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The hepatitis C virus (HCV) internal ribosome entry site (IRES) that directs cap-independent viral translation is a primary target for small interfering RNA (siRNA)-based HCV antiviral therapy. However, identification of potent siRNAs against HCV IRES by bioinformatics-based siRNA design is a challenging task given the complexity of HCV IRES secondary and tertiary structures and association with multiple proteins, which can also dynamically change the structure of this cis-acting RNA element. In this work, we utilized siRNA tiling approach whereby siRNAs were tiled with overlapping sequences that were shifted by one or two nucleotides over the HCV IRES stem-loop structures III and IV spanning nucleotides (nts) 277–343. Based on their antiviral activity, we mapped a druggable region (nts 313–343) where the targets of potent siRNAs were enriched. siIE22, which showed the greatest anti-HCV potency, targeted a highly conserved sequence across diverse HCV genotypes, locating within the IRES subdomain IIIf involved in pseudoknot formation. Stepwise target shifting toward the 5′ or 3′ direction by 1 or 2 nucleotides reduced the antiviral potency of siIE22, demonstrating the importance of siRNA accessibility to this highly structured and sequence-conserved region of HCV IRES for RNA interference. Nanoparticle-mediated systemic delivery of the stability-improved siIE22 derivative gs_PS1 siIE22, which contains a single phosphorothioate linkage on the guide strand, reduced the serum HCV genome titer by more than 4 log10 in a xenograft mouse model for HCV replication without generation of resistant variants. Our results provide a strategy for identifying potent siRNA species against a highly structured RNA target and offer a potential pan-HCV genotypic siRNA therapy that might be beneficial for patients resistant to current treatment regimens. [ABSTRACT FROM AUTHOR]

Abstract: Poly-γ-glutamic acid (γ-PGA) is an anionic polypeptide secreted by Bacillus sp. that has been shown to activate immune cells through interactions with toll-like receptor 4 (TLR4). However, its ability to induce the type I interferon (IFN) response has not yet been characterized. Here, we demonstrate that γ-PGA induces type I IFN signaling pathway via the TLR4 signaling pathway. The induction required both myeloid differentiation factor 2 (MD2) and the pattern-recognition receptor CD14, which are two TLR4-associated accessory proteins. The γ-PGA with high molecular weights (2000 and 5000 kDa) was able to activate the subsequent signals through TLR4/MD2 to result in dimerization of IRF-3, a transcription factor required for IFN gene expression, leading to increases in mRNA levels of the type I IFN-response genes, 2′–5′ OAS and ISG56. Moreover, γ-PGA (2000 kDa) displayed an antiviral activity against SARS coronavirus and hepatitis C virus. Our results identify high-molecular weight γ-PGA as a TLR4 ligand and demonstrate that γ-PGA requires both CD14 and MD2 for the activation of type I IFN responses. Our results suggest that the microbial biopolymer γ-PGA may have therapeutic potential against a broad range of viruses sensitive to type I IFNs. [Copyright &y& Elsevier]

Abstract: The hepatitis C virus (HCV) core protein is the primary protein component of the nucleocapsid that encapsidates the viral RNA genome. Besides its role as a viral structural protein, the core protein is implicated in HCV chronic infection-associated liver diseases by induction of reactive oxygen species (ROS) production and modulation of apoptosis. Here, we show that interaction of the core protein, through its N-terminal domain (amino acids 1–75), with heat shock protein (Hsp60) is critical for the induction of ROS production, leading to sensitization of core protein-expressing cells to apoptosis induced by tumor necrosis factor-α (TNF-α). Moreover, overexpression of Hsp60 rescued the core protein-expressing cells from cell death by reducing ROS production. Collectively, our results suggest that impairment of Hsp60 function through binding of HCV core protein contributes to HCV viral pathogenesis by ROS generation and amplification of the apoptotic effect of TNF-α. [Copyright &y& Elsevier]