2 and 3) Apart from being transcriptionally controlled by NF-κB,

2 and 3). Apart from being transcriptionally controlled by NF-κB, a subset of cytokines/chemokines contains AU-rich elements (AREs) in the 3′NTR of their mRNAs that regulate their expression selleck post-transcritionally

by controlling mRNA stability and/or translation. Among these cytokines/chemokines are IL-6, TNF-α, and MIP-1α,22 and so on, which were found to be induced in HCV-infected 7.5-TLR3 cells (Fig. 1). Although the TLR3-TRIF pathway is less capable of promoting the stabilization of ARE-containing mRNAs than are the myeloid differentiation primary response gene (88)–-dependent pathways,23 further studies are needed to investigate whether ARE-mediated enhancement Ipatasertib of mRNA stability/translation also contributes to TLR3-dependent up-regulation of specific proinflammatory mediators during HCV infection in hepatocytes. Our study defines the molecular features of HCV PAMP required for TLR3 activation as HCV dsRNA duplexes ≥80-100-bp, regardless of genome location or nucleotide composition. Although it is established that TLR3 senses dsRNA,24 whether TLR3 recognizes HCV RNA duplexes or the secondary structures present

in HCV RNA (such as the stem loops in the core- and NS5B-coding and 3′NTR regions) has not been determined previously. Our data demonstrate that HCV dsRNA duplexes generated during viral RNA replication are the ligands for TLR3 (Fig. 5), whereas the secondary structures present in either +ss or –ss HCV RNAs are not, as evidenced by the finding that neither strand of the NS-3′NTR RNA containing complex stem-loop structures in NS5B and 3′NTR sequences activated RANTES expression (Fig. 5B). Further supporting this, the +ss and –ss HCV RNAs derived from the HCV core-coding region, which contains two stem-loops,17 also failed to activate TLR3, unless the two RNA strands were annealed to form dsRNA duplexes (data not shown). We speculate that the highly structured HCV ssRNAs may not present the perfect

dsRNA structure required for binding to and/or form a stable complex with TLR3, as opposed to HCV dsRNA duplexes. RIG-I preferentially recognizes a segment of HCV 上海皓元 3′NTR RNA rich in poly-U/UC nucleotides.11 This is not the case for TLR3, because HCV dsRNA duplexes derived from core, E-p7, NS5A, and NS-3′NTR regions all activated TLR3 with similar potency (Fig. 5C), regardless of their nucleotide compositions. This suggests that the dsRNA duplex structure is the sole determinant for the HCV dsRNA recognition by TLR3, as long as the dsRNA meets the minimal length requirement (see below). We found that HCV dsRNA has to be at least 80-100-bp to reproducibly activate TLR3 and trigger chemokine induction (Fig. 6). This is consistent with the intracellular localization of TLR3 expressed in reconstituted 7.

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