More than two decades of intense research has provided a detailed understanding of hepatitis C virus (HCV), which chronically infects 2% of the world's population. This effort has paved the way for ...the development of antiviral compounds to spare patients from life-threatening liver disease. An exciting new era in HCV therapy dawned with the recent approval of two viral protease inhibitors, used in combination with pegylated interferon-α and ribavirin; however, this is just the beginning. Multiple classes of antivirals with distinct targets promise highly efficient combinations, and interferon-free regimens with short treatment duration and fewer side effects are the future of HCV therapy. Ongoing and future trials will determine the best antiviral combinations and whether the current seemingly rich pipeline is sufficient for successful treatment of all patients in the face of major challenges, such as HCV diversity, viral resistance, the influence of host genetics, advanced liver disease and other co-morbidities.
Hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases worldwide, but treatment options are limited. Basic HCV research required for vaccine and drug development has been ...hampered by inability to culture patient isolates, and to date only the JFH1 (genotype 2a) recombinant replicates spontaneously in hepatoma cells and releases infectious virus. A JFH1 chimera with the 5' end through NS2 from another genotype 2a strain, J6, had enhanced infectivity. However, the full-length J6 clone (J6CF), which we previously found to be fully functional in vivo, was replication incompetent in vitro. Through a systematic approach of culturing J6 with minimal JFH1 sequences, we identified three mutations in NS3, NS4A, and NS5B that permitted full-length J6 propagation and adaptation with infectivity titers comparable to JFH1-based systems. The most efficient recombinant, J6cc, had six adaptive mutations and did not accumulate additional changes following viral passage. We demonstrated that HCV NS3/NS4A protease-, NS5A- and NS5B polymerase-directed drugs respectively inhibited full-length J6 infection dose dependently. Importantly, the three J6-derived mutations enabled culture adaptation of the genetically divergent isolate J8 (genotype 2b), which differed from the J6 nucleotide sequence by 24%. The most efficient recombinant, J8cc, had nine adaptive mutations and was genetically stable after viral passage. The availability of these robust JFH1-independent genotype 2a and 2b culture systems represents an important advance, and the approach used might permit culture development of other isolates, with implications for improved individualized treatments of HCV patients and for development of broadly efficient vaccines.
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 of miRNA sites in vivo, leading to the discovery that miRNA 3'-end pairing is a general determinant of AGO binding specificity.
RNA-binding proteins (RBPs) are key players regulating RNA processing and are associated with disorders ranging from cancer to neurodegeneration. Here, we present a proteomics workflow for ...large-scale identification of RBPs and their RNA-binding regions in the mammalian brain identifying 526 RBPs. Analysing brain tissue from males of the Huntington's disease (HD) R6/2 mouse model uncovered differential RNA-binding of the alternative splicing regulator RBM5. Combining several omics workflows, we show that RBM5 binds differentially to transcripts enriched in pathways of neurodegeneration in R6/2 brain tissue. We further find these transcripts to undergo changes in splicing and demonstrate that RBM5 directly regulates these changes in human neurons derived from embryonic stem cells. Finally, we reveal that RBM5 interacts differently with several known huntingtin interactors and components of huntingtin aggregates. Collectively, we demonstrate the applicability of our method for capturing RNA interactor dynamics in the contexts of tissue and disease.
Background. Recently, a hepatitis C virus (HCV) cell-culture system was developed that employed strain JFH1 (genotype 2a), and JFH1-based intra- and intergenotypic recombinants now permit functional ...studies of the structural genes (Core, E1, and E2), p7, and NS2 of genotypes 1–4. The goal was to adapt the system to employ genotype 5. Methods. Huh7.5 cells infected with SA13/JFH1, containing Core-NS2 of strain SA13 (genotype 5a), were monitored for Core expression and for supernatant infectivity and HCV-RNA titers. Adaptive mutations of SA13/JFH1 were identified by sequence analysis of recovered genomes and reverse-genetic studies. Receptor blockage was performed with anti-CD81 and anti-SR-BI. For neutralization experiments, SA13/JFH1 or JFH1-based viruses of other genotypes were incubated with patient sera. Results. SA13/JFH1 with NS2 and NS3 mutations yielded infectivity titers >105 TCID50/mL. Infection with SA13/JFH1 was inhibited by CD81 blocking and SR-BI blocking, respectively, and by preincubation with genotype 5a chronic-phase patient sera. Such sera had varying cross-genotype neutralization potential. However, preincubation and treatment with homologous neutralizing antibodies could not control SA13/JFH1 infection in culture. Conclusion. The SA13/JFH1 culture permits genotype 5a-specific studies of Core-NS2 function and interfering agents. The ability of HCV to spread in vivo during treatment with neutralizing antibodies was confirmed in vitro.
The lack of a relevant, tractable, and immunocompetent animal model for hepatitis C virus (HCV) has severely impeded investigations of viral persistence, immunity, and pathogenesis. In the absence of ...immunocompetent models with robust HCV infection, homolog hepaciviruses in their natural host could potentially provide useful surrogate models. We isolated a rodent hepacivirus from wild rats (Rattus norvegicus), RHV‐rn1; acquired the complete viral genome sequence; and developed an infectious reverse genetics system. RHV‐rn1 resembles HCV in genomic features including the pattern of polyprotein cleavage sites and secondary structures in the viral 5′ and 3′ untranslated regions. We used site‐directed and random mutagenesis to determine that only the first of the two microRNA‐122 seed sites in the viral 5′ untranslated region is required for viral replication and persistence in rats. Next, we used the clone‐derived virus progeny to infect several inbred and outbred rat strains. Our results determined that RHV‐rn1 possesses several HCV‐defining hallmarks: hepatotropism, propensity to persist, and the ability to induce gradual liver damage. Histological examination of liver samples revealed the presence of lymphoid aggregates, parenchymal inflammation, and macrovesicular and microvesicular steatosis in chronically infected rats. Gene expression analysis demonstrated that the intrahepatic response during RHV‐rn1 infection in rats mirrors that of HCV infection, including persistent activation of interferon signaling pathways. Finally, we determined that the backbone drug of HCV direct‐acting antiviral therapy, sofosbuvir, effectively suppresses chronic RHV‐rn1 infection in rats. Conclusion: We developed RHV‐rn1‐infected rats as a fully immunocompetent and informative surrogate model to delineate the mechanisms of HCV‐related viral persistence, immunity, and pathogenesis. (Hepatology 2018).
Hepatitis C virus (HCV) uniquely requires the liver-specific microRNA-122 for replication, yet global effects on endogenous miRNA targets during infection are unexplored. Here, high-throughput ...sequencing and crosslinking immunoprecipitation (HITS-CLIP) experiments of human Argonaute (AGO) during HCV infection showed robust AGO binding on the HCV 5′UTR at known and predicted miR-122 sites. On the human transcriptome, we observed reduced AGO binding and functional mRNA de-repression of miR-122 targets during virus infection. This miR-122 “sponge” effect was relieved and redirected to miR-15 targets by swapping the miRNA tropism of the virus. Single-cell expression data from reporters containing miR-122 sites showed significant de-repression during HCV infection depending on expression level and site number. We describe a quantitative mathematical model of HCV-induced miR-122 sequestration and propose that such miR-122 inhibition by HCV RNA may result in global de-repression of host miR-122 targets, providing an environment fertile for the long-term oncogenic potential of HCV.
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•Genome-wide miRNA binding profiles were elucidated for HCV infection•HCV RNA functionally reduces miR-122 binding on endogenous mRNA targets•HCV miRNA sponging can be redirected by swapping viral miRNA tropism•Modeling validates single-cell measurements of HCV-induced mRNA de-repression
Hepatitis C virus uniquely requires the liver-specific tumor suppressor miRNA, miR-122, for its replication. During infection, viral RNA specifically sequesters miR-122 to de-repress its normal host targets, which may facilitate the long-term oncogenic potential of HCV.
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