Eiko Nishiuchi

microRNAs (miRNAs) act as sequence-specific guides for Argonaute (AGO) proteins, which mediate posttranscriptional silencing of target messenger RNAs. Despite their importance in many biological processes, rules governing AGO-miRNA targeting are only partially understood. Here we report a modified AGO HITS-CLIP strategy termed CLEAR (covalent ligation of endogenous Argonaute-bound RNAs)-CLIP, which enriches miRNAs ligated to their endogenous mRNA targets. CLEAR-CLIP mapped ∼130,000 endogenous miRNA-target interactions in mouse brain and ∼40,000 in human hepatoma cells. Motif and structural analysis define expanded pairing rules for over 200 mammalian miRNAs. Most interactions combine seed-based pairing with distinct, miRNA-specific patterns of auxiliary pairing. At some regulatory sites, this specificity confers distinct silencing functions to miRNA family members with shared seed sequences but divergent 3'-ends. This work provides a means for explicit biochemical identification...

Nonprimate hepacivirus (NPHV) is the closest known relative of hepatitis C virus (HCV) and its study could enrich our understanding of HCV evolution, immunity, and pathogenesis. High seropositivity is found in horses worldwide with ∼3% viremic. NPHV natural history and molecular virology remain largely unexplored, however. Here, we show that NPHV, like HCV, can cause persistent infection for over a decade, with high titers and negative strand RNA in the liver. NPHV is a near-universal contaminant of commercial horse sera for cell culture. The complete NPHV 3'-UTR was determined and consists of interspersed homopolymer tracts and an HCV-like 3'-terminal poly(U)-X-tail. NPHV translation is stimulated by miR-122 and the 3'-UTR and, similar to HCV, the NPHV NS3-4A protease can cleave mitochondrial antiviral-signaling protein to inactivate the retinoic acid-inducible gene I pathway. Using an NPHV consensus cDNA clone, replication was not observed in primary equine fetal liver...

Hepatitis C virus (HCV) is the most frequent cause of mixed cryoglobulinemia (MC), which is characterized by endothelial deposition of rheumatoid factor (RF)-containing immune complexes and end-organ vasculitis. MC is a lymphoproliferative disorder in which B cells express RF-like Ig, yet its precise antigenic stimulus is unknown. We have proposed that IgG-HCV immune complexes stimulate B cell expansion and somatic hypermutation (SHM)-induced affinity maturation in part via engagement of an RF-like B cell receptor. This study was undertaken to test the hypothesis that SHM augments RF activity. RFs cloned from single B cells from 4 patients with HCV-associated MC (HCV-MC) were expressed as IgM, IgG, or IgG Fab. Selected Ig were reverted to germline. RF activity of somatically mutated Ig and germline-reverted Ig was determined by enzyme-linked immunosorbent assay. Ig with SHM had RF activity, with the preference for binding being highest for IgG1, followed by IgG2 and IgG4, and lowest for IgG3, where there was no detectable binding. In contrast, reverted germline IgG exhibited markedly diminished RF activity. Competition with 1 μg/ml of protein A abrogated RF activity, suggesting specificity for IgG Fc. Swapping of mutated heavy-chain pairs and light-chain pairs also abrogated RF activity, suggesting that context-specific pairing of appropriate IgH and Igκ, in addition to SHM, is necessary for RF activity. SHM significantly contributes to RF activity in HCV-MC patients, suggesting that autoreactivity in these patients arises through antigen-dependent SHM, as opposed to nondeletion of autoreactive germline Ig.