tRNA-derived RNA fragments (tRFs) have emerged as a new class of functional RNAs implicated in cancer, metabolic and neurological disorders, and viral infection. Yet our understanding of their biogenesis and functions remains limited. In the present study, through analysis of small RNA profile we have identified a distinct set of tRFs derived from pre-tRNA 3′ trailers in the hepatocellular carcinoma cell line Huh7. Among those tRFs, tRF_U3_1, which is a 19-nucleotide-long chr10.tRNA2-Ser(TGA)-derived trailer, was expressed most abundantly in both Huh7 and cancerous liver tissues, being present primarily in the cytoplasm. We show that genetic loss of tRF_U3_1 does not affect cell growth and it is not involved in Ago2-mediated gene silencing. Using La/SSB knockout Huh7 cell lines, we demonstrate that this nuclear-cytoplasmic shuttling protein directly binds to the 3′ U-tail of tRF_U3_1 and other abundantly expressed trailers and plays a critical role in their stable cytoplasmic accumulation. The pre-tRNA trailer-derived tRFs capable of sequestering the limiting amounts of La/SSB in the cytoplasm rendered cells resistant to various RNA viruses, which usurp La/SSB with RNA chaperone activity for their gene expression. Collectively, our results establish the trailer-derived tRF-La/SSB interface, regulating viral gene expression.

No abstract

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B), an RNA-dependent RNA polymerase (RdRp), is the key enzyme for HCV RNA replication. We previously showed that HCV RdRp is phosphorylated by protein kinase C-related kinase 2 (PRK2). In the present study, we used biochemical and reverse-genetics approaches to demonstrate that HCV NS5B phosphorylation is crucial for viral RNA replication in cell culture. Two-dimensional phosphoamino acid analysis revealed that PRK2 phosphorylates NS5B exclusively at its serine residues in vitro and in vivo. Using in vitro kinase assays and mass spectrometry, we identified two phosphorylation sites, Ser29 and Ser42, in the ⌬1 finger loop region that interacts with the thumb subdomain of NS5B. Colonyforming assays using drug-selectable HCV subgenomic RNA replicons revealed that preventing phosphorylation by Ala substitution at either Ser29 or Ser42 impairs HCV RNA replication. Furthermore, reverse-genetics studies using HCV infectious clones encoding phosphorylation-defective NS5B confirmed the crucial role of these PRK2 phosphorylation sites in viral RNA replication. Molecular-modeling studies predicted that the phosphorylation of NS5B stabilizes the interactions between its ⌬1 loop and thumb subdomain, which are required for the formation of the closed conformation of NS5B known to be important for de novo RNA synthesis. Collectively, our results provide evidence that HCV NS5B phosphorylation has a positive regulatory role in HCV RNA replication. IMPORTANCEWhile the role of RNA-dependent RNA polymerases (RdRps) in viral RNA replication is clear, little is known about their functional regulation by phosphorylation. In this study, we addressed several important questions about the function and structure of phosphorylated hepatitis C virus (HCV) nonstructural protein 5B (NS5B). Reverse-genetics studies with HCV replicons encoding phosphorylation-defective NS5B mutants and analysis of their RdRp activities revealed previously unidentified NS5B protein features related to HCV replication and NS5B phosphorylation. These attributes most likely reflect potential structural changes induced by phosphorylation in the ⌬1 finger loop region of NS5B with two identified phosphate acceptor sites, Ser29 and Ser42, which may transiently affect the closed conformation of NS5B. Elucidating the effects of dynamic changes in NS5B phosphorylation status during viral replication and their impacts on RNA synthesis will improve our understanding of the molecular mechanisms of NS5B phosphorylation-mediated regulation of HCV replication.

No abstract

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.