent recognition of viral RNA

TLR7 recognizes the genome of ssRNA viruses such as Coxsackievirus B. Because TLR7 is expressed in intracellular compartments, viral RNA must be internalized before its recognition by TLR7. In this study, we define plasmacytoid dendritic cells (pDC) as peripheral blood mononuclear immune cells that respond to Coxsackievirus. pDC activation by Coxsackievirus B requires the presence of specific antiviral Abs. We show that Fc receptors mediate the recognition of virus-Ab complexes and that TLR7 is required for human and murine pDC production of cytokines. These data define a pathway by which intracellular TLR7 senses viral RNA and indicate a role for TLRs in association with Abs in sustaining virus-specific responses.     

Dual or triple activation of TLR7, TLR8, and/or TLR9 by single-stranded oligoribonucleotides

The toll-like receptors (TLRs) 7, 8, and 9 stimulate innate immune responses upon recognizing pathogen nucleic acids. Certain GU- or AU-rich RNA sequences were described to differentiate between human TLR7- and TLR8-mediated immune effects. Those single-stranded RNA molecules require endosomal delivery for stabilization against ribonucleases. We have discovered RNA sequences that preferentially activate TLR7, form higher ordered structures, and do not require specific cellular delivery. In addition, a dual activation of TLR8 and TLR9 without affecting TLR7 can be achieved by chimeric molecules consisting of GU-rich RNA and Cytosin (C) phosphordiester or phosphorthioat (p) guanine (CpG) motif DNA sequences. Such chimeras stimulate TLR9-mediated type I interferon (IFN) and TLR8-depending proinflammatory cytokine and chemokine production upon primary human cell activation. However, an RNA-dependent TLR7 IFN-α cytokine release is suppressed by the phosphorothioate DNA sequence contained in the chimeric molecule. To convert the immune response of a single-stranded RNA from TLR7/8 to TLR9, a simple chemical modification at the 5' end proves to be sufficient. Such 8-oxo-2'-deoxy-guanosine or 8-bromo-2'-deoxy-guanosine modifications of the first guanosine in GU-rich single-stranded RNAs convert the immune response to include TLR9 activation and demonstrate strong additive effects for type I IFN immune responses in human primary cells.     

Inosine-Mediated Modulation of RNA Sensing by Toll-Like Receptor 7 (TLR7) and TLR8

RNA-specific adenosine deaminase (ADAR)-mediated adenosine-to-inosine (A-to-I) editing is a critical arm of the antiviral response. However, mechanistic insights into how A-to-I RNA editing affects viral infection are lacking. We posited that inosine incorporation into RNA facilitates sensing of nonself RNA by innate immune sensors and accordingly investigated the impact of inosine-modified RNA on Toll-like receptor 7 and 8 (TLR7/8) sensing. Inosine incorporation into synthetic single-stranded RNA (ssRNA) potentiated tumor necrosis factor alpha (TNF-α) or alpha interferon (IFN-α) production in human peripheral blood mononuclear cells (PBMCs) in a sequence-dependent manner, indicative of TLR7/8 recruitment. The effect of inosine incorporation on TLR7/8 sensing was restricted to immunostimulatory ssRNAs and was not seen with inosine-containing short double-stranded RNAs or with a deoxy-inosine-modified ssRNA. Inosine-mediated increase of self-secondary structure of an ssRNA resulted in potentiated IFN-α production in human PBMCs through TLR7 recruitment, as established through the use of a TLR7 antagonist and Tlr7-deficient cells. There was a correlation between hyperediting of influenza A viral ssRNA and its ability to stimulate TNF-α, independent of 5′-triphosphate residues, and involving Adar-1. Furthermore, A-to-I editing of viral ssRNA directly enhanced mouse Tlr7 sensing, when present in proportions reproducing biologically relevant levels of RNA editing. Thus, we demonstrate for the first time that inosine incorporation into immunostimulatory ssRNA can potentiate TLR7/8 activation. Our results suggest a novel function of A-to-I RNA editing, which is to facilitate TLR7/8 sensing of phagocytosed viral RNA.     

TLR7 is involved in sequence-specific sensing of single-stranded RNAs in human macrophages

Human TLR7 and 8 (hTLR7/8) have been implicated in the sequence-dependent detection of RNA oligonucleotides in immune cells. Although hTLR7 sequence-specific sensing of short RNAs has been inferred from studies of murine TLR7, this has yet to be established for hTLR7. We found that different short ssRNA sequences selectively induced either TNF-alpha or IFN-alpha in human PBMCs. The sequence-specific TNF-alpha response to ssRNAs observed in PBMCs could be replicated in activated human macrophage-like (THP-1) cells pretreated with IFN-gamma. Surprisingly, suppression of hTLR7 expression by RNA interference in this model reduced sensing of all immunostimulatory ssRNAs tested. Modulation of the relative expression ratio of hTLR7 to hTLR8 in THP-1 cells correlated with differential sensing of immunostimulatory sequences. Furthermore, the sequence-specific IFN-alpha induction profile in human PBMCs was accurately modeled by a sequence-specific activation of murine TLR7 in mouse macrophages. Thus, we demonstrate for the first time that hTLR7 is involved in sequence-specific sensing of ssRNAs. We establish a novel cell model for the prediction of TNF-alpha induction by short RNAs in human macrophages. Our results suggest that differential sequence-specific sensing of RNA oligonucleotides between human and mouse macrophages is due to the modulation of TLR7 sensing by human TLR8.     

Activation of Autoreactive B Cells by Endogenous TLR7 and TLR3 RNA Ligands

The key step in the activation of autoreactive B cells is the internalization of nucleic acid containing ligands and delivery of these ligands to the Toll-like Receptor (TLR) containing endolysosomal compartment. Ribonucleoproteins represent a large fraction of autoantigens in systemic autoimmune diseases. Here we demonstrate that many uridine-rich mammalian RNA sequences associated with common autoantigens effectively activate autoreactive B cells. Priming with type I IFN increased the magnitude of activation, and the range of which RNAs were stimulatory. A subset of RNAs that contain a high degree of self-complementarity also activated B cells through TLR3. For the RNA sequences that activated predominantly through TLR7, the activation is proportional to uridine-content, and more precisely defined by the frequency of specific uridine-containing motifs. These results identify parameters that define specific mammalian RNAs as ligands for TLRs.     

LRV1 viral particles in Leishmania guyanensis contain double-stranded or single-stranded RNA.

The 32-nm-diameter spherical viral particles found in the cytoplasm of Leishmania guyanensis CUMC1-1A sediment at 130S and have a buoyant density of approximately 1.4 g/ml in cesium chloride gradients. These particles contain a 5.3-kb double-stranded RNA, while single-stranded RNA that corresponds to the viral positive strand is associated with less-dense particles. These results suggest a conservative and sequential mode of LRV1 viral RNA replication that is exemplified by the ScV L-A virus of yeast.     

Bioinformatic analysis and identification of single-stranded RNA sequences recognized by TLR7/8 in the SARS-CoV-2, SARS-CoV,and MERS-CoV genomes

During virus infection, host toll-like receptors (TLRs) can recognize different pathogen-associated molecular patterns and trigger the innate immune response. TLR7/8 can identify the single-stranded RNA (ssRNA) of the virus. This study aimed to search ssRNA sequences recognized by TLR7/8 from the SARS-CoV-2, SARS-CoV, and MERS-CoV whole genomes by a bioinformatic technique. The immunoinformatic approach showed that the SARS-CoV-2 genome has more ssRNA fragments that could be recognized by TLR7/8 than the SARS-CoV genome. These findings suggest innate immune hyperactivation by SARS-CoV-2. This activity is possibly able to provoke a robust proinflammatory response via TLR7/8 recognition and cause acute lung injury.     

The spinocerebellar ataxia 8 noncoding RNA causes neurodegeneration and associates with staufen in Drosophila

Spinocerebellar Ataxia 8 (SCA8) appears unique among triplet repeat expansion-induced neurodegenerative diseases because the predicted gene product is a noncoding RNA. Little is currently known about the normal function of SCA8 in neuronal survival or how repeat expansion contributes to neurodegeneration. To investigate the molecular context in which SCA8 operates, we have expressed the human SCA8 noncoding RNA in Drosophila. SCA8 induces late-onset, progressive neurodegeneration in the Drosophila retina. Using this neurodegenerative phenotype as a sensitized background for a genetic modifier screen, we have identified mutations in four genes: staufen, muscle-blind, split ends, and CG3249. All four encode neuronally expressed RNA binding proteins conserved in Drosophila and humans. Although expression of both wild-type and repeat-expanded SCA8 induce neurodegeneration, the strength of interaction with certain modifiers differs between the two SCA8 backgrounds, suggesting that CUG expansions alter associations with specific RNA binding proteins. Our demonstration that SCA8 can recruit Staufen and that the interaction domain maps to the portion of the SCA8 RNA that undergoes repeat expansion in the human disease suggests a specific mechanism for SCA8 function and disease. Genetic modifiers identified in our SCA8-based screens may provide candidates for designing therapeutic interventions to treat this disease.     

HIV envelope antibodies and TLR7 agonist partially prevent viral rebound in chronically SHIV-infected monkeys

A key challenge for the development of a cure to HIV-1 infection is the persistent viral reservoir established during early infection. Previous studies using Toll-like receptor 7 (TLR7) agonists and broadly neutralizing antibodies (bNAbs) have shown delay or prevention of viral rebound following antiretroviral therapy (ART) discontinuation in simian-human immunodeficiency virus (SHIV)-infected rhesus macaques. In these prior studies, ART was initiated early during acute infection, which limited the size and diversity of the viral reservoir. Here we evaluated in SHIV-infected rhesus macaques that did not initiate ART until 1 year into chronic infection whether the TLR7 agonist vesatolimod in combination with the bNAb PGT121, formatted either as a human IgG1, an effector enhanced IgG1, or an anti-CD3 bispecific antibody, would delay or prevent viral rebound following ART discontinuation. We found that all 3 antibody formats in combination with vesatolimod were able to prevent viral rebound following ART discontinuation in a subset of animals. These data indicate that a TLR7 agonist combined with antibodies may be a promising strategy to achieve long-term ART-free HIV remission in humans.     

Transfection of the Giardia lamblia double-stranded RNA virus into giardia lamblia by electroporation of a single-stranded RNA copy of the viral genome.

The development of a genetic vector for protozoan parasites is a major hurdle yet to be crossed in the study of the molecular and cellular biology of these parasites. We have identified and isolated a double-stranded RNA virus (G. lamblia virus [GLV]) from certain strains of the intestinal parasitic protozoan Giardia lamblia (A. L. Wang and C. C. Wang, Mol. Biochem. Parasitol. 21:269-276, 1986), which is capable of infecting other virus-free strains of G. lamblia (R. L. Miller, A. L. Wang, and C. C. Wang, Exp. Parasitol. 66:118-123, 1988). Here we demonstrate that G. lamblia can be infected with GLV by electroporating uninfected cells with purified single-stranded RNA (E. S. Furfine, T. C. White, A. L. Wang, and C. C. Wang, Nucleic Acids Res. 17:7453-7467, 1989) representing a full-length copy of one strand of the GLV double-stranded RNA genome. To the best of our knowledge, this is the first demonstration in vivo that a single-stranded RNA is a competent replicative intermediate for this class of double-stranded RNA virus. In addition, this result represents the first long-term transfection of a protozoan by a single species of RNA and will hopefully expedite the development of GLV as a genetic transfecting vector.     

ICOS-induced B7h shedding on B cells is inhibited by TLR7/8 and TLR9

We report in this study that B7h, the ligand for the ICOS costimulatory receptor, is rapidly shed from mouse B cells following either ICOS binding or BCR engagement. Shedding occurs through proteolytic cleavage that releases the extracellular ICOS-binding region of B7h. Prior exposure of B7h-expressing APCs to ICOS-expressing cells inhibits their subsequent ability to costimulate IFN-gamma and IL-4 production from CD4+ T cells. Shedding is regulated as TLR7/8 and TLR9 ligands inhibit B7h shedding. A shedding-resistant B7h mutant elicits greater costimulation of IFN-gamma production from CD4+ T cells than does wild-type B7h. These data define shedding of B7h as a novel mechanism for controlling costimulatory signaling by B7-CD28 family members that is regulated on B cells by TLR signaling.     

Mapping of herpesvirus saimiri proteins on the viral genome: proteins dependent and not dependent on viral DNA synthesis.

Hybrid selected translation was used to map the genome of herpesvirus saimiri, a lymphotropic and oncogenic herpesvirus. RNA extracted from virus-infected cells was hybridized to cloned genomic fragments, and the hybrid selected mRNAs were translated in vitro in a rabbit reticulocyte lysate. Forty-five virus-induced polypeptides were identified and correlated to their coding regions on the herpesvirus saimiri genome. Inhibition of the replication of viral DNA with phosphonoacetic acid showed that 22 of these polypeptides belong to the early group of herpesvirus saimiri gene products.     

A comparative study of human TLR 7/8 stimulatory trimer compositions in influenza A viral genomes

Background

Variation in the genomes of single-stranded RNA viruses affects their infectivity and pathogenicity in two ways. First, viral genome sequence variations lead to changes in viral protein sequences and activities. Second, viral genome sequence variation produces diversity at the level of nucleotide composition and diversity in the interactions between viral RNAs and host toll-like receptors (TLRs). A viral genome-typing method based on this type of diversity has not yet been established.

Methodology/Principal Findings

In this study, we propose a novel genomic trait called the “TLR stimulatory trimer composition” (TSTC) and two quantitative indicators, Score S and Score N, named “TLR stimulatory scores” (TSS). Using the complete genome sequences of 10,994 influenza A viruses (IAV) and 251 influenza B viruses, we show that TSTC analysis reveals the diversity of Score S and Score N among the IAVs isolated from various hosts. In addition, we show that low values of Score S are correlated with high pathogenicity and pandemic potential in IAVs. Finally, we use Score S and Score N to construct a logistic regression model to recognize IAV strains that are highly pathogenic or have high pandemic potential.

Conclusions/Significance

Results from the TSTC analysis indicate that there are large differences between human and avian IAV genomes (except for segment 3), as illustrated by Score S. Moreover, segments 1, 2, 3 and 4 may be major determinants of the stimulatory activity exerted on human TLRs 7 and 8. We also find that a low Score S value is associated with high pathogenicity and pandemic potential in IAV. The π value from the TSS-derived logistic regression model is useful for recognizing emerging IAVs that have high pathogenicity and pandemic potential.     

Sequence-specific potent induction of IFN-alpha by short interfering RNA in plasmacytoid dendritic cells through TLR7

Short interfering RNA (siRNA) is used in RNA interference technology to avoid non-target-related induction of type I interferon (IFN) typical for long double-stranded RNA. Here we show that in plasmacytoid dendritic cells (PDC), an immune cell subset specialized in the detection of viral nucleic acids and production of type I IFN, some siRNA sequences, independent of their GU content, are potent stimuli of IFN-alpha production. Localization of the immunostimulatory motif on the sense strand of a potent IFN-alpha-inducing siRNA allowed dissection of immunostimulation and target silencing. Injection into mice of immunostimulatory siRNA, when complexed with cationic liposomes, induced systemic immune responses in the same range as the TLR9 ligand CpG, including IFN-alpha in serum and activation of T cells and dendritic cells in spleen. Immunostimulation by siRNA was absent in TLR7-deficient mice. Thus sequence-specific TLR7-dependent immune recognition in PDC needs to be considered as an additional biological activity of siRNA, which then should be termed immunostimulatory RNA (isRNA).     

Antigen-specific T-cell responses to a recombinant fowlpox virus are dependent on MyD88 and interleukin-18 and independent of Toll-like receptor 7 (TLR7)- and TLR9-mediated innate immune recognition

Fowlpox virus (FWPV) is a double-stranded DNA virus long used as a live-attenuated vaccine against poultry diseases, but more recent interest has focused on its use as a mammalian vaccine vector. Here, in a mouse model system using FWPV encoding the nominal target antigen chicken ovalbumin (OVA) (FWPV(OVA)), we describe for the first time some of the fundamental processes by which FWPV engages both the innate and adaptive immune systems. We show that Toll-like receptor 7 (TLR7) and TLR9 are important for type I interferon secretion by dendritic cells, while TLR9 is solely required for proinflammatory cytokine secretion. Despite this functional role for TLR7 and TLR9 in vitro, only the adapter protein myeloid differentiation primary response gene 88 (MyD88) was shown to be essential for the formation of adaptive immunity to FWPV(OVA) in vivo. The dependence on MyD88 was confined only to the T-cell compartment and was not related to its contribution to TLR signaling, dendritic cell maturation, or the capture and presentation of FWPV-derived OVA antigen. We demonstrate that this is not by means of mediating T-cell-dependent interleukin-1 (IL-1) signaling, but rather, we suggest that MyD88 functions to support T-cell-specific IL-18 receptor signaling, which in turn is essential for the formation of adaptive immunity to FWPV-encoded OVA.     

The mRNA interferases, MazF-mt3 and MazF-mt7 from Mycobacterium tuberculosis target unique pentad sequences in single-stranded RNA

mRNA interferases are sequence-specific endoribonucleases encoded by toxin-antitoxin (TA) systems in bacterial genomes. Previously, we demonstrated that Mycobacterium tuberculosis contains at least seven genes encoding MazF homologues (MazF-mt1 to -mt7) and determined cleavage specificities for MazF-mt1 and MazF-mt6. Here we have developed a new general method for the determination of recognition sequences longer than three bases for mRNA interferases with the use of phage MS2 RNA as a substrate and CspA, an RNA chaperone, which prevents the formation of secondary structures in the RNA substrate. Using this method, we determined that MazF-mt3 cleaves RNA at UU˘CCU or CU˘CCU and MazF-mt7 at U˘CGCU (˘indicates the cleavage site). As pentad sequence recognition is more specific than those of previously characterized mRNA interferases, bioinformatics analysis was carried out to identify M. tuberculosis mRNAs that may be resistant to MazF-mt3 and MazF-mt7 cleavage. The pentad sequence was found to be significantly underrepresented in several genes, including members of the PE and PPE families, large families of proteins that play a role in tuberculosis immunity and pathogenesis. These data suggest that MazF-mt3 and MazF-mt7 or other mRNA interferases that target longer RNA sequences may alter protein expression through differential mRNA degradation, a regulatory mechanism that may allow adaptation to environmental conditions, including those encountered by pathogens such as M. tuberculosis during infection.