Recognition of double-stranded RNA by Toll-like receptor 3 (TLR3) will increase the production of cytokines and chemokines through transcriptional activation by the NF-B protein.Over 136 single-nucleotide polymorphisms (SNPs) in TLR3 have been identified in the human population. Of these, four alter the sequence of the TLR3 protein. Molecular modeling suggests that two of the SNPs, N284I and L412F, could affect the packing of the leucine-rich repeating units in TLR3. Notably, L412F is reported to be present in 20% of the population and is higher in the asthmatic population. To examine whether the four SNPs affect TLR3 function, each were cloned and tested for their ability to activate the expression of TLR3-dependent reporter constructs. SNP N284I was nearly completely defective for activating reporter activity, and L412F was reduced in activity. These two SNPs did not obviously affect the level of TLR3 expression or their intracellular location in vesicles. However, N284I and L412F were underrepresented on the cell surface, as determined by flow cytometry analysis, and were not efficiently secreted into the culture medium when expressed as the soluble ectodomain. They were also reduced in their ability to act in a dominant negative fashion on the wild type TLR3 allele. These observations suggest that N284I and L412F affect the activities of TLR3 needed for proper signaling.

Proteins that recognize pathogen-associated molecular patterns are key factors in the cascade of events from the detection to the elimination of an invading organism. This form of innate immunity is conserved in eukaryotes. For example, the Drosophila melanogaster Toll protein is responsible for resistance to fungal and bacterial infections (3, 4), and plants can encode disease-resistance proteins that are important in determining the outcome of infection (5). The vertebrate pathogendetecting proteins called Toll-like receptors (TLRs) 5 are key players in the activation of both the innate and adaptive arms of the immune system (6 -9).The TLRs and related pathogen sensors contain leucine-rich repeat motifs that form docking sites for pathogen ligands or adaptors that bind pathogen ligands, the binding of which will activate signal transduction pathway(s) (10 -12). TLR3 recognizes double-stranded RNA and may be a part of a redundant sensor system to detect viral infections (13-15). Although specific features in the ligands required to interact with TLR3 remain to be identified, TLR3 is activated by polyinosinepolycytidylic acid (poly(I:C)) and has been reported to be activated by RNAs extracted from necrotic cells (16).A number of issues concerning TLR3 structure and function remain to be elucidated. For example, TLR3 can apparently act both on the surface of the plasma membrane, as it does in fibroblasts, and by attaching to the membranes of intracellular vacuoles, where it is proposed to act in immature dendritic cells (17,18). The trafficking of TLRs should be influenced by glycosylation in general, and N-linked glycosylation of TLR2 and TLR4 has been shown to play essential roles in its localization (19,20). A significant recent advance in TLR3 was the elucidation of a 2.1 Å structure of the soluble ectodomain by Choe et al. (1). Bell et al. (2) independently elucidated a highly similar structure. In both studies, the crystallized ectodomains were produced in a baculovirus expression system and formed a horseshoe-shaped solenoid structure that was extensively decorated with glycosyl modifications, some of which were partially resolved in the structure. Whether the glycosylations are important in TLR3 localization and/or function were not directly addressed in these works (1, 2). However, de Bouteiller et al. (21) showed that a change of Asn-247 to an arginine in TLR3 negatively affected TLR3 activity.We expressed the extracellular domain (ECD) of the human TLR3 in human embryonic kidney cells (HEK 293T) and demonstrated that it was modified with N-linked glycosylations. Using the GlcNAc-transferase inhibitor tunicamycin, a concentration-dependent inhibition of TLR3 activity was observed. Systematic mutational analysis of the predicted N-linked glycosylation sites identified two asparagine residues in leucine-rich repeats 8 and 15 that are important for TLR3 activity. The mutant proteins remain expressed at levels similar to wild type. In addition, because our ectodomain was produced in human cells as opp...

BackgroundInterleukin-33 is a member of the IL-1 cytokine family whose functions are mediated and modulated by the ST2 receptor. IL-33-ST2 expression and interactions have been explored in mouse macrophages but little is known about the effect of IL-33 on human macrophages. The expression of ST2 transcript and protein levels, and IL-33-mediated effects on M1 (i.e. classical activation) and M2 (i.e. alternative activation) chemokine marker expression in human bone marrow-derived macrophages were examined.ResultsHuman macrophages constitutively expressed the membrane-associated (i.e. ST2L) and the soluble (i.e. sST2) ST2 receptors. M2 (IL-4 + IL-13) skewing stimuli markedly increased the expression of ST2L, but neither polarizing cytokine treatment promoted the release of sST2 from these cells. When added to naïve macrophages alone, IL-33 directly enhanced the expression of CCL3. In combination with LPS, IL-33 blocked the expression of the M2 chemokine marker CCL18, but did not alter CCL3 expression in these naive cells. The addition of IL-33 to M1 macrophages markedly increased the expression of CCL18 above that detected in untreated M1 macrophages. Similarly, alternatively activated human macrophages treated with IL-33 exhibited enhanced expression of CCL18 and the M2 marker mannose receptor above that detected in M2 macrophages alone.ConclusionsTogether, these data suggest that primary responses to IL-33 in bone marrow derived human macrophages favors M1 chemokine generation while its addition to polarized human macrophages promotes or amplifies M2 chemokine expression.

Modified nucleic acids, also called xeno nucleic acids (XNAs), offer a variety of advantages for biotechnological applications and address some of the limitations of first-generation nucleic acid therapeutics. Indeed, several therapeutics based on modified nucleic acids have recently been approved and many more are under clinical evaluation. XNAs can provide increased biostability and furthermore are now increasingly amenable to in vitro evolution, accelerating lead discovery. Here, we review the most recent discoveries in this dynamic field with a focus on progress in the enzymatic replication and functional exploration of XNAs.

Virology 132:26-37, 1984). Typically, these resistance mutations map to the thymidine kinase (TK) gene and render the virus TK deficient. To examine this process more closely, a plating efficiency assay was used to determine whether the frequencies of naturally occurring mutations in populations of the laboratory strains HSV-1 SC16, HSV-2 SB5, and HSV-2 333 grown in MRC-5 cells were similar when scored for resistance to penciclovir (PCV) and ACV. Our results indicate that (i) HSV mutants resistant to PCV and those resistant to ACV accumulate at approximately equal frequencies during replication in cell culture, (ii) the spontaneous mutation frequency for the HSV-1 strain SC16 is similar to that previously reported for HSV-1 laboratory strains KOS and Cl101, and (iii) spontaneous mutations in the laboratory HSV-2 strains examined were 9-to 16-fold more frequent than those in the HSV-1 strain SC16. These observations were confirmed and extended for a group of eight clinical isolates in which the HSV-2 mutation frequency was approximately 30 times higher than that for HSV-1 isolates. In conclusion, our results indicate that the frequencies of naturally occurring, or spontaneous, HSV mutants resistant to PCV and those resistant to ACV are similar. However, HSV-2 strains may have a greater propensity to generate drug-resistant mutants than do HSV-1 strains.The antiviral drug standard for the treatment of herpes simplex virus (HSV) infections including herpes labialis and genital herpes for almost 2 decades has been acyclovir (ACV) [9-(2-hydroxyethoxymethyl)guanine]. However, with the more recent introduction of penciclovir (PCV) (BRL 39123) [9-(4-hydroxy-3-hydroxymethylbut-1-yl)guanine] and its oral prodrug, famciclovir, the usage of antivirals alternative to ACV for the management of herpesvirus infections has also increased. Identical activation pathways and similar modes of action suggest that the mechanisms of HSV resistance to PCV and ACV are likely to be analogous (2, 40). An assumption that the frequency with which resistance in HSV arises is identical for PCV and ACV can be based on the biochemical similarities of the two compounds and the cross-resistance of thymidine kinase (TK)-negative mutants; however, direct genetic evidence is not available.A low level of replication errors is typically associated with DNA synthesis (10, 33). Resistance to PCV or ACV can arise by a single base mutation in the DNA encoding the HSV TK protein which activates the antiviral agent (6, 23, 29). These spontaneous mutations occur during DNA replication and are independent of the presence of antiviral drug (16). These errors, or random mutations, provide genetic diversity to facilitate the adaptation and evolution of an organism (15). Data from a study of the molecular evolution of HSV type 1 (HSV-1) show that its evolution is slow; the mutation rate was estimated to be 3.5 ϫ 10 Ϫ8 substitutions per site per year (36). Mispaired deoxyribonucleoside triphosphates are often removed by the HSV polymerase (Pol) through its associat...

The structure of the human Toll-like receptor 3 (TLR3) ectodomain (ECD) was recently solved by x-ray crystallography, leading to a number of models concerning TLR3 function ( The structure revealed four pairs of cysteines that are putatively involved in disulfide bond formation, several residues that are predicted to be involved in dimerization between ECD subunits, and surfaces that could bind to poly(I:C). In addition, there are two loops that protrude from the central solenoid structure of the protein. We examined the recombinant TLR3 ECD for disulfide bond formation, poly(I:C) binding, and protein-protein interaction. We also made over 80 mutations in the residues that could affect these features in the full-length TLR3 and examined their effects in TLR3-mediated NF-B activation. A number of mutations that affected TLR3 activity also affected the ability to act as dominant negative inhibitors of wild type TLR3. Loss of putative RNA binding did not necessarily affect dominant negative activity. All of the results support a model where a dimer of TLR3 is the form that binds RNA and activates signal transduction.

Toll-like receptor 3 (TLR3) can signal the production of a suite of cytokines and chemokines in response to double-stranded RNA (dsRNA) ligands or the dsRNA mimic poly(I-C). Using a human embryonic kidney 293T cell line to express human TLR3, we determined that poly(I-C)-induced signal could be significantly inhibited by single-stranded DNAs (ssDNAs), but not ssRNA or dsDNA. The ssDNA molecules that down-modulated TLR3 signaling did not affect TLR4 and do not require the hypomethylated CpG motif found in TLR9 ligands. The degree of modulation can be altered by the length, base sequence, and modification state of the ssDNAs. An inhibitory ssDNA was found to colocalize with TLR3 in transfected cells and in a cell line that naturally expresses TLR3. The inhibitory ssDNAs can compete efficiently with dsRNA for binding purified TLR3 ectodomains in vitro, while noninhibitory nucleic acids do not. The ssDNAs also decrease the levels of several cytokines produced by the human bronchial epithelial cell line BEAS-2B and by human peripheral blood mononuclear cells in response to poly(I-C) stimulation of native TLR3. These activities indicate that ssDNAs could be used to regulate the inflammatory response through TLR3.

To gain global pathway perspective of ex vivo viral infection models using human peripheral blood mononuclear cells (PBMCs), we conducted expression analysis on PBMCs of healthy donors. RNA samples were collected at 3 and 24 h after PBMCs were challenged with the Toll-like receptor-3 (TLR3) agonist polyinosinic acid-polycytidylic acid [poly(I:C)] and analyzed by internally developed cDNA microarrays and TaqMan PCR. Our results demonstrate that poly(I:C) challenge can elicit certain gene expression changes, similar to acute viral infection. Hierarchical clustering revealed distinct immediate early, early-to-late, and late gene regulation patterns. The early responses were innate immune responses that involve TLR3, the NF-kappaB-dependent pathway, and the IFN-stimulated pathway, whereas the late responses were mostly cell-mediated immune response that involve activation of cell adhesion, cell mobility, and phagocytosis. Overall, our results expanded the utilities of this ex vivo model, which could be used to screen molecules that can modulate viral stress-induced inflammation, in particular those mediated via TLRs.