Viral Double-Strand RNA-Binding Proteins Can Enhance Innate Immune Signaling by Toll-Like Receptor 3

Abstract

Toll-like Receptor 3 (TLR3) detects double-stranded (ds) RNAs to activate innate immune responses. While poly(I:C) is an excellent agonist for TLR3 in several cell lines and in human peripheral blood mononuclear cells, viral dsRNAs tend to be poor agonists, leading to the hypothesis that additional factor(s) are likely required to allow TLR3 to respond to viral dsRNAs. TLR3 signaling was examined in a lung epithelial cell line by quantifying cytokine production and in human embryonic kidney cells by quantifying luciferase reporter levels. Recombinant 1b hepatitis C virus polymerase was found to enhance TLR3 signaling in the lung epithelial BEAS-2B cells when added to the media along with either poly(I:C) or viral dsRNAs. The polymerase from the genotype 2a JFH-1 HCV was a poor enhancer of TLR3 signaling until it was mutated to favor a conformation that could bind better to a partially duplexed RNA. The 1b polymerase also co-localizes with TLR3 in endosomes. RNA-binding capsid proteins (CPs) from two positive-strand RNA viruses and the hepadenavirus hepatitis B virus (HBV) were also potent enhancers of TLR3 signaling by poly(I:C) or viral dsRNAs. A truncated version of the HBV CP that lacked an arginine-rich RNA-binding domain was unable to enhance TLR3 signaling. These results demonstrate that several viral RNA-binding proteins can enhance the dsRNA-dependent innate immune response initiated by TLR3.

LL-37 Peptide Enhancement of Signal Transduction by Toll-like Receptor 3 Is Regulated by pH

“…Cells and Reagents-The BEAS-2B cell line was from the American Type Culture Collection and cultured in BEGM media containing supplements (11,59). Proteasome inhibitors MG132 and lactacystin (both from Calbiochem) were dissolved in ethanol and DMSO, respectively.…”

“…Poly(I:C) and lipopolysaccharide (LPS) were from Invivogen. Reovirus dsRNA S4 was prepared by in vitro transcription as described in Lai et al (11). All peptides, including ones with covalently attached fluorophores, were custom-synthesized (AnaSpec) and purified to Ͼ95% purity.…”

Proteolytic Processing Regulates Toll-like Receptor 3 Stability and Endosomal Localization

“…Poly(A:U) was from Invivogen. Purified Reovirus double-stranded (ds) genomic RNAs were extracted from purified virions, and Reovirus S4 was made by in vitro transcription of the sense and antisense strands followed by annealing of a 1:1 ratio of the two RNAs as described in Lai et al (29).…”

“…In Huh7.5 cells, TLR9 signaling was also reduced in the presence of z-FA-FMK, whereas the effect on TLR3 was modest and not statistically different from the control treatments (supplemental Table 1). The human lung epithelial BEAS-2B cells express endogenous TLR3 and can secrete IL6 when induced with poly(I:C) (28,29). Poly(I:C)-activated BEAS-2B cells treated with z-FA-FIGURE 6.…”

The Human Antimicrobial Peptide LL-37, but Not the Mouse Ortholog, mCRAMP, Can Stimulate Signaling by Poly(I:C) through a FPRL1-dependent Pathway

“…LL-37 binding to lipopolysaccharides (LPS) can down-regulate signaling by Toll-like receptor 4 (TLR4), 2 binding to ssRNA can up-regulate TLR7 and TLR8 signaling, and interaction with flagellin can activate TLR5 signaling (3,7,8). Most relevant to this work, LL-37 binds doublestranded (ds) RNA, including poly(I:C) to enhance TLR3 signaling above the level observed with poly(I:C) alone (7,9,10). How LL-37 functions in concert with dsRNA to affect TLR3 signaling remains to be better understood.…”